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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 25, 1988 through August 22, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Well documented study conducted in compliance with GLP and using a method similar to OECD guideline 408 with only minor deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Animals dosed with test substance daily for 5 days a week and not 7, only checked for mortality once a day instead of twice, no analytical data, and analytical purity is unknown in the report.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): 5601-57-2
- Physical state: clear colorless liquid
- Analytical purity: responsibility of the Sponsor
- Stability under test conditions: no apparent change in the physical state of the test article during administration
- Storage condition of test material: no data
- Other:specific gravity= 1.203 gm/ml

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Wilmington, Massachusetts
- Age at study initiation: 40 days
- Fasting period before study: fasted overnight prior to the time of sacrifice.
- Housing: Individually in stainless steel 1/2" wire mesh cages.
- Diet: Purina Certified Rodent Lab Meal, ad libitum.
- Water: fresh tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 degree C +/- 3 degree C
- Humidity (%): 30 to 70%
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: deionized water (5 mL/kg)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test article was administered as received (undiluted) on a mg/kg basis

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily, 5 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
3 000 mg/kg bw/day (nominal)
Dose / conc.:
5 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3 groups of 30 rats (15 male and 15 female)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were based upon a 28 Day Dose Range Finding Study and were specified by the sponsor.
- Post-exposure observation period: Additional groups of control and high dose observed for 28 days post dosing
Positive control:
no

Examinations

Observations and examinations performed and frequency:
- All pharmacological and toxicological signs were documented daily including their onset, degree and duration.
- Mortality checks were made daily and recorded.
- Measurement was made of food consumption weely.

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations: included but were not limited to changes in skin and fur, eyes and mucous membranes as well as respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behavior pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: weighed initially and weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the administration of the test substance at approximately Day 30, Day 90 and prior to termination of the study
- Dose groups that were examined: all rats

HAEMATOLOGY: Yes
- Time schedule for collection of blood: no data
- Anaesthetic used for blood collection: no data
- Animals fasted: Yes
- How many animals: determinations made on five or seven randomly selected rats/sex/group at the scheduled sacrifice periods of the study.
- Parameters checked: hemoglobin, hematocrit, erythrocyte count, total and differential leucoyte counts, platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals: determinations made on five or seven randomly selected rats/sex/group at the scheduled sacrifice periods of the study.
- Parameters checked: calcium, phosphorus, chloride, sodium, potassium, fasting glucose, serum alanine aminotransferase, serum aspartate aminotransferase, gamma glutamyl transpeptidase, urea nitrogen, albumin, globulin, blood creatinine, total bilirubin, total serum protein measurements

Sacrifice and pathology:
GROSS PATHOLOGY: Yes; examination of the external surface of the body, all orifices, and the cranial, thoracic, abdominal and pelvic cavities and their contents. The liver, adrenal glands, kidneys, gonad, and brain were weighed.

HISTOPATHOLOGY: Yes; A full histopathologic evaluation was performed in all control and high dose animals for the 90 day sacrifice to the following organs and tissues: all gross lesions, brain-including sections of medulla/pons, cerebellar cortex and cerebral cortex, pituitary, thyroid/parathyroid, thymus, lungs, trachea, heart, sternum with bone marrow, salivary glands, liver, spleen, kindneys/adrenals, pancreas, gonads, uterus, accessory genital organs (epididymides, protate, and if present, seminal vesicles), aorta, skin, esophagus, nasal turbinates, stomach, duodenum, jejunum, ilem, cecum, colon, rectum, urinary bladder, representative lymph node, mammary gland, thigh musculature, peripheral nerve, eyes, femur-including articular surface, spinal cord at three levels-cervical, midthoracic and lumbar, exorbital lachrymal glands.
- All animals which died prior to completion of the study were also subjected to a full histopathologic evaluation.
Statistics:
One-way analysis of variance was performed to ensure that no statistical significance was present between groups, followed by Dunett's or Tukey's tests.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no pharmacotoxic signs observed in the control groups during the study. Clinical signs which were observed in the control and treated groups included alopecia, scab formation and alopecia with scab formation. Chromodacryorrhea and dislodged upper incisors were observed in the mid and high dose males. Swelling with ulceration in the ventral cervical region was observed in a high dose rat. These observations were considered not to be related to test article administration. Pharmacotoxic signs which were observed in the test substance groups included immediate post-dose salivation, rales, immediate post-dose rales, abnormal gait, abnormal stance, decreased activity and dyspnea.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Five high dose rats died on study. Three males and two females from the recovery group died during test article administration prior to the 28-day recovery period. The deaths were not considered to be test article related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Body weight: Statistically significant reductions in the group mean male body weights when compared to the recovery control group were observed in the high dose (5 g/kg) recovery group on Day 28 and Days 35 through 84. There were no significant differences noted for the treated female animals.
- Body weight gain: The daily body weight gains were significantly reduced in the high dose (5 g/kg) group mean male recovery group on Day 28 and significantly increased on Days 98 and 112, when compared to the recovery controls. High dose female recovery group body weight gains were significantly increased on Day 14 of the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The daily food consumption for the high dose recovery males was significantly reduced on Day 7, Days 28 through 42 and Days 56 through 84, when compared to the recovery control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The examination evaluated the adnexa, conjunctiva, sclera, cornea, anterior chamber, lens and posterior segment (vitreous, retina and optic disc). Eighteen rats exhibited abnormal ocular defects prior to study initiation. These animals were replaced with normal animals prior to the initiation of the study. On Day 30, an interim ocular examination was performed on all rats. Two animals exhibited lesions. Striated retinopathy was detected in the left eye of a low dose (1 g/kg) male and anterior synechia was detected in the right eye of a high dose (5 g/kg) female. These lesions were probably sequelae of previous infection with sialodacryoadenitis virus and were not considered to be related to test article treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A significant decrease was observed in the high dose male mean corpuscular volume at the Day 90 interim necropsy. At terminal necropsy, a significant decrease was observed in the high dose male mean corpuscular volume. Significant increases were observed in the female high dose red blood cell counts, hematocrit and hemoglobin values.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant increases were observed in the group mean high dose male albumin, creatinine and chloride values at the Day 30 necropsy. Total bilirubin was signicantly increased in the male low dose group. Significant increases noted for the high dose females at the Day 30 necropsy were albumin and calcium. A statistically significant decrease was observed in the high dose female phosphorus values. At the Day 90 interim sacrifice, significant differences in blood chemistry parameters were observed for both males and females. Statistically significant increases were noted in the mid and high dose male phosphorus values and high dose male chloride values. Significant increases noted for the females included total bilirubin and phosphorus in the low dose group and sodium levels in the high dose group. Statistically significant decreases were observed in the low and high dose glucose levels. At terminal necropsy statistically significant decreases were noted in the high dose male and female total protein and albumin values. Significant decreases were also noted in high dose male GGPT and globulin values and high dose female calcium levels.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- absolute organ weights: A statistically significant increase in the high dose male absolute brain weight was observed at the Day 30 interim necropsy. No significant differences were noted for the female absolute organ weights at Day 30, or male and female organ weights at Day 90. At the Day 118 terminal necropsy, the male high dose kidney weights were significantly reduced. No significant differences were noted for the females.
- relative organ to brain wieghts: At Day 118 terminal necropsy, the high dose male testes to body weights were significantly larger.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- The majority of observations noted at the Day 30, Day 90 or terminal sacrifice were non-specific, low in incidence and without dosage-trend relationship. These observations included enlarged cervical lymph nodes, submandibular mass, enlarged and firm salivary glands with a white mass, kidneys which were mottled and pitted, pale surface and dilated pelvis, urinary bladder with a thickened wall and calculi in the lumen or proteinaceous plug present, ureter distended and fluid-filled, lungs with mottled red foci, purple areas and scattered white foci or dark pink apical lobe, small adrenals, small ovaries, bilateral chromolacrimation of the exorbital lacrimal glands, red foci on the thymus, small or fluid-filled uterine horns, skin alopecia and a small right testis. These observations are common findings noted at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
When compared to the male and female rats in the vehicle control group (Group I), no test article related lesions were present in any of the tissues evaluated from the male and female rats receiving 5.0 g/kg/day, 5 days per week for 90 days.
Incidental changes were present in the lungs and tracheas of both the vehicle control and test rats which were compatible with the early microscopic changes of chronic murine respiratory disease. These changes were characterized by perivascular/bronchial mononuclear cell infiltrations, multifocal areas of pneumonitis or foci of foamy macrophages in the lung, and/or multifocal areas of chronic tracheitis. The kidneys of individual rats had changes comparable with the early stages of chronic progressive nephropathy. These consisted of multifocal nonsuppurative nephritis, tubular regeneration, and/or tubular dilatation. Incidental changes in the liver were nonsuppurative hepatitis and/or foci of mononuclear cells.
The heart of an occasional rat had areas of multifocal nonsuppurative myocarditis consistent with the cardiomyopathy found in Sprague-Dawley rats. A few rats had iatrogenic lesions in the heart considered to be related to the percultaneous cardiocenthesis used for blood sampling at the time of necropsy. These consisted of focal or multifocal areas of myocardial fiber degeneration (cardiomyopathy) and/or focal hemorrhage.
The degree of spermatogenesis in the testes of the high dose males and the ovarian activity of the high dose females were similar to the vehicle control rats. The degree of hematopoiesis and pigment deposition in the spleen varied in individual rats within groups and between sexes but was considered to be within normal limits for the age and strain of rats. The bone marrow activity was comparable between the high dose and vehicle control rats.
A variety of other microscopic findings were observed in individual rats and occurred as incidental findings or in such small numbers as to have no apparent relationship to the test material.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 5 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Statistically significant differences were observed between the control and treated animals (primarily high dose) in the group body weights, daily body weight gains, daily food consumption determinations, absolute organ weights, relative organ to body weights and relative organ to brain weight data. Significant differences were also noted in the evaluated blood chemistry and hematology parameters.
However, none of the above noted changes were dose-dependent, and there were no histomorphologic alterations attributed to the oral administration of the test substance of a 90 day period. Therefore, the NOAEL was established as being greater than 5000 mg/kg bw/day. The test substance is not to be classified as STOT RE according to criteria described in the CLP regulation.