1,2,4-Oxadiazole, 3-phenyl-5-(2-thienyl)-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 22, 2010 to December 16, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 408 and OPPTS Guideline 870.3100, in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-phenyl-5-(thiophen-2-yl)-1,2,4-oxadiazole

EC Number:

810-533-8

Cas Number:

330459-31-9

Molecular formula:

C12H8N2OS

IUPAC Name:

3-phenyl-5-(thiophen-2-yl)-1,2,4-oxadiazole

Test material form:

solid: pellets

Details on test material:

- Name of test material (as cited in study report): MON 102100

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: Approximately 7 weeks old
- Weight at study initiation: 163 g to 198 g (males); 144 g to 169 g (females)
- Housing: 2-3 per cage by sex for approximately one day. Thereafter, all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board.
- Diet: Certified Rodent LabDiet #5002 (meal), fed ad libitum except during FOB and locomotor activity assessments and during the period of fasting prior to clinical pathology blood collection when food, but not water, was withheld.
- Water: Reverse osmosis treated drinking water, delivered by an automatic watering system were provided ad libitum.
- The results of the diet and water analyses revealed no contaminants at concentrations sufficient to interfere with the objectives of this study.
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature: 71 ± 5°F
- Humidity (%): 50 ± 20%
- Air changes (per h): 10 (air handling units were set to provide a minimum of 10 fresh air changes per h).
- Photoperiod: 12 h light/12 h dark. The 12 h light/12 h dark photoperiod was interrupted as necessary to allow for the performance of protocol specified activities. The light status (on or off) was recorded once every 15 min.

- Individual body weights and food consumption were recorded and detailed physical examinations were performed periodically during the pretest period. Ophthalmic examinations were performed prior to randomization. Functional observational battery and locomotor activity data were also recorded for pretest animals prior to the initiation of diet administration.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: Normal feed

Details on oral exposure:

Continuous exposure via oral diet for 13 weeks

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability of test diet admixtures at concentrations ranging from 3 ppm to 10,000 ppm was 10 d when stored at room temperature or frozen.

- Prior to the initiation of diet administration, samples were collected from 10 and 1,500 ppm diet preparations. Samples for concentration analysis were collected weekly from the middle stratum of each dosing formulation (including the control group) and stored frozen. One duplicate set from the dietary preparations collected during study weeks 0, 3, 7, and 12 was analysed and the remaining duplicate set was frozen and retained as backup samples. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated gas chromatography method using flame ionization detection.

The analysed dietary formulations were homogeneous (i.e., met the protocol specified criteria of an RSD ≤10% and a mean concentrations within ±15% of the target concentrations). The analysed dietary formulations were found to contain 90.7% to 113% of the target test substance concentration in the diet which met the WIL Research SOP requirements (85% to 115%). The test substance was not detected in the analysed basal diet formulation that was administered to the control group (group 1).

Average test substance consumption (mg/kg bw/day), were determined based on target dietary concentrations of the test substance.

Duration of treatment / exposure:

90 d

Frequency of treatment:

Continuously in feed

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 50, 250, 750 and 1,500 ppm test substance (i.e., equivalent to 1, 3, 16, 47 and 91 mg a.i./kg bw/day for the males and 1, 4, 19, 55 and 113 mg a.i./kg bw/day for the females)
Basis:
nominal in diet

No. of animals per sex per dose:

10 males and 10 females per dose group.

Control animals:

yes, plain diet

Details on study design:

Rationale for animal assignment: Seven days prior to the initiation of diet administration, all available rats were weighed and examined in detail for physical abnormalities. The animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure based on body weight stratification in a block design. Individual body weights at randomization were within ±20% of the mean for each sex. Each group (groups 1-6) consisted of 10 males and 10 females. These animals were then randomized into five study replicates to allow for the reasonable conduct of the functional observational battery and locomotor activity assessments. Each group and sex were approximately equally represented within each study replicate.

Examinations

Observations and examinations performed and frequency:

SURVIVAL:
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Animals found dead were examined macroscopically as soon as possible to ensure that tissues were not lost due to autolysis.

CLINICAL OBSERVATIONS:
Clinical examinations were performed once daily. The absence or presence of findings was recorded for individual animals at the scheduled intervals. Observations included, but were not limited to, changes in the skin, fur, eyes and mucous membranes; respiratory, circulatory, autonomic, and central nervous systems function; somatomotor activity; and behaviour patterns. Once daily clinical observations were not performed on days when detailed physical examinations were conducted. Detailed physical examinations, including removal from home cage and placement in a standard arena for observations for changes in gait, posture, clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), and permanent or semi-permanent signs, such as skin lesions and hair loss, were conducted on all animals approximately weekly throughout the study, beginning at pretest initiation and including the day of randomization, and prior to the scheduled necropsy. A separate computer protocol was used to record any treatments administered.

BODY WEIGHTS:
Individual body weights were recorded at pretest initiation (presented as study week -2), at randomization (presented as study week -1), approximately weekly throughout the study, and on the day of the scheduled necropsy (fasted). During study week 12, body weights were recorded twice weekly. Mean body weights and mean body weight changes were calculated for the corresponding intervals.

FOOD AND TEST SUBSTANCE CONSUMPTION:
Individual food consumption was recorded for two weeks during pretest (study week -2 to -1 and study week -1 to 0) and approximately weekly during the dosing period. During study week 12, food consumption was recorded twice weekly. Food intake was calculated as g/animal/day and g/kg/day for the corresponding body weight intervals. Food efficiency (body weight gained as percent of feed consumed) was also calculated for each interval. The mean amounts of test substance consumed (mg/kg bw/day) by each sex per diet group were calculated from the mean food consumed (g/kg of mean body weight/day) and the appropriate target concentration of test substance in the food (mg/kg of diet).

FUNCTIONAL OBSERVATIONAL BATTERY (FOB) ASSESSMENTS:
FOB assessments were recorded for all animals prior to the initiation of diet administration and during study week 12. Testing was performed by the same technicians, whenever possible, without knowledge of the animals group assignments.
All animals were observed for following.
HOME CAGE OBSERVATIONS: Posture, convulsions/tremors, faeces consistency, biting and palpebral (eyelid) closure
HANDLING OBSERVATIONS: Ease of removal from cage, lacrimation/chromodacryorrhea, piloerection, palpebral closure, eye prominence, red/crusty deposits, ease of handling animal in hand, salivation, fur appearance, respiratory rate/character, mucous membranes/eye/skin colour and muscle tone
OPEN FIELD OBSERVATIONS: Mobility, rearing, convulsions/tremors, grooming, bizarre/stereotypic behaviour, time to first step (seconds), gait, arousal, urination/defecation, gait score and backing
SENSORY OBSERVATIONS: Approach response, startle response, pupil response, forelimb extension, air righting reflex, touch response, tail pinch response, eye blink response, hind limb extension and olfactory orientation
NEUROMUSCULAR OBSERVATIONS: Hindlimb extensor strength, hindlimb foot splay, grip strength-hind and forelimb and rotarod performance
PHYSIOLOGICAL OBSERVATIONS: Catalepsy, body temperature and body weight

LOCOMOTOR ACTIVITY: Locomotor activity was assessed for all animals prior to the initiation of diet administration and during study week 12. Total locomotor activity was defined as a combination of fine locomotor skills (i.e., grooming; interruption of a single photobeam) and ambulatory locomotor activity (e.g., interruption of two or more consecutive photobeams).

CLINICAL PATHOLOGY:
Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected from all animals on the day of the scheduled necropsy (study week 13). Haematology and coagulation parameters evaluated were total leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration (MCHC), platelet count (PLATELET), prothrombin time (PT), activated partial thromboplastin time (APTT), reticulocyte count percent (RETIC), absolute (RETIC ABSOLUTE), mean platelet volume (MPV), differential leukocyte count, red cell distribution width (RDW), haemoglobin distribution width (HDW), platelet estimate, red cell morphology (RBC Morphology).

SERUM CHEMISTRY: Parameters evaluated were albumin, total protein, globulin [by calculation], albumin/globulin ratio (A/G Ratio), total bilirubin (total Bile), urea nitrogen, Creatinine, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT), glucose, total cholesterol (cholesterol), calcium, chloride, phosphorus, potassium, sodium, triglycerides (triglyceride) and sorbitol dehydrogenase (SDH)
URINALYSIS: Parameters evaluated were specific gravity (SG), pH, urobilinogen (URO), total volume (TVOL), colour (COL), clarity (CLA), protein (PRO), glucose (GLU), ketones (KET), bilirubin (BIL), occult blood (BLD), leukocytes (LEU), nitrites (NIT) and microscopy of sediment.

OPHTHALMIC EXAMINATIONS:
Ocular examinations were conducted on all animals prior to randomization (study week 2) and near the end of the treatment period (study week 12). All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.

Sacrifice and pathology:

MACROSCOPIC EXAMINATION:
A complete necropsy was conducted on all animals. Animals surviving to the scheduled necropsy were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The tissues and organs were collected for analysis were adrenals, aorta, bone with marrow, femur, sternum, bone marrow smear, brain, cerebrum level, cerebrum level, cerebellum with medulla/pons, cervix, epididymides, eyes with optic nerve, gastrointestinal tract, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, heart, kidneys, larynx, liver (sections of 2 lobes), lungs (including bronchi, fixed by inflation with fixative), lymph nodes, nasal cavity, ovaries with oviducts, pancreas, peripheral nerve (sciatic), peyer’s patches, pituitary, pharynx, prostate, salivary glands, seminal vesicles, skeletal muscle (rectus femoris), skin (with mammary gland), spinal cord (cervical, thoracic,lumbar), spleen, testes, thymus, thyroid (with parathyroids, if present), trachea, urinary bladder, uterus, vagina, gross lesions.

ORGAN WEIGHTS:
The organs weighed from all animals at the scheduled necropsy were adrenals, brain, epididymides, heart, kidneys, liver, ovaries with oviducts, spleen, testes, thymus, thyroid with parathyroids and uterus.

SLIDE PREPARATION AND MICROSCOPIC EXAMINATION: Microscopic examination was performed on tissues listed under macroscopic examination from the 1,500 ppm group female found dead and all animals in the control and 1,500 ppm groups at the scheduled necropsy.

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. All statistical tests were performed using WTDMS™. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 5% and 1%, comparing each test substance treated group to the control group by sex.

All repeated measures analysis of variance (RANOVA) statistical analyses for total and ambulatory locomotor activity counts recorded during pretest and after dosing were conducted by BioSTAT Consultants, Inc., Portage, MI, using SAS version 9.1 software (SAS Institute, Inc., 2002-2003).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

SURVIVAL:
There was no test substance related effect on mortality. One female from the 1,500 ppm group was found dead on study day 77. Haemorrhage within the soft tissue and bone peripheral to the nasal cavity were suggestive of trauma. Haemorrhage was also a component of extensive nasal exudation that likely resulted in impaired respiration, aerophagia, and the gas filled gastrointestinal tract noted at necropsy. In the absence of other noteworthy findings, the cause of death was attributed to traumatic injury unrelated to the test substance.

CLINICAL OBSERVATIONS:
There were no test substance related clinical observations. Low incidences of brown material around the anogenital area were noted in the 1,500 ppm group males at the time of detailed physical examinations and during the daily clinical observations, noted sporadically between study day 63 and 91. All other clinical findings in the test substance treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose related manner, and/or were common findings for laboratory rats of this age and strain.

BODY WEIGHTS:
Test substance related lower body weight gains were noted in the 750 ppm group females and 1,500 ppm group males and females. Test substance related lower mean cumulative body weight gains were noted throughout the study in the 750 ppm group females and 1,500 ppm group males and females (sometimes statistically significant) when compared to the control group. During the study, mean body weights were up to 10.0% lower in the 750 ppm group females and up to 7.5% and 10.5% lower in the 1,500 ppm group males and females, respectively, when compared to the control group. At end of the study (study week 12), mean body weights were 10.0% lower in the 750 and 1,500 ppm group females and 3.0% lower in the 1,500 ppm group males when compared to the control group. There were no test substance related effects noted on body weights in the 10, 50, or 250 ppm groups or 750 ppm group males. Lower mean body weights were noted in the 50 ppm group males and females during the study; however, these differences lacked a dose response trend and were preceded by significantly lower mean body weight gain, compared to the control group, during study week -1 to 0. Some additional statistically significant differences were noted when the test substance treated and control groups were compared; however, these differences were incidental and not considered test substance related.

FOOD AND TEST SUBSTANCE CONSUMPTION:
Test substance related lower food consumption was noted in the 1,500 ppm group males and 750 and 1,500 ppm group females generally throughout the study. Test substance related lower food efficiency was noted in the 1,500 ppm group males and females during study week 0 to 1. Lower mean food consumption values (g/animal/day and g/kg/day) were noted beginning at study week 0 and generally throughout the study, when compared to the control group. Slightly lower mean food consumption values (g/animal/day and g/kg/day) were also noted, to a lesser extent, in the 750 and 1,500 ppm group females beginning at study week 4 and intermittently throughout the study. Statistically significantly lower mean food efficiency values were noted from study week 0 to 1 in the 1,500 ppm group males and females and from study week 1 to 2 in the 750 ppm group females when compared to the control group. Food efficiency values thereafter were generally similar to control group values. The correlating effects in food consumption and food efficiency in males during study week 0 to 1, along with a lack of effect in food consumption in females suggests toxicity, rather than palatability issues.
There were no test substance related effects noted in food consumption or food efficiency values in the 10, 50, 250, or 750 ppm group males or females. Some statistically significant differences were noted when the test substance-treated and control groups were compared; however, these differences were not considered test substance related.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
HOME CAGE OBSERVATIONS: Home cage observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated group males and females were compared to the control group at the study week 12 evaluation.
HANDLING OBSERVATIONS: Handling observations were unaffected by test substance administration. There were no statistically significant differences when the test substance treated group males and females were compared to the control group at the study week 12 evaluation.
OPEN FIELD OBSERVATIONS: Open field observations were unaffected by test substance administration. There were no statistically significant differences when the test substance treated group males and females were compared to the control group at the study week 12 evaluation.
SENSORY OBSERVATIONS: Sensory observations were unaffected by test substance administration. There were no statistically significant differences when the test substance treated group males and females were compared to the control group at the study week 12 evaluation.
NEUROMUSCULAR OBSERVATIONS: Neuromuscular observations were unaffected by test substance administration. Statistically significantly lower mean rotarod performances were noted in the 50, 250, and 750 ppm group females when compared to the control group at the study week 12 evaluation; however, because mean rotarod performance for females in all test substance treated groups was generally equivalent to or higher than during pretest, and because the control group mean was higher than the WIL Research historical control reference range, these differences were not considered test substance related. There were no other statistically significant differences when the test substance treated group males and females were compared to the control group at the study week 12 evaluation.
PHYSIOLOGICAL OBSERVATIONS: Test substance related lower body weights were noted in the 750 ppm group females and 1,500 ppm group males and females at study week 12 when compared to the control group.
All other physiological observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated group males and females were compared to the control group at the study week 12 evaluation.
LOCOMOTOR ACTIVITY: Locomotor activity patterns (total and ambulatory activity counts) were unaffected by test substance administration. At study week 12, statistically significantly, non-dose-related higher ambulatory motor activity counts were noted over the 60-min test session in the 10 ppm group males, when compared to the control group. In addition, lower total motor activity counts were noted from 21-30 min at the study week 12 evaluation in the 750 and 1,500 ppm group females, when compared to the control group. Due to inconsistent trends and the lack of concurrent differences between ambulatory and total motor activity counts, the statistically significant differences noted at study week 12 were not considered test substance-related. There were no other statistically significant changes for the test substance-treated group males and females when compared to the control group at the study week 12 evaluation. With the exception of those differences listed above, values obtained from the six epochs evaluated (0-10 minutes, 11-20 minutes, 21-30 min, 31-40 min, 41-50 min, and 51-60 min) and the overall 60-minute test session were comparable to the concurrent control values. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the animals were evaluated on study week 12.

HEMATOLOGY AND COAGULATION:
Test substance-related lower red blood cell counts (RBC), haemoglobin (HGB) levels, and haematocrit (HCT) levels were noted in the 1,500 ppm group females. Test substance-related alterations in haematology parameters were limited to statistically significantly lower mean RBC counts, HGB levels, and HCT levels in the 1,500 ppm group females compared to the control group mean. Although the magnitudes of change were small and, with the exception of two females with haemoglobin concentrations slightly lower than the WIL Research historical control reference range, all other values were within the historical control reference ranges. A similar effect was not observed in the test substance-treated male groups. Although not statistically significantly different from the control group, a higher mean total white blood cell count was noted in the 1,500 ppm group males; this variation also was due primarily to a higher mean absolute lymphocyte count. The variations for both parameters were attributed to high individual values for one male in the 1,500 ppm group, with total white blood cell and lymphocyte counts that exceeded the WIL Research historical control reference ranges. With the exclusion of these individual values, a dose response would not have been apparent in the 1,500 ppm group males, and therefore, the higher mean white blood cell and lymphocyte counts were considered to be incidental and unrelated to test substance administration. There were no test substance-related or statistically significant alterations in coagulation parameters. There were no test substance-related effects on haematology parameters noted in the 10, 50, 250, or 750 ppm group males or females. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. These findings included higher mean white blood cell and absolute lymphocyte counts in the 750 ppm group females. In the absence of a dose response, these variations were considered to be incidental and unrelated to test substance administration.

SERUM CHEMISTRY:
Test substance-related higher cholesterol levels were noted in the 1,500 ppm group males and females. Test substance-related alterations in serum chemistry parameters were limited to high mean cholesterol levels in the 1,500 ppm group males and females. The variation was statistically significant in the 1,500 ppm group females when compared to the control group. An effect of test substance administration on cholesterol levels in the 1,500 ppm group males was suggested by the magnitude of change and dose response similar to that noted in the females. With the exception of one male in the 1,500 ppm group, all individual animal cholesterol levels were within the historical control reference range. There were no test substance-related effects noted on serum chemistry parameters in the 10, 50, 250, or 750 ppm group males or females.

URINALYSIS:
Test substance-related lower urine pH was noted in the 1,500 ppm group males and variable urine colour (yellow, dark yellow, and/or red) was noted in the 1,500 ppm group males and females and 750 ppm group females. Test substance-related lower mean urine pH values were statistically significantly in the 1,500 ppm group males when compared to the control group. In addition, macroscopic evaluation of the urine revealed test substance-related variations in urine colour (yellow, dark yellow, and red) in the 1,500 ppm group males and females and in the 750 ppm group females. There were no test substance-related effects on urinalysis parameters in the 10, 50, or 250 ppm group males or females or the 750 ppm group males.

OPHTHALMIC EXAMINATIONS:
No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

MACROSCOPIC EXAMINATION:
Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance.

ORGAN WEIGHTS:
Test substance-related alterations in mean liver weights were noted in the 1,500 ppm groups. Statistically significantly higher mean liver relative to final body weights were noted in the 1,500 ppm group males and females. There were no test substance-related differences in organ weights noted in the 10, 50, 250, or 750 ppm group males or females. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. Mean kidney relative to final body weights and absolute testes weights were higher in the 1,500 ppm group males. The higher kidney relative to final body weight was a function of a slightly higher mean absolute kidney weight and slightly lower mean final body weight resulting in a ratio statistically significantly different from that noted in the control group males. Although statistically significantly different from the control group mean, mean absolute testes weights were only 7.8% higher in the 1,500 ppm group. All but two of 10 individual absolute testes weights for the 1,500 ppm group were within two standard deviations of the WIL Research historical control mean. Therefore, due to the small magnitudes of change and absence of histologic correlations for the organ weight differences, all of the statistically significant variations noted in the 1,500 ppm group males were considered to be incidental and unrelated to test substance administration. As was noted in the males, there were multiple statistically significantly higher mean relative organ weight variations due to slightly higher absolute organ weights in conjunction with the 10.5% lower final body weight noted in the 1,500 ppm group females. These higher organ relative to final body weight ratios were noted with the brain, kidney, and liver in the 1,500 ppm group females. Due to the small magnitudes of change and absence of histologic correlations for the organ weight differences, all of the statistically significant variations noted in the 1,500 ppm group females were considered to be incidental and unrelated to test substance administration. A higher mean thymus relative to final body weight ratio was noted in the 750 ppm group females and a lower mean absolute thyroid/parathyroid weight was noted in the 50 ppm group females. In the absence of a dose response, these variations were also considered to be incidental and unrelated to test substance administration.

MICROSCOPIC EXAMINATION:
Test substance related histopathologic findings were noted in the femoral bone (metaphyseal hyperostosis) at ≥750 ppm and kidneys (foreign material, tubular hyperplasia, and brown pigment) at ≥250 ppm group males and females. In the femur, hyperostosis was characterized by increased amounts of metaphyseal trabeculae filling the marrow space. This change was most obvious in the 1,500 ppm group males. In the control group males, the metaphyseal trabeculae typically did not extend into the diaphysis, the trabeculae were oriented parallel to the bone with minimal lateral anastomoses or branching, and this bone occupied <50% of the marrow cavity within the metaphysis. Hyperostosis in the males was characterized by the extension of trabeculae into the diaphysis with increased amounts of ‘lateral branching’ and occupation of >50% of the marrow cavity. Normal baseline morphology of the femur in the control group females was similar to minimal to mild hyperostosis in the males with trabeculae extending well into the diaphysis, lateral branching was common and trabeculae occupied up to approximately 75% of the marrow cavity in the metaphysis. Minimal hyperostosis was diagnosed in the females when this lateral branching was so prominent as to nearly span the diameter of the metaphysis. Hyperostosis was diagnosed in one female each from the control, 10, and 50 ppm groups which was attributed to the arbitrary subjective assessment of ‘normal’ and some variations in tissue morphology due to sectioning. Hyperostosis noted in the 750 ppm group males and females was considered to represent a test substance-related effect. In the kidney, there was a foreign material within the tubular lumina in the cortex (minimal), and rarely, the outer stripe of the outer medulla (mild). This material was brown to gray, granular, and formed small aggregates; the surrounding tubular epithelium appeared normal. The composition of this material was unknown but was inconsistent with tubular cell or inflammatory cell debris and was noted at dietary concentrations ≥250 ppm in both sexes. These findings associated with the foreign material were not considered adverse and the foreign material was not in the same location as the renal tubular hyperplasia noted. Renal tubular hyperplasia was characterized by small aggregates of basophilic tubules with epithelial cells that were enlarged and often piled upon each other. These tubules were consistently in the outer stripe of the outer medulla and were morphologically distinct from chronic progressive nephropathy (CPN) and basophilic tubules considered to be the precursors of CPN. This finding was noted only in the 1,500 ppm group males and females.
Two males in the 1,500 ppm group and one male from the 750 ppm group had small amounts of a coarsely granular brown pigment within tubular epithelial cells. Although this material was considered to be consistent with lipofuscin, a special staining procedure (AFIP method for lipofuscin) was negative. Therefore, the identity and toxicological importance of this pigment were undetermined. There were no other definitive test substance related histologic changes. The female in the 1,500 ppm group that died on study day 77 had minimal multifocal and bilateral renal tubular necrosis of the cortex. It was unclear if this finding was secondary to the animal’s moribund condition prior to death or was related to test substance administration.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

16 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: Metaphyseal hyperostosis noted microscopically in the femoral bone

2

Dose descriptor:

NOAEL

Effect level:

19 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: Metaphyseal hyperostosis noted microscopically in the femoral bone

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this 90 d dietary study, the NOAEL for systemic toxicity of the test substance in rat was considered to be 250 ppm (equivalent to 16 and 19 mg/kg bw/day for males and females, respectively).

Executive summary:

A 90 d dietary study in Sprague-Dawley rats was conducted at concentrations of 0, 10, 50, 250, 750 and 1,500 ppm test substance (i. e., equivalent to 0, 1, 3, 16, 47 and 91 mg a.i./kg bw/day for males and 0, 1, 4, 19, 55 and 113 mg a.i./kg bw/day for females) according to OECD Guideline 408 and OPPTS 870.3100, in compliance with GLP. This included evaluation of potential neurotoxicity by functional observational battery (FOB) and motor activity assessments. Ten males and 10 females per dose group were used. Adverse effects included haematological differences noted at 1,500 ppm (females only), tubular hyperplasia noted microscopically at 1,500 ppm, and metaphyseal hyperostosis noted microscopically in the femoral bone at ≥750 ppm. Under the conditions of this study, the NOAEL for systemic toxicity of the test substance in rat was considered to be 250 ppm (equivalent to 16 and 19 mg/kg bw/day for males and females, respectively) (Kirkpatrick JB, 2013).