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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and OECD Guideline study For justification of read-across please refer to section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 83-1 (Chronic Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8.5 to 9.5 months old
- Weight at study initiation: body weights ranged from 7.5 to 13.5 kg for the males and 6.8 to 9.7 kg for the females.
- Housing: individually housed in elevated stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 3 weeks prior to initiation of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 59 to 88
- Humidity (%): 11 to 96
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES:
From: 14 June 1989
To: 14 June 1990

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): A mixture of test material and canine diet was prepared weekly, portions of the diet mixture were diluted 9:1 (water:feed) daily for dose
administration.
- Mixing appropriate amounts with (Type of food): Purina® Certified Canine Diet Meal
- Storage temperature of food: Room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dosing slurries that were prepared prior to the initiation of the study were analyzed for test article homogeneity and for Day 0 stability analyses. These samples were obtained from the top, middle, and bottom of dosing slurries prepared for each treatment group.
Samples of the dosing slurries prepared from dietary mixes 7 and 14 days after preparation of the dietary mixes from the pretreatment diet mix were evaluated for stability.
Samples of the test article/diet mix prepared pretreatment and weekly during the study were retained frozen. Samples of the dosing slurries for each group for Days 1, 8, 15, 22, 50, 78, 106, 134, 162, 190, 218, 246, 274, 302, 330, and 358 were analyzed for verification of concentration. Samples of the dosing slurries from Groups 1-3 for Days 2 and 3 were analyzed for verification of concentration because the values obtained on Day 1 were more than 10 % below target.
The analytical method used for the test material in a 9:1 water:feed slurry was UV-VIS spectrophotometry.
Results from the homogeneity analyses revealed that the dosing slurries were homogeneous having a % relative standard deviation (RSD) less than 5 %. Stability data also indicate that the test material was stable in feed for at least 14 days. Analyses of the prepared dosing slurries prepared on Day 1 revealed low concentrations of the test material for Groups 2 and 3 which were 66.5 and 86.2 % of target, respectively. These levels were then remixed on Day 2 and the resultant dosing slurries were found to still be low (80.6 and 81.6 % of target for Groups 2 and 3, respectively). These levels were again remixed on Day 3 and concentrations were then within acceptable limits. Verification of concentrations results for the remainder of the study indicated that the dosing slurries were within acceptable limits (+/- 10 % of target) with the exception of the Day 15 and Day 50 Group 3 mixes which were slightly lower (88.1 and 89.8 % of target, respectively).
Duration of treatment / exposure:
52 weeks
Frequency of treatment:
Two daily doses, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 3, 10 and 30 (changed to 20 on Day 36) mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
4/sex
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: It was anticipated that at the higher dosage level(s), some toxicological or pharmacological effect(s) would be observed and that at the lower dosage level(s) no treatment-related effects would be seen
- Rationale for animal assignment: Random
- Rationale for selecting satellite groups: None
- Post-exposure recovery period in satellite groups: None
- Section schedule rationale: Random
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations and physical examinations were performed once each week. Physical examinations were performed weekly by laboratory personnel and at least once every 3 months by a staff veterinarian.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights data were collected weekly for all animals for the first 14 weeks and then every 2 weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule: Food consumption data were collected weekly for all animals for the first 14 weeks and then every 2 weeks thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: Ophthalmic examinations were performed prior to treatment and prior final sacrifice
- How many animals: Ophthalmoscopic examinations were performed on all animals prior to treatment and prior to termination using indirect ophthalmoscopy on all animals. A 1 % Mydriacyl solution was used for pupil dilation.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to treatment and at termination. Blood samples were collected via the jugular vein.
- Anaesthetic used for blood collection: Yes (sodium thiamylal)
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked:
cell morphology
corrected leukocyte count
erythrocyte count
hematocrit
hemoglobin
platelet
mean cell volume
leukocyte count
leukocyte differential
mean cell hemoglobin
mean cell hemoglobin concentration
reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to initiation of dosing and at Weeks 13, 26, and 52
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked:
alanine aminotransferase
albumin (ALBUMIN)
aspartate aminotransferase
blood urea nitrogen
calcium
chloride
creatine kinase
creatinine
gamma glutamyltransferase
globulin
glucose
inorganic phosphorus
potassium
sodium
total bilirubin
total cholesterol
total protein

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to initiation of dosing and at Weeks 13, 26, and 52
- Collection of urine: Samples were collected from cagepans overnight
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked:
appearance
urine volume
specific gravity
occult blood
bilirubin
urobilinogen
microscopic examination of
sediment
fecal flotation (O & P; performed once prior to the initiation of dosing)
protein
pH
glucose
reducing substances
ketones
nitrites (
Sacrifice and pathology:
SACRIFICE AND GROSS PATHOLOGY
After 52 weeks of treatment, all surviving animals were weighed, anesthetized with sodium thiamylal, and exsanguinated. Necropsies were performed on each animal (including Group 2 male No. 26729 which was found dead) by trained personnel under the direct supervision of a pathologist.
Findings were recorded.
The necropsy included examination of the following:
external surface
all orifices
cranial cavity
carcass
external surface of the brain and spinal cord and the cut surfaces of the spinal cord (at necropsy); the cut surfaces of the brain were examined at the time of tissue trimming
nasal cavity and paranasal sinuses
thoracic, abdominal, and pelvic cavities and their viscera
cervical tissues and organs

ORGAN WEIGHTS
For each terminally sacrificed animal, the following organs (when present) were weighed following careful dissection and trimming to remove fat and other contiguous tissue in a uniform manner:
adrenals
brain with brainstem
heart
kidneys
liver with drained gallbladder
ovaries
pituitary
spleen
testes with epididymides
thyroid/parathyroids
Using these values, the organ-to-body-weight ratios were calculated.

TISSUE PRESERVATION
The following tissues (when present) from each animal were preserved in 10 % neutral-buffered formalin.
adrenals
aorta
brain with brainstem (medulla/pons, cerebellar cortex, and cerebral cortex)
mandibular and mesenteric lymph nodes
mid-thoracic spinal cord
ovaries
pancreas
cervical spinal cord
colon, cecum, rectum, duodenum, jejunum, ileum
esophagus
eyes (including optic nerve, eyes were fixed in a glutaraldehyde fixative)
femur including articular surface
gall bladder
heart
kidneys
lesions
liver (representative section from each lobe)
lumbar spinal cord
lung with mainstem bronchi (Lungs were inflated with formalin via the trachea)
mammary gland (female only)
pituitary
prostate
mandibular salivary glands
sciatic nerve
skin
skeletal muscle (thigh)
spleen
sternum with bone marrow
stomach
testes with epididymides
thymus
thyroid with parathyroids
trachea
urinary bladder (urinary bladders were inflated with formalin for examination after fixation)
uterus

HISTOPATHOLOGY
The aforementioned tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically from all animals.
Other examinations:
None.
Statistics:
Parametric variables were intercompared for the dose and control groups using Levene’s test for homogeneity of variance and by analysis of variance. Non-parametric data were transformed by log10, square, square root, reciprocal, angular (arcsine), or rank transformation. Dunnett’s test was used to compare significant results from the analysis of variance.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were generally higher incidences of emesis, salivation, and soft/mucoid/liquid faeces in the two higher dose groups than in the low dose group and control. The incidence of these clinical signs was high at 30 mg/kg bw/day but decreased to tolerable levels when dosage was lowered to 20 mg/kg bw/day. One animal in the 3 mg/kg bw/day died on Day 42 due to gavage error. All other animals survived to study termination.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were generally higher incidences of emesis, salivation, and soft/mucoid/liquid faeces in the two higher dose groups than in the low dose group and control. The incidence of these clinical signs was high at 30 mg/kg bw/day but decreased to tolerable levels when dosage was lowered to 20 mg/kg bw/day. One animal in the 3 mg/kg bw/day died on Day 42 due to gavage error. All other animals survived to study termination.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg bw/day, mean bw changes for Weeks 0-4 were significantly decreased for males and females compared to control groups. When the dose level was decreased, the body weight changes generally became comparable to or greater than the control values.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean total food consumption was significantly decreased in Week 1 for the 3 mg/kg bw/day males and females and in Weeks 1-4 for females only.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Slight, non-significant decreases in erythrocyte count, haemoglobin and haematocrit were observed in high dose males and females at 13, 26 and 52 weeks. No other differences from control were considered to be treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Total cholesterol was significantly decreased in high dose group females at 13 weeks. Total protein was significantly decreased in high dose males at Week 52 and albumin was significantly decreased in this group.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: systemic effects
Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion