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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-21 to 2013-01-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): OS48604M
- Physical state: Light brown, clear, viscous liquid
- Lot/batch No.: 1X4 LT
- Stability under test conditions: Considered stable under these conditions
- Storage condition of test material: room temperature, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Crl:CD(SD) rats were received in good health from Charles River Laboratories, Inc. on 22 May 2012; 75 females received from the Raleigh, NC facility, 38 males were received from the Portage, MI facility, and 37 males were received from the Kingston, NY facility.

The animals were approximately 63 days old upon receipt.
The animals were housed for an acclimation period of 10 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior, and the testes of all males were palpated at least once.

Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week. The 5 rats/sex in the control and high-dosage groups assigned to the post-treatment period were not paired and remained in clean, stainless steel wire-mesh cages until euthanasia. The breeding phase rats (12/sex/group) were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding. The dams and their litters were housed in these cages until euthanasia on PND 4 (pups) or lactation day 5 (dams). Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until post-cohabitation or post-mating day 25.

Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study, with the following exception. All males and lactation day 5 females in the treatment phase were fasted prior to clinical pathology blood collection when food, but not water, was withheld.

All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively. Actual mean daily temperature ranged from 70.1°F to 71.4°F (21.2°C to 21.9°C) and mean daily relative humidity ranged from 45.5% to 54.4% during the study.

Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod.

Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): stability and solubility
- Concentration in vehicle: 0, 20, 40, 100, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): 2AF0861 and 2AJ0197, exp. dates: 1 June 2013 and 14 September 2013
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and 8-day room temperature stability were established at concentrations of 50 and 200 mg/mL in a previous study (Toot, Draft, WIL-168199). Prior to the initiation of dose administration, samples for homogeneity determination were collected from the top, middle, and bottom strata of the 20 mg/mL dosing formulation. In addition, samples for stability and resuspension homogeneity determinations were collected from the top and bottom strata of this same dosing suspension following room temperature storage for 8 days. Samples for concentration analysis were collected weekly from the middle stratum of each dosing formulation (including the control group). One set of samples from the first and last dosing formulations was subjected to the appropriate analyses. The remaining set of samples was stored frozen (approximately -20°C) until discarded following acceptance of the analytical results. Following the completion of the dosing period, additional formulations were prepared at 20 and 200 mg/mL to further assess stability of the test item in corn oil. Samples were collected from the middle stratum of these formulations on the day of preparation (time zero) and following 13 days of frozen storage (approximately -20°C).

Validated gas chromatography method with flame ionization detection.
A GC method using FID for the determination of OS48604M concentration in formulations containing corn oil and test item ranging in concentration from
10.0 to 400 mg/mL was validated in a previous study (Toot, Draft, WIL-168199). In the present study. A formulation prepared at a target test item concentration of 20 mg/mL was analyzed to assess test item homogeneity/concentration acceptability and, following 8 days of room temperature storage, resuspension homogeneity and stability.
Additionally, formulations prepared at target test item concentrations ranging from 20 to 200 mg/mL, following 13 days of frozen (approximately -20°C) storage, were analyzed to assess test item stability. Finally, formulations used for dose administration were analyzed to verify test item concentration acceptability.

Method
Instrument: Varian CP-3800 GC equipped with an flame ionization detector, autosampler, and Dionex Chromeleion® software version 6.8 or Varian MS Workstation software
Column: Zebron ZB-5HT Inferno, 30 m × 0.25 mm, 0.25-μm film-thickness
Injector Temperature: 200°C
Injection Volume: 1.0 μL, split
Split Ratio: 10:1
Carrier Gas: Helium
Flow Rate: 1.5 mL/minute
Temperature Program: Initial temperature 50°C, hold for 3.0 minutes
Ramp at 25°C/minute to 300°C, hold for 2.0 minutes
Detector: Flame ionization detector
Detector Temperature: 310°C
Retention Time: Approximately 6.1 minutes for OS48604M
Run Time: 15.0 minutes
Retention time of the test item was approximately 6.1 minutes

Calibration
A calibration stock solution was prepared at a concentration of 1.00 mg OS48604M/mL as follows. Approximately 10 mg of OS48604M (WIL ID no. 120017; no correction for purity) was accurately weighed in a tared glass weigh funnel and transferred to a 10-mL volumetric flask with rinses of acetone. The preparation was mixed as necessary to achieve complete dissolution of the test item. Additional acetone was added to obtain the desired concentration.
Calibration standards: 100, 125, 150, 175, and 200 μg/mL

QC
QC samples were prepared to simulate the processing of formulation samples at concentrations of 10.0, 200, and 400 mg/mL (nominal QC concentrations) by combining aliquots of the QC stock solution, vehicle (corn oil), and acetone in polypropylene tubes. The processed samples were mixed with vortex action. Portions of the samples were further diluted as necessary with acetone in amber autosampler vials.

Formulation sampling
Quadruplicate formulation samples were collected using a syringe and dosing cannula and placed in polypropylene tubes. Two samples from each quadruplicate set were processed for analysis. The remaining 2 samples (back-up samples) were stored frozen (approximately -20°C). Formulation samples were processed by adding acetone and mixing with vortex action. Samples were further diluted with acetone in amber autosampler vials.

Specificity
Assay specificity/selectivity was confirmed when GC/FID analysis of processed vehicle samples revealed that there were no significant peaks (with S/N >10) at or near the retention time for the test item (approximately 6.1 minutes)

Homogeneity
The analyzed formulation met the acceptance criteria for homogeneity, i.e., the RSD for the mean concentration was ≤10% at a concentration within the acceptable limits (85% to 115% of the target concentration), concentration acceptability for suspension formulations, i.e., the analyzed concentration was 85% to
115% of the target concentration, and resuspension homogeneity, i.e., the RSD for the mean concentration was ≤10%.

Test item stability
The mean post-storage concentration was 94.5% of the pre-storage value.
The 13-day frozen (approximately -20°C) mean post-storage concentrations ranged from 92.2% to 95.0% of the pre-storage values.

Test item concentrations

Mean Concentration, mg/mL (% of Target)
Date of Preparation Group 1 Group 2 Group 3 Group 4 Group 5
(0 mg/mL) (20 mg/mL) (40 mg/mL) (100 mg/mL) (200 mg/mL)
31 May 2012 ND 21.4 (107) 43.8 (109) 112 (112) 214 (107)
12 July 2012 ND 20.8 (104) 42.6 (107) --- 203 (101)
19 July 2012 --- --- --- 98.2 (98.2) ---
No test item was detected in the analyzed vehicle administered to the control group
Duration of treatment / exposure:
The males selected for mating were dosed during study days 0-30 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a
total of 31 doses.
The females selected for mating were dosed during study days 0 through 2 days prior to euthanasia (14 days prior to pairing through lactation day 3) for a
total of 39-51 doses; however, these females should also have been dosed on lactation day 4 but were not administered the test item.
Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or postcohabitation day 25) for a total of 39-52 doses.
Males not selected for pairing remained on study for a 14-day non-dosing post-treatment period following 28 doses.
Females not selected were dosed through study day 39 (the day prior to the first necropsy of paired females on lactation day 5), for a total of 40 doses.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 rats/sex in the control and high-dosage groups assigned to the post-treatment period
Breeding phase rats: 12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Random

Dose selection:
Dosage levels were selected based on the results of previous studies, in which an acute oral toxicity study conducted in albino rats produced an LD 50 at 2 g/kg, as well as a 14-day dietary range-finding oral toxicity study (Toot, Draft, WIL-168199), and a 7-day repeat dose range-finding oral toxicity study (Toot, Draft, WIL-168200). In the 14-day dietary study (Toot, Draft, WIL-168199), mean body weight losses and lower mean body weight gains and/or food consumption were noted for males and females at all dietary concentrations (5000, 10,000, and 15,000 ppm). Corresponding findings of decreased defecation and/or small feces were in these groups. Lower heart, spleen, and/or thymus weights (absolute and/or relative to final body weight) were noted at ≥ 10,000 ppm in
males and/or females. Lower mean absolute and relative adrenal gland and thymus weights were noted at ≥ 10,000 ppm group for females, and higher mean absolute and relative thyroid/parathyroid and liver weights were noted at ≥ 5000, 10,000, and/or 15,000 ppm for females.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, as part of neurobehavioural observations.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon, for moribundity and mortality
Individual detailed physical examinations were recorded weekly (prior to test item administration during the treatment period). Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration.
Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded weekly, beginning 1 week prior to test item administration, on the first day of dose administration, and weekly beginning 1 week prior to test item administration thereafter until euthanasia. Individual female body weights were recorded weekly, beginning 1 week prior to test item administration, until evidence of copulation was observed for females selected for pairing. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 0 (when possible), 1, and 4. Weekly body weights continued to be recorded for those females with no evidence of mating and those that were not selected for pairing.

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Individual food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. Following mating, food consumption for females with no evidence of mating and for all post-treatment phase males and females was measured on a weekly basis until the scheduled euthanasia

OPHTHALMOSCOPIC EXAMINATION: No

CLINICAL PATHOLOGY
Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 6 animals/sex/group at the scheduled necropsies (study day 31, males and lactation day 5, females) and from 5 animals/sex/group in the control and high-dosage groups following a 14-day non-dosing post-treatment period (study day 42 for males and study day 54 for females). The same animals selected for FOB and motor activity assessments were selected for clinical pathology assessment at the end of the treatment period.
All animals (including those males not scheduled for clinical pathology assessments) were fasted overnight prior to blood collection, while in metabolism cages
for urine collection. Blood for serum chemistry and hematology was collected from the retro-orbital sinus following isoflurane anesthesia.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked:
Total leukocyte count (WBC)
Erythrocyte count (RBC)
Hemoglobin (HGB)
Hematocrit (HCT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin
(MCH)
Mean corpuscular hemoglobin
concentration (MCHC)
Platelet count (PLATELET)
Prothrombin time (PT)
Activated partial thromboplastin time
(APTT)
Reticulocyte count
Percent (RETIC)
Absolute (RETIC ABSOLUTE)
Mean Platelet Volume (MPV)
Red cell distribution width
(RDW)
Hemoglobin Distribution Width
(HDW)
Differential leukocyte count -
Percent and absolute
-Neutrophil (NEU)
-Lymphocyte (LYMPH)
-Monocyte (MONO)
-Eosinophil (EOS)
-Basophil (BASO)
-Large unstained cell (LUC)
Platelet estimatea
Red cell morphology
(RBC Morphology)

CLINICAL CHEMISTRY: Yes
- Parameters checked:.
Albumin
Total protein
Globulin
Albumin/globulin ratio (A/G Ratio)
Total bilirubin (Total Bili)
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)
Bile acidsa
Appearance

URINALYSIS: Yes
- Parameters checked:
pH
Urobilinogen (URO)
Total volume (TVOL)
Color (COL)
Clarity (CLA)
Protein (PRO)
Glucose (GLU)
Ketones (KET)
Bilirubin (BIL)
Occult blood (BLD)
Leukocytes (LEU)
Nitrites (NIT)
Osmolality a
Microscopy of sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: Recorded for 6 animals/sex/group during study week 4 (prior to dose administration; males) and on lactation day 4 (females)
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Home cage observations, handling, open field
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy included examination of the external surface, all orifices and the cranial cavity, external surfaces of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.
Organs/tissues examined microscopically:
Adrenal glands (2) Ovaries and oviducts (2), Aorta, Pancreas, Bone with marrow (sternebrae), Peripheral nerve (sciatic), Brain (forebrain, midbrain, hindbrain), Pituitary gland, Coagulating glands (2), Prostate gland, Eyes with optic nerve (2), Salivary gland (mandibular [2]), Gastrointestinal tract, Seminal vesicles (2), Esophagus, Skeletal muscle (rectus femoris), Stomach Skin with mammary gland, Duodenum Spinal cord (cervical), Jejunum, Spleen, Ileum, Testes with epididymidesd (2), Cecum and vas deferens, Colon, Thymus gland, Rectum Thyroids [with parathyroids, Heart if present (2)],Kidneys (2), Trachea, Liver (sections of 2 lobes), Urinary bladder, Lungs (including bronchi, fixed Uteruse with cervix and vagina by inflation with fixative), All gross lesions, Lymph node (axillary [2], mesenteric, and mandibular [2])
Organ weights: Adrenal glands, Ovaries with oviducts, Brain, Spleen, Epididymidesa, Testesa, Heart, Thymus gland, Kidneys, Thyroids with parathyroids, liver.

HISTOPATHOLOGY: Yes
Microscopic examination was performed on all tissues listed previously from all animals that were found dead, euthanized in extremis, and from control and 1000 mg/kg/day group males and females that were selected for pairing at the scheduled treatment period necropsies. In addition, all gross lesions from all groups and the liver and thyroid glands from all animals in the low- and mid-dosage groups and all animals in the post-treatment phase were examined microscopically.
Statistics:
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Statistical analyses were not conducted if the number of animals was 2 or less. Data obtained from non-gravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group by sex.
Mean parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, absolute and relative organ weights, clinical pathology values, and FOB data values were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test item-treated groups to the control group. FOB parameters that yield scalar or descriptive data and histopathological findings in the test item-treated groups were compared to the control group using Fisher’s Exact test (Steel and Torrie, 1980). If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test item-treated groups to the control group.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Mortality:
mortality observed, treatment-related
Description (incidence):
See details on results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See details on results
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
One male each in the 500 and 1000 mg/kg/day groups was found dead. The male in the 1000 mg/kg/day group was found dead on study day 8 prior to dose administration and had an unkempt appearance, red material on the urogenital area, forelimbs, nose, mouth, and ventral abdomen, yellow material on the urogenital area, and decreased defecation within 3 days of death. This male lost 114 g of body weight and consumed only 5 g/day of feed during study days 0-7. Pale, enlarged thyroid glands were noted for this male at necropsy. In the 500 mg/kg/day group, one male was found dead on study day 31 prior to dose administration. The only clinical finding for this male consisted of clear material around the mouth observed 1 hour following dose administration 9 days prior to death. Dark red areas in the lungs were noted for this male at necropsy; there was no evidence of esophageal perforation. Test item-related microscopic findings for these males (hepatocellular vacuolation [both males] and hepatocellular cytomegaly and thyroid follicular cell hyperplasia [1000 mg/kg/day male]) were similar to those observed in the 1000 mg/kg/day group males at the scheduled necropsy. Therefore, the deaths in the 500 and 1000 mg/kg/day group males were attributed to the test item.
One female in the 500 mg/kg/day group and one female in the 1000 mg/kg/day group were euthanized in extremis on gestation day 22 and lactation day 0, respectively, due to dystocia; coagulative necrosis of the liver was noted microscopically and contributed to the condition of these females. Pale, cool body was noted during parturition for the female in the 500 mg/kg/day group on the day of euthanasia. Dystocia, also noted in other females and was attributed to the test item. In addition, microscopic findings for these females included test item-related coagulative necrosis of the liver; this finding was considered the cause of moribundity. Two females in the control group were found dead on lactation days 5 and 0, respectively; a cause of death could not be determined for either female. No clinical findings were noted prior to death for these females. At necropsy, dark red areas in the lungs were noted for one control group female and lungs that were not fully collapsed, dark red contents in the jejunum, and dark red areas in the thymus gland were noted for the other control group female. There was no evidence of esophageal perforation for either of these females.
All other animals survived to the scheduled necropsies.

Test item-related clinical findings were noted in males and females in all test item-treated groups at the time of dosing and/or 1 hour following dose administration; the onset (earlier at higher dosage levels), number of affected animals, and frequency of findings generally occurred in a dose-related manner. The most frequently observed findings (red/clear material around the mouth/nose and yellow material around the urogenital area) were generally noted throughout the treatment period, at least sporadically, at ≥200 mg/kg/day once observed. Salivation noted at ≥200 mg/kg/day or evidence thereof (clear material around the mouth/nose) at ≥100 mg/kg/day were attributed to potentially irritating properties of the test item and were not considered adverse. Red material around the mouth at ≥ 200 mg/kg/day and red material around the nose, and yellow material on the urogenital area at 1000 mg/kg/day were not considered adverse. Soft stool at 1000 mg/kg/day was considered adverse. Although males only received 31 doses (treatment period males) or 28 doses (1000 mg/kg/day post-treatment period males), more males were affected and/or had generally earlier onset of findings compared to the females, which received 39-51 doses.
Following the 14-day post-treatment period, clinical observations noted in the 1000 mg/kg/day group males and females (hair loss or scabbing on the neck or forelimbs and red material around the nose) were noted in single animals and similarly in the control group.

BODY WEIGHT AND WEIGHT GAIN, FOOD CONSUMPTION
Mean body weight losses and/or lower mean body weight gain and food consumption were observed in the 500 and 1000 mg/kg/day group males generally throughout the treatment period. As a result, mean body weights in these males were 9.2% and 13.4% lower, respectively, than the control group on study day 30. Mean body weights and body weight gain in the 1000 mg/kg/day during the post-treatment period remained lower than the control; however, mean body weight gain was slightly improved. No test item-related effects on mean body weights, body weight gains, or food consumption were noted in the 100 and 200 mg/kg/day group males. No test item-related effects on food consumption were noted in females at any dosage level during the pre-mating period.
Lower mean food consumption was noted in the 1000 mg/kg/day group females generally throughout gestation, and lower mean body weight gains were noted only late in gestation (days 14-20), resulting in a mean body weight that was 5.8% lower than the control group on gestation day 20. The effects on mean body weight gain late in gestation in the 1000 mg/kg/day group females were attributed to the test item-related lower mean number of pups and an increase in the mean number of unaccounted-for sites, rather than maternal toxicity. No test item-related effects on mean body weights and body weight gain were noted in these females during lactation or in the 1000 mg/kg/day group post-treatment phase females. No test item-related effects on mean body weights, body weight gains, or food consumption were noted in the 100, 200, and 500 mg/kg/day group females throughout the treatment period.

CLINICAL CHEMISTRY, HAEMATOLOGY, URINALYSIS
Test item-related clinical pathology findings in male rats included the following. Longer mean prothrombin and activated partial thromboplastin times and higher mean urine volumes and lower urine osmolality were noted at >100 mg/kg/day. Minimally to slightly higher mean albumin, total protein, and alkaline phosphatase levels were noted at 500 and 1000 mg/kg/day. Slightly lower mean erythrocyte and eosinophil counts, triglyceride, chloride, phosphorus, and potassium levels and slightly higher mean reticulocyte counts and red blood cell distribution width, mean globulin, total bilirubin, creatinine, and cholesterol, were noted at 1000 mg/kg/day. Test item-related findings were considered to be adverse for prothrombin at >200 mg/kg/day and for activated partial thromboplastin time at 1000 mg/kg/day. These findings resolved in the 1000 mg/kg/day group males during the recovery period with the exception of slightly lower mean erythrocyte and eosinophil counts and alanine aminotransferase level. Nonadverse, test item-related findings in female rats consisted of minimally to slightly higher mean albumin levels at >100 mg/kg/day, higher mean cholesterol levels and lower mean alanine transferase levels at >200 mg/kg/day, lower erythrocyte counts, and mean hemoglobin and hematocrit levels and urine osmolality and higher mean total bilirubin at >500 mg/kg/day, and slightly higher mean red blood cell distribution width, and alkaline phosphatase levels, and slightly lower mean triglyceride levels at 1000 mg/kg/day.
Following the post-treatment period, percent and absolute mean reticulocyte counts were slightly lower than the control group in the 1000 mg/kg/day females. The mean hemoglobin distribution width in the 1000 mg/kg/day females was slightly lower than controls on study day 54 and was considered to be secondary to the lower mean reticulocyte counts.

NEUROBEHAVIOUR
No test item-related effects were noted at any dosage level during the FOB or motor activity evaluations during study week 4 (males) or on lactation day 4 (females).

ORGAN WEIGHTS
Test item-related effects on organ weights consisted of lower mean thymus in the 200, 500, and 1000 mg/kg/day group males, lower mean spleen and heart weights in the 500 and 1000 mg/kg/day group males, lower brain weight in the 1000 mg/kg/day group males, higher mean liver weights in the 500 and 1000 mg/kg/day group males, and higher thyroid/parathyroid weights in 500 and 1000 mg/kg/day group males and females at the treatment period necropsy. Mean liver and thyroid gland weights remained higher in the 1000 mg/kg/day group females at the post-treatment necropsy.

GROSS PATHOLOGY
No test item-related macroscopic findings were noted at the necropsy of F0 animals that were found dead, euthanized in extremis, or at the scheduled necropsies.

HISTOPATHOLOGY
Test item-related microscopic findings were noted in the liver and thyroid of the 200, 500, and 1000 mg/kg/day groups. These findings corresponded to higher organ weights and consisted of midzonal hepatocellular vacuolation, hepatocellular cytomegaly, and thyroid follicular cell hyperplasia. Liver lesions were more severe in males than in females for all test item-treated groups. The severity of hepatocellular vacuolation and thyroid follicular cell hyperplasia in the 1000 mg/kg/day group males and females was less at the post-treatment period necropsy.

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: An expert review of the data indicates that the effects observed at 200 mg/kg/day are adaptive with animals in the recovery groups showing a reduction in symptoms.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The analyzed dosing formulations were within WIL Research’s SOP range for suspensions (85% to 115%), the 20 mg/mL dosing formulation was homogeneous, and the 20 and 200 mg/mL dosing formulations were stable after 13 days of frozen storage.

The test item was not detected in the vehicle formulation that was administered to the control group (Group 1).

See attached Document: Analyses of dosing formulations.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this screening study, the NOAEL for systemic toxicity was 100 mg/kg/day based on effects on organ weights (lower thymus gland weights at ≥200 mg/kg/day, lower spleen and heart weights at ≥ 500 mg/kg/day, lower brain weight at 1000 mg/kg/day, and higher liver and thyroid gland weights at ≥500 mg/kg/day) and the presence of test item-related microscopic findings at ≥200 mg/kg/day. The microscopic findings were still present at the
post-treatment phase necropsy, however, at lesser severity.

The NOAEL of 100 mg/kg bw/day as suggested by the Study Director is based on minor and reversible effects on organ weights and microscopic findings at 200 mg/kg bw/day whereas more significant toxic effects were noted at 500 and 1000 mg/kg bw/day. Consequently it is considered more appropriate to propose a LOAEL of 500 mg/kg bw/day, a NOAEL of 200 mg/kg bw/day and 100 mg/kg bw/day as the NOELfor systemic toxicity
Executive summary:

Test Guidance

OECD 422: A Combined 28-Day Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity

Screening Test in Rats, with Recovery.

Method and material

The test item, OS48604M, in the vehicle (corn) was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and 2 mid-dosage groups (Groups 2, 3, and 4, respectively) each consisted of 12 males and 12 females, and the high-dosage

group (Group 5) consisted of 17 males and females. Dosage levels were 100, 200, 500, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group (Group 1) of 17 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 10 weeks of age at the beginning of test item administration. Twelve males/group selected for pairing received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 31 doses. Twelve females/group selected for pairing received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-51 doses; however, these females should also have been dosed on lactation day 4 but were not administered the test item. The females that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 39-52 doses. The extra 5 rats/sex in the control and high-dose groups were not used for mating but were treated on a comparable regimen beginning on study day 0; following 28 doses for the males and 40 doses for the females, these animals were assigned to the post-treatment period and remained on study for a 14-day non-dosing period.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and locomotor activity data were recorded for 6 males/group following approximately 28 days of dose administration and for 6 females/group on lactation day 4. All F0 females were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (hematology, serum chemistry, and urinalysis) were performed on 6 F0 animals/sex/group at the treatment period necropsy and on all animals at the post-treatment period necropsy. The F0 males were euthanized following completion of the mating period, and the F0 females were euthanized on lactation day 5. The 5 rats/sex in the control and high-dosage groups assigned to the post-treatment period were euthanized following the 14-day non-dosing period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dosage groups at the treatment period necropsy. In addition, the liver and thyroid glands from all animals in the low- and mid-dosage groups and all animals in the post-treatment phase were examined microscopically.

Results

One male each in the 500 and 1000 mg/kg/day groups was found dead on study days 31 and 8, respectively. The 1000 mg/kg/day group male lost 114 g of body weight and had an unkempt appearance, red material on the urogenital area, forelimbs, ventral abdomen, nose and mouth, and decreased defecation prior to death. Findings for the 500 mg/kg/day group male were limited to red material around the mouth 1 week prior to death. These deaths were attributed to the test item. One female in each of these groups was euthanized in extremis during the treatment period due to test item-related dystocia; pale, cool body was noted for the female in the 1000 mg/kg/day group. Microscopic findings for these females included test item-related coagulative necrosis of the liver, which was considered the cause of moribundity. This finding was also noted in 2 males in the 200 mg/kg/day group and 1 female each in the control, 100, and 500 mg/kg/day groups at the treatment period necropsy. All other animals survived to the scheduled necropsies.

Test item-related clinical findings noted in the 100, 200, 500, and 1000 mg/kg/day group males and females consisted of salivation prior to dose administration or clear material around the mouth and/or nose. These findings were attributed to potentially irritating properties of the test item and were not considered adverse. Other test item-related clinical findings noted in the 200, 500, and 1000 mg/kg/day groups consisted of red material around the mouth. In the 1000 mg/kg/day group males and females, yellow material on the urogenital area, red material around the nose, and soft stool were also noted. Soft stool was considered adverse. Mean body weight losses and/or lower mean body weight gain and food consumption were observed in the 500 and 1000 mg/kg/day group males generally throughout the treatment period. As a result, mean body weights in these males were 9.2% and 13.4% lower, respectively, than the control group on study day 30. Mean body weights and body weight gain in the 1000 mg/kg/day during the post-treatment period remained lower than the control; however, mean body weight gain was slightly improved. No test item-related effects on mean body weights, body weight gains, or food consumption were noted in the 100 and 200 mg/kg/day group males. No test item-related effects on food consumption were noted in females at any dosage level during the pre-mating period. Lower mean food consumption was noted in the 1000 mg/kg/day group females generally throughout gestation, and lower mean body weight gains were noted only late in gestation (days 14-20), resulting in a mean body weight that was 5.8% lower than the control group on gestation day 20. The effects on mean body weight gain late in gestation in the 1000 mg/kg/day group females were attributed to the test item-related lower mean number of pups and an increase in the mean number of unaccounted-for sites, rather than maternal toxicity. No test item-related effects on mean body weights and body weight gain were noted in these females during lactation or in the 1000 mg/kg/day group post-treatment phase females. No test item-related effects on mean body weights, body weight gains, or food consumption were noted in the 100, 200, and 500 mg/kg/day group females throughout the treatment period.

No test item-related effects were noted at any dosage level during the FOB or motor activity evaluations during study week 4 (males) or on lactation day 4 (females). Test item-related clinical pathology findings in male rats included the following. Longer mean prothrombin and activated partial thromboplastin times and higher mean urine volumes and lower urine osmolality were noted at >100 mg/kg/day. Minimally to slightly higher mean albumin, total protein, and alkaline phosphatase levels were noted at 500 and 1000 mg/kg/day. Slightly lower mean erythrocyte and eosinophil counts, triglyceride, chloride, phosphorus, and potassium levels and slightly higher mean reticulocyte counts and red blood cell distribution width, mean globulin, total bilirubin, creatinine, and cholesterol, were noted at 1000 mg/kg/day. Test item-related findings were considered to be adverse for prothrombin at >200 mg/kg/day and for activated partial thromboplastin time at 1000 mg/kg/day. These findings resolved in the 1000 mg/kg/day group males during the recovery period with the exception of slightly lower mean erythrocyte and eosinophil counts and alanine aminotransferase level. Nonadverse, test item-related findings in female rats consisted of minimally to slightly higher mean albumin levels at >100 mg/kg/day, higher mean cholesterol levels and lower mean alanine transferase levels at >200 mg/kg/day, lower erythrocyte counts, and mean hemoglobin and hematocrit levels and urine osmolality and higher mean total bilirubin at >500 mg/kg/day, and slightly higher mean red blood cell distribution width, and alkaline phosphatase levels, and slightly lower mean triglyceride levels at 1000 mg/kg/day. Following the post-treatment period, percent and absolute mean reticulocyte counts were slightly lower than the control group in the 1000 mg/kg/day females. The mean hemoglobin distribution width in the 1000 mg/kg/day females was slightly lower than controls on study day 54 and was considered to be secondary to the lower mean reticulocyte counts. One and 2 females in the 500 and 1000 mg/kg/day groups, respectively, were noted with dystocia. This finding was attributed to the test item. No test item-related macroscopic findings were noted at the necropsy of F0 animals that were found dead, euthanized in extremis, or at the scheduled necropsies. Test item-related effects on organ weights consisted of lower mean thymus in the 200, 500, and 1000 mg/kg/day group males, lower mean spleen and heart weights in the 500 and 1000 mg/kg/day group males, lower brain weight in the 1000 mg/kg/day group males, higher mean liver weights in the 500 and 1000 mg/kg/day group males, and higher thyroid/parathyroid weights in 500 and 1000 mg/kg/day group males and females at the treatment period necropsy. Mean liver and thyroid gland weights remained higher in the 1000 mg/kg/day group females at the post-treatment necropsy. Test item-related microscopic findings were noted in the liver and thyroid of the 200, 500, and 1000 mg/kg/day groups. These findings corresponded to higher organ weights and consisted of midzonal hepatocellular vacuolation, hepatocellular cytomegaly, and thyroid follicular cell hyperplasia. Liver lesions were more severe in males than in females for all test item-treated groups. The severity of hepatocellular vacuolation and thyroid follicular cell hyperplasia in the 1000 mg/kg/day group males and females was less at the post-treatment period necropsy.

Conclusions

Under the conditions of this screening study, the NOAEL for systemic toxicity was 100 mg/kg/day based on effects on organ weights (lower thymus gland weights at ≥200 mg/kg/day, lower spleen and heart weights at ≥ 500 mg/kg/day, lower brain weight at 1000 mg/kg/day, and higher liver and thyroid gland weights at ≥500 mg/kg/day) and the presence of test item-related microscopic findings at ≥200 mg/kg/day. The microscopic findings were still present at the post-treatment phase necropsy, however, at lesser severity.

The NOAEL of 100 mg/kg bw/day as suggested by the Study Director is based on minor and reversible effects on organ weights and microscopic findings at 200 mg/kg bw/day whereas more significant toxic effects were noted at 500 and 1000 mg/kg bw/day. Consequently it is considered more appropriate to propose a LOAEL of 500 mg/kg bw/day, a NOAEL of 200 mg/kg bw/day and 100 mg/kg bw/day as the NOELfor systemic toxicity