Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-03-26 - 1998-12-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted on 27th July 1995
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Substance Law 1987 (December 9, 1986) by Environmental Agency (no. 700), MHW (10-93) and MITI (1014)
Deviations:
not specified
Principles of method if other than guideline:
Guideline study (OECD TG 407) performed under GLP. With regard to the current version of the guideline, prescribed wet weighing of coagulating glands, prostate and seminal vesicles were not performed. Furthermore, some optional parameters (hormones measurement) to detect endocrine disrupters were not determined. However, mandatory histopathological endpoints (gonads, accessory sex organs, adrenal, thyroid, and vagina) and some optional histopathological endpoints (mammary gland and pituitary) recommended for the detection of endocrine disruptors were included in this reliable study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan (received on May 14, 1998)
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 182 - 204 g; females 152 - 177 g (at day -2, randomization)
- Housing: individually in suspended stainless steel wire mesh cages, except during urine collection, when they were transferred to urine collection cages
- Diet: PMI Certified Rodent Chow #5002 (Purina Mills, Inc.), ad libitum
- Water: Municipal tap water, ad libitum
- Acclimation period: 14 days prior to in-life initiation (animals were examined upon receipt and daily following separation from gang-housing (day -10) for signs of physical or behavioral abnormalities. General health/mortality and moribundity checks were performed twice daily. Individual body weights were recorded following separation from gang-housing (day -10) and prior to randomization (day -2)).
- Recovery period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 65 - 79 °F [approximately 18 - 26 °C] (recorded daily)
- Humidity: 30 - 70 % (recorded daily)
- Air changes (per hr): 10 – 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES:
- From: 1998-05-28 To: 1998-07-08 (28-day study)
- From: 1998-04-01 To: 1998-04-15 (14-day range-finding study)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Mazola corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
An appropriate amount of the test item was weighed into a beaker. A sufficient quantity of corn oil was added to the beaker to achieve the desired concentration, the mixture was placed on a heated stir plate, heated to approximately 50°C and stirred until homogenous. Each dosing mixture was prepared fresh weekly, dispensed into daily aliquots and stored refrigerated. During each preparation, an appropriate amount of the vehicle was also dispensed into daily aliquots and stored refrigerated for administration to control animals. The physical state of the vehicle and each test item dosing mixture was recorded during each preparation. The vehicle control was a clear yellow liquid, the low- and mid-dose mixtures were clear yellow solutions and the high-dose mixture was a light amber solution (28-day study) or a yellow suspension (14-day range-finding study). Dally aliquots of the dosing mixtures were removed from the refrigerator and allowed to equilibrate to room temperature prior to dispensing. The mixtures were stirred continuously following dispensing until dosing was complete.

TREATMENT:
- Oral by gavage, single dose daily

VEHICLE AND DOSE VOLUME:
- Corn oil (Mazola corn oil)
- 5 mL/kg body weight and treatment day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to initiation of the 28-day study, homogeneity and stability analyses were performed on concentrations of the test item in the vehicle which encompassed the low- and high-doses administered in this study. Homogeneity analyses were performed on duplicate samples taken from the top, middle and bottom of the two mixtures. Stability of the test item in the vehicle was evaluated on duplicate samples at 1, 5 and 9 days following preparation and refrigerated storage. Concentration verification analysis was performed on the vehicle and each test item dosing mixture on weeks 1, 2, 3 and 4.
The test item was homogeneously distributed and stable in the vehicle for up to eight days post-preparation when stored refrigerated. Analysis of dosing mixtures resulted in average test item recoveries ranging from 93.0 to 104.7%, indicating that the mixtures were accurately prepared.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days per week.
Doses / concentrationsopen allclose all
Dose / conc.:
50 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:
toxicity testing: 5/sex/dose (0, 50, 200, 1000 mg/kg bw/d)
recovery testing: 5/sex/dose (0, 1000 mg/kg bw/d)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based upon data from a 14-day oral toxicity (range-finding) study (3 rats/sex/dose level, doses: 0, 125, 250, 500, 1000, 1500 mg/kg bw/d)
- Randomization: Computer-generated random algorithm
- Post-exposure recovery period in satellite groups: 14-day recovery period

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- General health/mortality and moribundity checks were performed twice daily, in the morning and afternoon.
- A (detailed) clinical observation was performed for each animal dally, between one-half hour and two hours following dosing and dally during the recovery phase.

BODY WEIGHT: Yes
- Individual body weights were recorded on days -2, 8, 15, 22 and 28 (during the treatment phase) and days 35 and 41 (during the recovery phase). In addition, a terminal body weight was recorded on the day of scheduled necropsy (days 29/30 or 42) for calculation of relative organ weight data.

FOOD CONSUMPTION: Yes
- Food consumption was recorded on days 1, 8, 15, 22 and 28 (during the treatment phase) and days 35 and 41 (during the recovery phase). Food consumption was reported as grams/animal/day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- near the end of the treatment phase (day 26) and recovery phase (day 40). Pupils were dilated using 0.5% Mydriacyl ophthalmic solution prior to biomicroscopic and indirect ophthalmoscopic examination.

CLINICAL PATHOLOGY
Blood was collected from all animals on the day of scheduled euthanasia at the end of the treatment phase (day 29 for males/day 30 for females) or the recovery phase (day 42 for males/females) for evaluation of selected hematology, coagulation and biochemistry parameters. The blood samples were obtained via the orbital plexus while the animals were under light Isoflurane anesthesia. If values could not be obtained for coagulation analysis on day 29/30, additional blood samples were collected from the vena cava at necropsy, when possible. For day 42 coagulation analysis, all blood samples were collected from the vena cava at necropsy. Feed was withheld overnight prior to blood collection, however, water was available.

HAEMATOLOGY: Yes
- Parameters examined: Erythrocyte count (RBC), Hematocrit (Hct), Hemoglobin concentration (Hgb), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Mean corpuscular volume (MCV), Platelet count, Total and differential leukocyte counts, Coagulation: Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Parameters examined: Alanine aminotransferase (ALT), Albumin, Albumin/globulin ratio (calculated), Alkaline phosphatase, Aspartate aminotransferase (AST), Calcium, Cholesterol, Blood creatinine, Globulin (calculated), Glucose, Electrolytes (sodium, potassium and chloride), Phosphorus, Total bilirubin, Total serum protein, Urea nitrogen

URINALYSIS: Yes
- Urine samples were collected from all animals overnight prior to scheduled euthanasia at the end of the treatment phase (day 29 for males/day 30 for females) or the recovery phase (day 42 for males/females). Each rat was housed in a urine collection cage without food, however, the animals were allowed access to water.
- Parameters examined: Overnight volume, Colour and appearance, pH, Specific gravity, Protein, Glucose, Ketones, Urobilinogen, Nitrites, Bilirubin, Occult blood, Leukocytes, Microscopy of spun deposit

DETAILED CLINICAL OBSERVATIONS: yes
- Functional Observation Battery (FOB): An abbreviated functional observation battery (home cage, removal from home cage and open field) was performed on days - 1 , 6, 13 and 20 for each animal. A full FOB (home cage, removal from home cage, open field, manipulative tests and motor activity) was performed on days 27/28 and 40/41. All FOB assessments (except the pretest) were performed blind (animals were not identified by group). FOB involved Home Cage Observations (e.g. body posture), Removal from Home Cage Observations (e.g. reactivity to being handled), Open Field Observations (e.g. mobility score), Manipulative Tests (e.g. touch response), and Motor Activity.
Sacrifice and pathology:
GROSS PATHOLOGY/NECROPSY: Yes
All animals were subjected to a complete gross examination at scheduled necropsy (days 29/30 or 42). The necropsy examination included evaluation of the external surfaces of the body and all viscera. At the end of the treatment phase, five males/group were necropsied on day 29 and five females/group were necropsied on day 30. At the end of the recovery phase, the remaining five males and five females in the control and high-dose groups were necropsied on day 42. The animals were killed by carbon dioxide inhalation followed by exsanguination. The animals were fasted overnight prior to necropsy.
Fresh organ weights were obtained at scheduled necropsy for the liver, kidneys, adrenal glands, testes with epididymides, ovaries, spleen, thymus, brain and heart of all animals. Paired organs were weighed together. The following organs and tissues were preserved in 10% neutral buffered formalin: Accessory genital organs (epididymides, seminal vehicles, and prostate or uterus and vagina), Adrenals, All gross lesions, Aorta, Brain (Including sections of medulla/pons, cerebellar cortex, and cerebral cortex), Cecum, Colon, Duodenum, Esophagus, Exorbital lachrymal glands, Eyes with optic nerve, Femur (including articular surface) and bone marrow, Heart, Ileum, Jejunum, Kidneys, Liver (3 sections collected), Lungs (Infused with formalin) with bronchi, Mammary gland, Mandibular lymph node, Mediastinal lymph node, Mesenteric lymph node, Pancreas, Peripheral nerve (sciatic), Pituitary, Rectum, Skeletal muscle (thigh), Skin, Spinal cord (cervical, midthoracic and lumbar), Spleen, Sternum with bone marrow, Stomach (glandular/nonglandular), Submaxillary salivary gland, Testes/ovaries, Thymus, Thyroid/parathyroid, Tongue, Trachea, Urinary bladder.

HISTOPATHOLOGY: Yes
All tissues and organs collected at necropsy (days 29/30 or 42) from animals in the control and high-dose groups were processed for histopathological examination. The tissues were trimmed, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Histology was performed by HistoTechnlques, Powell, Ohio.
Statistics:
Data, including body weights, weight gain, food consumption, hematology, coagulation, biochemistry, organ weights and appropriate urinalysis parameters were analyzed by one-way analysis of variance (ANOVA). When significance was observed with ANOVA, group by group comparisons were performed using the Tukey-Kramer method. All tests were two-tailed with a minimum significance level of 5%.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A slight increase In the incidence of increased activity was noted for males and females in the 1000 mg/kg bw/d group during the treatment and recovery phases. No other remarkable clinical signs were observed for males or females in any of the study groups.
Mortality:
no mortality observed
Description (incidence):
All animals survived to scheduled necropsy at the end of the 28-day phase (day 29/30) or the recovery phase (day 42).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant or toxicologically meaningful differences in mean body weights or body weight gain among the groups during the treatment or recovery phases.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no statistically significant or toxicologically meaningful differences in mean food consumption among the groups during the treatment or recovery phases.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test item-related ocular abnormalities were noted in any of the study animals.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically meaningful differences in hematology or coagulation data among the groups during the treatment or recovery phases. Erythrocyte counts for males in the 1000 mg/kg bw/d group were statistically decreased at the end of the recovery phase (day 42). The decreased erythrocyte counts were not considered toxicologically meaningful since the value was within the range of historical control data and a similar decrease did not occur at the end of the treatment phase (day 29/30). In addition, similar decreases were not observed in other red cell parameters evaluated and there was no correlative organ pathology. No other statistically significant differences were noted in the hematology or coagulation parameters evaluated at the end of the treatment or recovery phases.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically meaningful differences In biochemistry data among the groups during the treatment or recovery phases. Globulin for males in the 1000 mg/kg bw/d group was statistically increased at the end of the treatment phase (day 29). The increased globulin was not considered toxicologically meaningful since the value was within the range of historical control data. In addition, similar differences were not observed In related biochemistry parameters and there was no correlative organ pathology. No other statistically significant differences were rioted in the biochemistry parameters evaluated at the end of the treatment or recovery phases.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no toxicologically meaningful differences among the groups in the urinalysis parameters evaluated at the end of the treatment or recovery phases.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There was no evidence of neurological effects following treatment with the test article, the functional observation battery, which included home cage, removal from home cage, open field, manipulative testing, and motor activity, indicated comparable results among the treated and untreated groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically meaningful differences in absolute or relative organ weights among the groups at the end of the treatment or recovery phases. Mean absolute and relative kidney weights for females in the 1000 mg/kg bw/d group were statistically decreased at the end of the recovery phase (day 42). The decreased kidney weights were not considered toxicologically meaningful since similar decreases did not occur at the end of the treatment phase (day 29/30) and the decreases did not correlate with any abnormal biochemistry or pathological changes in this organ. No other statistically significant differences in absolute or relative organ weights were noted among the groups at the end of the treatment or recovery phases.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No remarkable gross necropsy observations were noted for males or females at the end of the treatment or recovery phases.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test item-related microscopic changes were observed in any of the organs or tissues examined from rats In the 1000 mg/kg bw/d group. All microscopic changes were considered spontaneous and unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

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 test item related effects were observed.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

RANGE-FINDER

In a previous subacute 14-day range-finding study dosages of 0, 125, 250, 500, 1000, and 1500 mg test item/kg bw/d were administered to rats by gavage. No mortality occurred during the preliminary study. Clinical signs were limited to scabs on the right lateral neck of one male each in the 125, 1000 and 1500 mg/kg bw/d groups, hair loss in one male each from the 125, 500 and 1500 mg/kg bw/d groups, dark material around the nose of one male each in the 500 and 1500 mg/kg/day groups, and fecal stain in one male of the 1500 mg/kg bw/d group. No clinical signs were observed for females in any of the study groups. There were no consistent dose-related differences in mean body weights or body weight gain among the groups during the study. No remarkable gross necropsy findings were observed in any of the study animals. Thus, based on the results, dosage levels of 50, 250 and 1000 mg/kg bw/d were selected for the 28-day oral toxicity study in rats.

Applicant's summary and conclusion

Conclusions:
Based on the above results, a dosage level of 1000 mg/kg/day was considered the no-observed-adverse-effect level (NOAEL) following 28-day oral administration of the test article to rats.
Executive summary:

The potential toxicity of the test item ws investigated when administered orally, by gavage, to rats for a minimum of 28 consecutive days followed by a 14-day recovery phase. The study design consisted of a control group and three treatment groups with ten animals per sex In the control and high-dose groups and five animals per sex In the low- and mid-dose groups. The test article was administered as a single dally dose at dosage levels of 50, 250 and 1000 mg/kg/day for a minimum of 28 consecutive days. Control animals received corn oil under the same experimental conditions. A detailed clinical observation was performed daily, between one-half hour and two hours following dosing and dally during the recovery phase. A functional observation battery was performed on days - 1 , 6, 13, 20, 27/28 for each animal; and on day 40/41 for animals euthanized following the recovery phase. Individual body weights were recorded on days -2, 8, 15, 22 and 28 (during the treatment phase) and days 35 and 41 (during the recovery phase). In addition, a terminal body weight was recorded on the day of scheduled euthanasia (days 29/30 or 42). Food consumption was recorded on days 1, 8, 15, 22 and 28 (during the treatment phase) and days 35 and 41 (during the recovery phase). Blood and urine samples were obtained from all animals on the day of scheduled euthanasia (days 29/30 or 42) for evaluation of selected clinical pathology parameters. Ophthalmology examinations were performed on all animals on day 26 and for recovery animals on day 40. Each rat was subjected to a complete gross necropsy examination at scheduled euthanasia (days 29/30 or 42). At the end of the treatment phase, five males/group were euthanized on day 29 and five females/group were euthanized on day 30. The remaining five males and five females In the control and high-dose groups were euthanized at the end of the recovery phase (day 42). Fresh organ weights were obtained for all animals and selected tissues were preserved from all rats. All tissues and organs collected at necropsy from animals In the control and high-dose groups were examined microscopically. Results: All animals survived to scheduled euthanasia at the end of the 28-day phase (day 29/30) or the recovery phase (day 42). A slight Increase in the incidence of increased activity was noted for males and females in the 1000 mg/kg/day group during the treatment and recovery phases. No other remarkable clinical signs of toxicity were observed for males and females In any of the study groups. There were no toxicologically meaningful differences among the groups with respect to the functional observation battery, body weights, body weight gain, food consumption, hematology, coagulation, biochemistry, urinalysis or organ weights during the treatment or recovery phases. No test article-related ocular abnormalities were noted In any of the study animals and no remarkable gross necropsy observations were noted for males or females at the end of the treatment or recovery phases. No test article-related microscopic changes were observed in any of the organs or tissues examined. All microscopic changes were considered spontaneous arid unrelated to treatment. Based on the above results, a dosage level of 1000 mg/kg/day was considered the no-observed-adverse-effect level (NOAEL) following 28-day oral administration of the test article to rats.