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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 06, 2013 - March 24, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Nopcote 1661
- Physical state: Light yellow, clear liquid (solution)
- Analytical purity: 46.3% w/w (in water solution)
- Lot/batch No.: 79389
- Expiration date of the lot/batch: 11 July 2013
- Storage condition of test material: Controlled room temperature (15-25 C, below 70 RH%)
- Stability under test conditions: A non-GLP 24-hours stability of the test item in the vehicle assessed, CiToxLAB study code: 12/331-929AN

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Laboratories, Research Models and Services, Germany GmbH- Age at study initiation: Approx. 10 weeks at starting and 12 weeks at mating.- Weight at study initiation: Males: 362–391 g, Females: 195-225 g - Housing: up to 5 animals per sex and cage type II and/or III polycarbonatem, with lignocel bedding.- Diet (e.g. ad libitum): Ad libitum- Water (e.g. ad libitum): Ad libitum- Acclimation period: 6 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 18.5-23.6 ºC- Humidity (%): 33-68 %- Air changes (per hr): 15-20 air chainges per hour- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(distilled)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:The test item was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected. Formulations were prepared fresh prior to administration to animals. The amount of the dose volumes was formulated considering the density of the test item and then they were corrected with the purity of the test item in water solution.VEHICLE- Amount of vehicle (if gavage): 1.71 mL/kg bw- Lot/batch no. (if required): 3450611 / 4310612
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the formulations for concentration and homogeneity was performed using validated spectrophotometric method (CiToxLAB study code 12/331-316AN). Control, top, middle and bottom duplicate samples were taken from test item formulations on 3 occasions, during the first and last weeks and approximately midway during the treatment, one set to analyse and the other for back-up. No test item was identified in the control samples. The test item formulation appeared to be homogenous and had actual concentrations of 99-105% of the nominal concentrations, within the 100±10% acceptable range. These results were considered suitable for the study purposes.
Duration of treatment / exposure:
2 weeks before mating, during the mating, and continued up to and including the day before the necropsy.
Frequency of treatment:
Daily, 7 days per week.
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 animals per sex and per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based the previous repeated dose range finding study in the rat (CiToxLAB study code 12/331-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Test item administrated to Wistar rats at 62.5, 250 and 1000 mg/kg bw/day, daily for 7 consecutive days, was not associated with any overt adverse effects that could be clearly ascribed to test item administration other than a transient body weight and food intake effect in the first few days of treatment in males at the High dose. Based on the results, the dose levels selected for the main study were 0, 62.5, 250, 1000 mg/kg bw/day.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: Twice daily for morbidity and mortalityOnce daily for general clinical observations and behavious.DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: Once before the first exposure, and at least weekly.- Observations: skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were observed during the study. BODY WEIGHT: Yes- Time schedule for examinations: before treatment for randomization purposes, on Day 0, afterwards at least weekly and at termination.FOOD CONSUMPTION:- Animal food consumption was determined by re-weighing the non-consumed diet on Day 7 and then at least weekly.HAEMATOLOGY: Yes- Time schedule for collection of blood: Immediately prior to scheduled necropsy.- Anaesthetic used for blood collection: Yes, pentorbital anaesthesia.- Animals fasted: Yes- How many animals: 5 rats per sex and per dose (subgroup B)- Parameters checked: Red Blood Cell (erythrocyte), White Blood Cell (leukocyte), Haemoglobin concentration, Haematocrit (relative volume of erythrocytes), Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Haemoglobin, Mean Corpuscular (erythrocyte) Haemoglobin Concentration, Red Cell (erythrocyte) volume, Platelet (thrombocyte) count, Mean Platelet Thrombocyte volume, Reticulocyte count, Neutrophil, Lymphocyte, Monocyte, Basophil, Eosinophil, Large Unstained Cells, Activated Partial Thromboplastin Time, Prothrombin Time.CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: Immediately prior to scheduled necropsy.- Anaesthetic used for blood collection: Yes, pentorbital anaesthesia.- Animals fasted: Yes- How many animals: 5 rats per sex and per dose (subgroup B)- Parameters checked: Blood sugar concentration, Total Bilirubin concentration, Urea concentration, Cholesterol concentration, Creatinine concentration, Phosphorus concentration, Sodium concentration, Potassium concentration, Calcium concentration, Chloride concentration, Total Protein concentration, Albumin concentration, Alb/glob ration, Alanine Aminotransferase activity, Alkaline. Phosphatase – activity, Gamma Glutamyltransferase -activity, Bile acids. URINALYSIS: Yes- Time schedule for collection of urine: Prior to scheduled necropsy- Metabolism cages used for collection of urine: Yes (16 hours)- Animals fasted: Yes- How many animals: 5 rats per sex and per dose (subgroup B)- Parameters checked: Leukocyte, Nitrite, pH, Protein, Glucose, Urobilinogen, Bilirubin, Ketones, ERY Blood, Erythrocytes, Specific Gravity, Sediment, Volume, Colour/Appearance.NEUROBEHAVIOURAL EXAMINATION: Yes - Time schedule for examinations: During the last exposure week (males on Day 24, females on PND 4).- Dose groups that were examined: 5 rats per sex and per dose (subgroup A)- Battery of functions tested: sensory activity / grip strength / motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: YesAll animals were necropsied. External appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.BODY WEIGHT AND ORGAN WEIGHTS: YesIn all animals, uterus (with and without cervix), vagina, testes, epididymides, prostate, seminal vesicles with coagulating glands, brain. ovaries and pituitary were measured. For 5 rats per sex and group (subgroup B), heart, kidneys, liver, spleen, thymus and adrenals were also weighted. Paired and individual organ weights were summarised. Relative organ weight (to body and brain weight) were calculated.HISTOPATHOLOGY: YesThe weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. For 5 rats per sex and group (subgroup B), the following organs and tissues were preserved: Gross findings, Adrenal glands, Animal identification, Aorta (thoracic and abdominal), Brain, Clitoral gland / Preputial gland, Epididymes, Eye with the optic nerve, Oesophagus, Femur with marrow incl. joint, Heart, Kidney, Large intestine, External lachrymal gland, Harderian gland, Liver, Lungs with bronchi, Lymph nodes, Larynx, Nasopharynx, Ovaries with oviduct, Pancreas, Pituitary, Prostate, Salivary glands, Sciatic nerve, Seminal vesicles (with coagulating glands), Skeletal muscle (quadriceps), Skin/subcutis with mammary, gland area, Small intestine, Spinal cord (cervical, lumbar, and thoracic levels), Spleen, Sternum with marrow, Stomach, Testis, Thymus, Thyroid with parathyroid gland, Tongue, Trachea, Urinary bladder, Uterus, Vagina.Detailed histological examination were performed in the control and high dose groups and all macroscopic findings from all animals.
Statistics:
The statistical evaluation was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs related to treatment.
Mortality:
no mortality observed
Description (incidence):
There was no mortality during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males treated at 1000 mg/kg, transient lower body weight and body weight gain values were observed during the pre-mating period. The mean body weight value at termination was comparable to the control. The mean body weight values of females in all test item treated groups were comparable to the control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Compared to control, lower food consumption values, attaining statistical significance were noted for males at 1000 mg/kg (High dose) during the first week of the treatment. These changes were generally correlated with the body weight and body weight gain values. In addition, slightly lower food consumption was observed during the mating period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant differences between the control and any of treated groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Minor changes at 1000 mg/kg bw/day. Higher bile acid concentration, attaining statistical significance were noted in High dose male and female animals. Compared to the control higher potassium and serum urea concentration (Urea) were observed in Mid and High dose female animals. No signs of kidney injury were observed during pathology evaluation.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Minor changes at 1000 mg/kg bw/day. Urobilinogen and urinary bilirubin, calcium oxalate crystals as well as dark yellow discoloration of the urine was presented in high dose males. In high dose females and in one from the mid dose, calcium oxalate crystals were also presented in the urine. Dark yellow discoloration of the urine was observed in one mid dose female. Semi quantitative measurement of protein in the urine showed a slight elevation in high dose, when compared to the control.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects on organ weights.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Single or multiple ulcers of the non-glandular mucosa of the stomach (4/12 High dose males and 4/12 High dose females) were observed as treatment-related macroscopic findings. Other minor changes were considered incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related ulcers of the non-glandular mucosa of the stomach were microscopically observed in 4/7 High dose males and 4/7 High dose females, and were in correlation with necropsy. There was no evidence of test item-related histological findings in the High dose animals or macroscopic observations from all groups in the reproductive organs.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Basis for effect level: minor changes in clinical chemistry and urinalysis parameters and ulcers of the non-glandular mucosa of the stomach at 1000 mg/kg bw/day.

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the minor changes in clinical chemistry and urinalysis parameters and the ulcers of the non-glandular mucosa of the stomach at 1000 mg/kg bw/day dose level, the no adverse effect level (NOAEL) was determined to be 250 mg/kg bw/day in male and female rats after at least 28 days of test item treatment by oral gavage.
Executive summary:

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed in accordance with OECD Guideline 422 following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels, 62.5, 250 and 1000 mg/kg bw/day. The control animals were treated with the vehicle only (distilled water). 12 Male and female Wistar rats per group were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period, up to and including postpartum/lactation Day PPD4. Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. At termination, necropsy with macroscopic examination was performed. Selected organs were subjected to histopathology examination. No mortality was observed during the study. There were no clinical signs related to treatment. There was no effect of treatment noted during evaluation of the functional observation battery, grip strength, foot splay or motor activity. In males treated at 1000 mg/kg, transient lower body weight and body weight gain values were observed during the pre-mating period. The mean body weight value at termination was comparable to the control. The mean body weight values of females in all test item treated groups were comparable to the control. Single or multiple ulcers of the non-glandular mucosa of the stomach (4/12 High dose males and 4/12 High dose females) were observed as treatment-related macroscopic findings. There was no effect of treatment on organ weights. Test item related microscopic findings were found at 1000 mg/kg bw/day (High dose). In the stomach, ulcers of the non-glandular mucosa were microscopically observed in 4 High dose males and 4 High dose females. There were no microscopic findings related to treatment at 62.5 or 250 mg/kg bw/day. Lower food consumption values were noted for males at 1000 mg/kg during the first week of the treatment. These changes generally correlated with the body weight and body weight gain values. There was no effect of treatment on haematology and blood clotting parameters. Increases were noted in potassium and/or urea and bile acids in high dose males and females. Calcium oxalate crystals were noted in high dose males and females, with increases in urobilinogen and bilirubin in males.

Based on the minor changes in clinical chemistry and urinalysis parameters and the ulcers of the non-glandular mucosa of the stomach at 1000 mg/kg bw/day dose level, the no adverse effect level (NOAEL) was determined to be 250 mg/kg bw/day in male and female rats after at least 28 days of test item treatment by oral gavage.