2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridine carboxylate; imazapyr (ISO)

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-02-13 to 1987-02-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: American Cyanamid Company, AC 4866-62

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, in the dark, dry area in a fiber drum lined with a plastic bag.
- Stability under test conditions: stable

Test animals

Species:

dog

Strain:

Beagle

Details on species / strain selection:

purebred

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Laboratory Research Enterprises, Inc., Kilamazoo, Michigan, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 6 months
- Weight at study initiation: males: 7.37 - 7.55 kg; females: 7.18 - 7.4 kg
- Housing: individually in stainles steel cages
- Diet: 1 hour per day (approx. 400 g of Purina Certified Canine Chow, 5007 Meal)
- Water: ad libitum via automatic watering
- Acclimation period: approx. 2 weeks

DETAILS OF FOOD AND WATER QUALITY: Feed and water were monitored for contaminants. Feed was monitored and certified by the supplier. Water was monitored by the lab every six months by means of a sample submitted for analysis to Lancaster Labs, Lancaster, PA.

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

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The justification for this route of exposure was the fact that humans and/or animals would be exposed to the test substance via nutrition, if such exposure would occur.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: Purina-certified canine chow
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate 20 g portions of each canine meal sample were assayed by HPLC Method M-1465 described in another study report. The described method is validated for test substance in canine meal from 1000 to 10000 ppm.
The test substance concentration in initial samples from the 1000 ppm batch ranged from 927 to 959 ppm with a mean of 943 ppm and standard deviation (SD) of 13 ppm, corresponding to 94.3 % with a coefficient of variation (CV) of 1.3 % of nominal. Initial samples at 10000 ppm ranged from 8982 to 9891 ppm vith a mean of 9275 SD 345 ppm or 92.8 CV 3.7 % of nominal. With both CVs below 10 % , mixing was acceptable.
Test substance concentrations in diet after 1 and 24 hours in exposed feeders and after 7 and 14 days in lidded, opaque plastic pails in the dog room were 957, 985, 940 and 974 ppm (95.7, 98.5, 94.0 and 97.4% of nominal) for the 1000 ppm batch. Corresponding values at 10000 ppm were 9638, 9371, 9560 and 10500 ppm (96.4, 93.7, 95.6 and 105.0 % of nominal). It was concluded that stability was adequate.

Duration of treatment / exposure:

1 year

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

corresponding to 1000 ppm

Doses / concentrations 2

Dose / conc.:

140 mg/kg bw/day (nominal)

Remarks:

corresponding to 5000 ppm

Doses / concentrations 3

Dose / conc.:

286 mg/kg bw/day (nominal)

Remarks:

corresponding to 10000 ppm

No. of animals per sex per dose:

6

Control animals:

yes, plain diet

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: mortality, general appearance, behaviour, gait and excretory function

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during pretest quarantine period, at 6 months and prior to termination of the study
- Dose groups that were examined: all animals were examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at pretest, at 6 weeks and at 3, 6 and 12 months
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, approx. 18 hours
- How many animals: all animals
- Parameters checked: erythrocyte count (RBC), erythrocyte morphology, hematocrit (Hmct), hemoglobin (Hgb), differential leukocyte count, total leukocyte count (WBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (Plat) and reticulocyte count (if signs of anemia are present)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at pretest, at 6 weeks and at 3, 6 and 12 months
- Animals fasted: Yes, approx. 18 hours
- How many animals: all animals
- Parameters checked: albumin (A), albumin/globulin (A/G) ratio, serum creatinine (CREAT), blood urea nitrogen (BUN), carbon dioxide (CO2), creatine phosphokinase (CPK), gamma-glutamyl transpeptidase (GGTP), globulin (G), glucose or fasting blood sugar (FBS), serum alkaline phosphatase (SAP), serum electrolytes (sodium (Na), calcium (Ca), potassium (K), phosphorus (P) determined as phosphates (PO4), chloride (Cl)), serum aspartate aminotransferase (SGOT), serum alanine aminotransferase (SGPT), serum lactic dehydrogenase (LDH), total and direct bilirubin (TB/DB-direct, if total is elevated), total cholesterol (Chol), and total protein (TP)

URINALYSIS: Yes
- Time schedule for collection of urine: at pretest, at 6 weeks and at 3, 6 and 12 months
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes, approx. 18 hours
- Parameters checked: appearance and color, specific gravity, pH, qualitative tests (bilirubin, glucose, ketones, occult blood, protein, urobilinogen) and microscopic examination of sediment

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All survivors were euthanized and the following organs were examined and weighed:
brain, gonads (combined weight), heart, kidneys (combined weight), liver, lungs, pituitary, thymus and thyroids/parathyroids (combined weight)

HISTOPATHOLOGY: Yes
The following organs were examined microscopically:
adrenals, aorta, bone and marrow (sternal), brain (fore-, mid- and hindbrain), cecum, colon, duodenum, epididymis, esophagus, eyes, gallbladder, heart, duodenum, ileum, jejunum, kidneys (both), liver, lungs with mainstem bronchi (left and right), lymph nodes (mediastinal and mesenteric), mammary gland, optic nerve, ovaries (both), pancreas, parathyroids (both), prostate, salivary gland, sciatic nerve, skeletal muscle, skin (mafmary area), spinal cord (cervical, thoracic and lumbar), spleen, stomach, testes (both), thymus, thyroids (both), tongue, trachea, urinary bladder, uterus (corpus and cervix), vagina, and any other tissue(s) with gross lesions.

Statistics:

Data collected during the study were statistically evaluated by one-way analysis of variance (ANOVA) using F-test for variance comparison. If significant differences among the means were indicated, then the Dunnett's t-test was used to determine which means were significantly different from the control at the 95 % confidence level. The following parameters were subject to this analysis: mean body weight data, mean feed consumption data, mean clinical chemistry values, mean hematological values, mean organ weights, mean organ-to-body weight ratios and mean organ-to-brain weight ratios.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results"

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results"

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results"

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):

Please refer to "details on results"

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results"

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results"

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results"

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results"

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

Clinical signs and mortality
Clinical signs observed consisted primarily of skin lesions, characterized as alopecia, nodules, cuts or abrasions (lesions, rashes) and wart-like structures probably due to the caging system. White mucoid material was observed occasionally in the feces of one or more dogs in each group except low and high-dose males and mid-dose females. Salivation was observed in mid-dose male #2435; vomiting was observed in low-dose female #2418. High-dose male #2447 exhibited prolapsed penis frequently during the study but the problem was considered minimal and did not result in evident stress to the animal requiring euthanasia. All dogs otherwise appeared normal and survived to the terminal necropsy.

Ophthalmology
Pretest examinations revealed ocular anomalies in four dogs; none of the abnormalities seen precluded use of the animals on test. However, two of these animals, male temporary #25 and female temporary #15, were not placed on study. Male temporary #18 had single retinal folds in each eye and was placed on study as high-dose male #2443. Male temporary #3 had a non-pigmented fundus in both eyes and was placed on study as high-dose male #2445. In males, at 6 months, ocular anomalies described included a granuloma in the non-tapetal fundus which were seen in control male #2409, corneal scar in low-dose male #2423, mild pigment loss around the optic nerve head in the right eye of mid-dose males #2427 and #2435, visible choroidal vessels in the non-tapetal retina with some pigment lightening in mid-dose male #2437, three clumps of pigment in the tapetal retina of high-dose male #2443, non-pigmented fundus in high-dose #2445 (as in the pretest) and perinuclear rings in the lens of both eyes of high-dose male #2447. In the latter animal, the ophthalmologist noted that this change was the only anomaly suggestive of a potential pathological condition.
In the females at 6 months, pigment clumps were seen in the tapetal area of low dose #2 as well as changes in choroidal vessels and in pigment of control females #2404, #2410, #2412 and #2414. These observations were considered to be within normal variation. Furthermore, there were no ocular histopathological findings observed.
At termination of the study, an ocular anomaly was described in only one dog, high-dose female #2440; this anomaly was characterized as a fullness to the optic nerve that seemed somewhat more excessive than normally expected. The ophthalmologist, however, commented that this was not thought to be related to the test chemical and that the optic nerve would probably be normal upon histological examination. There were no other ocular anomalies described at termination. The perinuclear rings in the lens of high-dose male #2447 were not observed at necropsy.
Results, therefore, of ophthamological examinations revealed no consistent ocular abnormalities that can be attributed as an adverse effect of the test chemical.

Body weight
Mean weekly body weights of males at all dose levels of test chemical used in this study were consistently equal to or exceeded that of the control group throughout the study. Mean weekly body weight of high-dose males at week 47 was significantly greater than that of the controls; statistically significant increased mean body weight is not toxicologically significant. The test chemical, therefore, did not have an adverse effect on mean body weights of males at dietary levels used in this study.
With females, mean weekly body weights of high-dose animals were generally lower than that of the controls after week four, but the differences between control and high-dose females were never statistically significant. In the mid-dose group, mean body weights were essentially similar to or slightly exceeded that of the controls and there were no statistically significant differences observed during this 52-week experimental period. In the low-dose group, mean weekly body weights of treated females were slightly lower than that of the controls (and also the mid-dose animals) but these differences from control were never statistically significant. Results, therefore, indicate no adverse effect of the test chemical on mean body weights of female dogs.
Results, therefore, indicate no adverse effect of the test chemical on mean body weights of male or female Beagles at dose levels as high as 10000 ppm.

Feed consumption
Mean weekly feed consumption for treated male dogs at all dietary levels used in this study were generally similar to or exceeded that of the controls throughout the study. The only statistically significant differences between control and compound-treated males were increases in feed consumption, which were seen frequently; statistically significant increased feed consumption is not toxicologically significant.
In females, feed consumption of high-dose animals was slightly lower than that of controls during approximately the first eight weeks but these differences were never statistically significant and consumption in the high-dose animals is considered similar to the controls. In low and mid-dose groups, mean weekly feed consumption was generally similar to that of the controls throughout the study and there were no statistically significant differences.
Results, therefore, indicated that the test chemical at dietary levels used in this study did not produce an adverse effect on feed consumption of treated males or females.

Clinical chemistry
Mean clinical chemistry parameter values for males assigned to the various groups when determined during the pretest period were similar in all groups. There were no statistically significant differences in the mean clinical chemistry parameters when determined at 6 weeks; mean LDH values in low and high-dose animals were slightly higher than in controls but the differences were not statistically significant. At 3 months, mean fasting blood sugar (FBS) levels in low and high-dose animals were slightly but significantly higher than that of the controls; mean values in all groups, however, were within the range expected in this laboratory and the significant increases in low and high-dose groups are considered to be random occurrences unrelated to treatment with the test chemical.
At 6 months, mean potassium (K) levels in all treated groups were slightly greater than that in the control group and all increases were statistically significant because the mean value of the control group was slightly lower than expected; the mean values, however, were within the range normally expected and the significant increases are considered to be random occurrences unrelated to the test chemical. Prior to termination of the study, mean total protein (TP) in high-dose males was slightly lower than in the controls and was statistically significant; likewise, the mean potassium (K) value in high-dose males was slightly lower than in the controls and was also statistically significant.
However, the mean total protein (TP) and potassium (K) values were in the normal range expected and the statistically significant decreases are considered to be random occurrences.
In females, the pretest A/G ratio calculated for high-dose dogs was slightly greater than in the control group and was statistically significant; this is a random occurrence that is frequently obtained. At week 6, the mean cholesterol (Chol) value in the mid-dose group was higher than in the controls and was statistically significant whereas the mean values in the low and high-dose groups were similar to the controls. At month 3, the mean albumin (A) value of mid-dose animals and the mean potassium (K) value of low-dose animals were lower than in the controls and were statistically significant; the mean values, however, were within the normal expected ranges. At month 6, the only statistically significant differences between control and treated values consisted of elevated mean cholesterol (Chol) in mid-dose females and slightly decreased mean calcium (Ca) in low-dose animals. There were no statistically significant differences between control and compound treated females prior to termination of the study. Results, therefore, obtained in the females revealed occasional statistically significant differences between control and compound-treated animals but the differences were not time-consistent nor dose-dependent and are considered to be random occurrences unrelated to treatment with the test chemical.
The test chemical, therefore, at dietary levels used in this study did not produce any consistent changes in blood biochemical parameters that can be attributed to a toxicological effect of the test chemical. Occasional statistically significant differences between control and compound-treated males and females are considered to be random occurrences unrelated to the test chemical.

Hematology
In males, there were no statistically significant differences at any interval in any of the hematological parameters determined in compound-treated animals when compared to the controls. All values were within the normal range expected at this laboratory.
In females, mean pretest values were similar for animals assigned to control and compound-treated groups. At week 6, the band cells (%) in the differential counts of high-dose animals were higher than in the controls and the increased values were statistically significant. Likewise, mean platelet counts (Plat) in mid-dose animals were higher than in the controls and were statistically significant but an increased platelet count is not toxicologically significant.
At month 3, band cells in the white blood cell differential counts of high-dose animals were significantly higher than in the controls as was seen at week 6; mean hemoglobin (Hgb) and hematocrit (Hct) values in all treated groups were lower than in the controls and the decreased values in low- and mid-dose groups were statistically significant. All values, however, were within the normal range and the statistically significant differences are considered to be random occurrences.
At month 6, mean hemoglobin (Hgb) values in all groups were slightly lower than in the controls and the decreased values in low and high-dose groups were statistically significant. At month 12, there were no statistically significant differences between control and compound-treated animals at any dose level. Results, therefore, indicated occasional statistically significant differences between compound-treated and control females; the lack of consistent statistical differences, dose dependency and lack of effect at twelve months suggest that results obtained in females were random occurrences unrelated to treatment with the test chemical.
The test chemical, therefore, at dietary levels used in this study did not produce consistent dose-dependent differences in hematological parameters between control and compound-treated animals; occasional statistically significant differences are considered random occurrences unrelated to treatment with the test chemical.

Urinalysis
Review of urinalysis parameters of male and female dogs in control and compound-treated groups revealed no consistent differences that can be attributed to an effect of the test chemical. The distribution of various parameters between control and compound-treated groups was indicative of normal renal function; occasional dogs, males and females, in control and treated groups had low specific gravities but the distribution of animals with low values was similar in control and compound-treated groups. Determination of mean pH revealed no essential differences between control and compound-treated groups.
Results, therefore, of urinalysis on male and female dogs revealed no responses that could be attributed to an effect of the test chemical at dietary levels used in this study.

Organ weights and weight ratios
In males, there were no statistically significant differences between mean absolute organ weights or organ-to-body weight and organ-to-brain weight ratios in compound-treated and control animals.
In females, there were no statistically significant differences between mean absolute organ weights or in the mean organ-to-body weight and organ-to-brain weight ratios of compound-treated and control females.
The test chemical, therefore, at dietary levels used in this study did not produce any statistically significant differences in mean organ weights and organ weight ratios.

Gross pathology
There were no gross lesions observed in compound-treated males or females that can be considered an effect of the test chemical. The most frequently observed lesions were found in the cervical lymph nodes. These lesions were characterized primarily as dark or congested cervical lymph nodes seen in equal incidence in control and all compound-treated males and in control, low and mid-dose females; in females, nodal findings characterized as enlarged and/or congested were also seen in mediastinal, axillary and inguinal lymph nodes of an occasional animal in control, low and mid-dose groups. The distribution of these findings clearly indicates that they were not related to the test chemical. Lesions observed in the heart were characterized as valvular nodules and white foci or slight thickening of ventricular epithelium; these lesions were observed in one dog each in control and compound-treated male groups and in control and low-dose females and in two mid-dose females but was not seen in high-dose females. Mammary gland congestion was seen in one control and one mid-dose female and in three high-dose females a finding conmonly seen in sexually mature females. Other gross findings in males and females were seen only in an occasional animal. None of the gross lesions were considered an effect of the test chemical.

Histopathology
None of the microscopic findings were compound-related and these findings, for the most part, confirmed the gross lesions seen at necropsy. Other lesions were seen only in an occasional animal and, as such, were considered random findings. Microscopic lesions seen most frequently consisted of atrophy of cervical mesenteric lymph nodes and pigmented cervical lymph nodes as well as subendocardial valvular hemorrhagic nodules; these lesions occurred either in near equal incidence in control and compound-treated animals or were found principally in control and mid-dose animals. The pigment in the cervical lymph nodes most likely came from the ear tattoo numbers.
Other lesions observed in an occasional male consisted of a cerebral organizing hemorrhage in a low-dose male; focal interstitial pulmonary pneumonia, a non-malignant subpleural parasitic granuloma and an interdigital abscess, each in one mid-dose male; and a pituitary Rathke's Pouch cyst in a high-dose male. In females, other lesions seen in only one animal in a group consisted of mediastinal lymph node acute periadenitis, renal chronic interstitial nephritis, ovarian cysts and brain metastatic calcification in controls; pulmonary small parasitic granulomata, subpleural granuloma, ovarian cysts, pituitary Rathke's Pouch cyst, axillary lymph node follicular hyperplasia and interdigital abscess in the mid-dose group; and a pulmonary small parasitic granuloma, chronic cholecystitis, pituitary cysts and an organizing spinal cord hemorrhage in the high-dose group. No ocular histopathological changes were observed in any of the dogs, male or female. All microscopic lesions are considered random findings incidental to chemical treatment.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 286 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Results of this study revealed no consistent responses that would be considered as adverse effects of the test chemical.

Target system / organ toxicity

Key result

Critical effects observed:

no