Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-10-22 to 2016-06-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat (Rattus norvegicus) has been selected by the Sponsor to satisfy the regulatory equirements for toxicity testing as the preferred rodent species, according to the test guideline.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Canada Inc. (St-Constant, Quebec, Canada)
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 177 to 237 g; females: 137 to 221 g
- Housing: Animals were housed individually in stainless steel rodent cages equipped with an automatic watering system. Following group assignment, all cages were clearly labeled with a color-coded cage card indicating at least the following: study number and animal number.
- Diet: A standard certified commercial chow (ENVIGO Teklad Certified Global Rodent Diet #2018C) was provided to the animals ad libitum.
- Water: Municipal tap water (which was exposed to ultraviolet light and purified by reverse osmosis) was provided to the animals ad libitum, except during designated procedures, via an automatic watering system or water bottles when considered necessary.
- Acclimation period: An acclimation period of 14 or 15 days was allowed between receipt of the animals and the start of dosing to accustom the animals to the laboratory environment.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 50 ± 20 %
- Air changes: 10 - 15 per h
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route is one of the potential routes of human exposure to this test item and was considered suitable to provide the systemic exposure required on this toxicology study. The oral route is not the deliberate route of human exposure of the test item, but needs to be taken into account for an occupational exposure risk assessment.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item and reference item/vehicle dosing formulations were prepared weekly. The test item was weighed and transferred into an appropriate sized container (or weighed directly into the container). The vehicle (corn oil, selected for its ability to solubilize the test item) was added (Q.S.), followed by stirring. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stir plate. The dosing formulations were stored at room temperature prior to administration.

VEHICLE
- Justification for use and choice of vehicle: solubility of test item
- Concentration in vehicle: 5; 20; 75 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Supplier: Sigma-Aldrich
- Lot/batch no.: MKBV2080V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
To verify the concentration and homogeneity of the test item in the formulations, representative samples (5 mL, in duplicate) were taken from the top, middle, and bottom of each concentration (including control) at the start of the study, and from the middle of a preparation conducted at Week 11 (concurrently with the FOB) and towards the end of the treatment period. An additional 15-mL sample was collected from the middle of each concentration at the start of the study for density measurement (discarded after measurement). Samples were stored frozen (set to -20°C) pending shipment (first set) or discard (second set). One of the two sets of the duplicate samples was shipped on dry ice to CiToxLAB Hungary. The second set of the duplicates was retained at CiToxLAB North America pending discard. The analysis was performed by the analytical laboratory by gas chromatography (15/347-316AN) within the expected 1-month stability data period. Acceptance criteria for the formulations were ± 15 % of nominal. The analytical report was provided by the Principal Investigator to the Study Director for inclusion in the study report. Any analytical samples not employed in the primary analysis or required following completion of the primary analysis were discarded prior to issuance of the audited final report. The absence of test item concentration in Vehicle samples (Group 1) was demonstrated. All formulations were shown to be homogeneous. Agreement with nominal concentration were inside a ± 10% range from nominal, ranging from 89 to 100% of nominal concentration.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected by the Sponsor based on available data and previous experiments performed by the Sponsor in rats, including Harlan Study Number D53274 (Combined Repeated Dose Toxicity 28-day / Reproduction/Developmental Toxicity Screening Test, OECD 422). In that study, Han Wistar rats were administered Neononylacetate at dose levels of 40, 125 and 400 mg/kg bw/day. All males survived until the scheduled necropsy. The No-Observed-Adverse-Effect-Level (NOAEL) was determined to be 400 mg/kg/day (males), the No-Observed-Effect-Level (NOEL) to be 40 mg/kg bw/day (females), and the reproduction and development NOEL to be 40 mg/kg bw/day as well. Mortality of females was observed at the end of the gestation period at dose levels of 125 and 400 mg/kg bw/day (2 and 8 females, respectively), possibly due to complications during the last days of pregnancy and/or difficult parturition. No clinical effects were observed at 40 and 125 mg/kg bw/day. Moderately decreased food intake was noted at 400 mg/kg bw/day (both males and females) at the beginning of the pre-pairing period, but was reversible thereafter. A body weight loss was noted at 400 mg/kg bw/day (both genders) at dosing start, but it recovered. There were no adverse test item-related histopathological changes. Therefore, a high dose level of 300 mg/kg bw/day was selected for this study. The low and mid dose were selected to provide a graded dose response.

- Post-exposure recovery period in satellite groups: 28 days
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during all phases of the study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to animal assignment, then weekly starting on Day -8, and including the day prior to necropsy (or up to 2 days prior, given the necropsies of Main animals were staggered over 2 days). On the day of scheduled necropsy, a fasted body weight was recorded, for the calculation of relative organ weight.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined: Yes
Individual food intake was recorded weekly throughout the study starting one week prior to initiation of dosing.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during pre-dose period; once at week 13
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Cell morphology; Platelet count; Hematocrit; Plateletcrit / thrombocrit; Hemoglobin; Red blood cell count; Hemoglobin distribution width; Red cell distribution width; Mean corpuscular hemoglobin; Reticulocyte counts (absolute and relative); Mean corpuscular volume; White blood cell count (WBC); Mean corpuscular hemoglobin concentration; WBC differential (absolute and relative)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters examined: A/G ratio (calculated); Creatinine; Alanine aminotransferase; Globulin (calculated); Albumin; Glucose; Alkaline phosphatase; Phosphorus (inorganic); Aspartate aminotransferase; Potassium; Bile acids; Sodium; Bilirubin (total); Total protein; Total Calcium; Triglycerides; Chloride; Urea; Cholesterol (total)

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (16 +/- 1 hours)
- Animals fasted: Yes
- Parameters examined: Bilirubin; pH; Blood Protein; Color and appearance; Specific gravity; Glucose; Urobilinogen; Ketones; Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during pre-treatment period and once during week 11
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other: please refer to "Functional Observation Battery" below

OTHER:
- Functional Observation Battery:
The Functional Observation Battery (FOB), consisting of a series of observations, was performed for each Main and Recovery animal on 2 occasions: once during the pre-treatment period (also including spare animals) and once during Week 11, 2 to 3 hours post-dosing. All observations were performed by a technician that was blinded to the dose level of the observed animals. The observations included the following:
Open-field evaluation: Involuntary motor movements (such as convulsion and tremors); Respiration; Rearing; Urination; Defecation; Gait; Arousal; Mobility; Abnormal or stereotyped behavior
Manipulative evaluation: Ease of removal from the cage; Handling reactivity; Palpebral closure; Approach response; Auditory test (Click response); Tail pinch response; Touch response; Pupil response to light; Extensor thrust reflex; Cornea reflex; Salivation; Piloerection; Eye observations (eg., lacrimation, exophthalmus) Physiological evaluation: Body temperature
Neuromuscular evaluation: Grip strength (forelimbs and hindlimbs); Landing foot splay

- Estrous Cycle:
The estrous cycles of all Main and Recovery females were determined by daily examination of the vaginal lavage smears for 14 days prior to their respective necropsy, and at scheduled necropsy. The estrous cycle was evaluated as described in CiToxLAB North America standard operating procedures.

- Thyroid Hormone Determination:
Blood samples (target of 1.1 mL) were collected from the jugular vein of all animals once pre-treatment and during Weeks 12 (Main animals only) and 16 (Recovery animals only), approximately at the same time during the day, into tubes containing K3-EDTA as anticoagulant. Samples were kept on wet ice for a maximum of 30 minutes pending centrifugation. Samples were centrifuged under refrigeration (set to +4°C at 1500 g RCF) for targeted 10 minutes. Plasma samples were transferred into 3 separate tubes (at least 125 μL in the first 2 tubes and the remaining plasma in the third tube) and placed on dry ice pending storage in a freezer set to maintain -70°C (pending shipment). The first two sets of samples (the first and second tubes) were shipped on dry ice, after each occasion, except for both terminal occasions (Weeks 12 and 16) which were shipped together as soon as possible after collection of Week 16 samples. Samples were shipped to Joachim Decorde, DEST, at CiToxLAB France. The third set of tubes was retained frozen (set to -70°C) at CiToxLAB North America pending discard (pending). Excursions from the target volume were documented in the raw data. In the event samples had to be re-centrifuged, the times of sample processing were documented. Samples from Main and Recovery animals were analyzed for measurement of TSH, T3 and T4 using a validated LC-MS/MS method (T3 and T4) or by Luminex (Millipex kit, for TSH). The method numbers were 39971RDR.MET.V04 for T3 and T4 and 40045RD.MET.V02 for TSH. Samples collected at pre-treatment from spare animals not assigned to the study were discarded.

- Male Reproduction Assessment:
Male reproductive assessments, using the right epididymis, were performed on control and high dose animals only, except for the motility which was performed on all animals. For low and mid dose animals, slides were prepared and the epididymis retained for possible future evaluation of spermatozoa count and morphology (to be determined prior to finalization). These assessments were not performed on animals found dead or preterminally euthanized.
a) Sperm Motility: The motility of the sperm was assessed as follows following dispersion of the sperm from the epididymis into medium:
- No motility
- Small number of motile sperm
- Moderate number of motile sperm
- Large number of motile sperm
- Some wave motion
- Fast wave motion
b) Spermatozoa Count: Sperm concentration (millions/gram of epididymis) was assessed from 2 counts of sperm obtained from the cauda epididymis.
c) Morphology: Two spermatozoa smears were prepared and the percent of abnormal sperm (out of an approximate 200 sperm cells/animal) was assessed.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Tissues intended for histological examination were prepared by embedding in paraffin wax, sectioning and staining with hematoxylin and eosin-phloxin. Liver tissue slides were stained with Oil-red O (ORO) for identification of fat vacuoles. For the ORO staining, fresh tissues were snap frozen in liquid nitrogen then embedded in OCT.
Histopathological examination was performed by the Study Pathologist, on the tissues identified under the tissue preservation section from all Main and Recovery animals of Groups 1 (Control) and 4 (High Dose), from the identified target organs (i.e. liver and kidneys) from Main and Recovery animals of intermediate groups, as well as unscheduled deaths or euthanasia and any gross abnormalities from animals of all groups.
Statistics:
for detailed information see "Any other information"

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item-related clinical signs were limited to salivation, wet and/or stained fur at the urogenital area and brown tail skin. Due to the overall incidence and/or severity of these signs, they were considered tolerable and not adverse. Salivation (occasionally associated with wet fur at lower jaw, muzzle and/or abdomen) was noted mainly at the high dose (Group 4, 300 mg/kg bw/day) in both sexes. Clinical signs of fur wet at the urogenital area (including sacrum and/or abdominal) was observed mainly at the high dose (Group 4, 300 mg/kg bw/day) in both sexes, but also in two mid dose animals (Group 3, 80 mg/kg bw/day), and was accompanied with yellow fur staining at similar body areas (i.e. urogenital, sacrum, abdominal and/or scrotum) at dose levels ≥ 80 mg/kg bw/day, although it was also observed once in a control female. These clinical signs were likely associated with the increased urinary output observed in test item-treated animals at end of treatment urinalysis. Brown staining of skin at the tail region (recorded as ‘skin brown at the tail’) was noted at all dose levels including control males, but with a greater incidence in Groups 3 and 4 males (≥ 80 mg/kg bw/day). In females, it was limited to one animal per test item-treated group (≥ 20 mg/kg bw/day). As for the wet fur, this increased incidence of brown skin staining was possibly linked with the increased urinary output, at least in a few individual animals. Thin fur cover noted mainly on the paws and/or limbs (fore and/or hind) was at all dose levels, including controls. Although this stress-linked clinical sign is common in laboratory-housed rats, the incidence of this observation was greater in test item-treated groups (≥ 20 mg/kg bw/day) compared to control, indicating likely increased stress in individual animals secondary to test item treatment. Other clinical signs (broken nail or teeth; thinness; ocular or penile discharge (red, brown or white); fur dull and/or stained (black, brown or red); skin dry, flaking, blue, red, yellow, scab, lump; swelling firm or soft; tail kinked, prominent ring or partly missing) were considered incidental since they occurred across groups (generally including control animals), are common in laboratory-housed members of this species, and/or showed no relationship to dose levels in incidence or severity.
Mortality:
mortality observed, treatment-related
Description (incidence):
There was a single animal death in the high dose (1 of 30 total high dose animals) that was of uncertain origin, but in the absence of other possible procedural causes, and since this animal received the high dose of test item, a relationship to treatment with the test item could not be discounted. There was also an early humane sacrifice of a control animal. Two female animals were preterminally euthanized or found dead during the Main phase of the study. High dose female No. 4602 was found dead approximately 6 hours post-dose on Day 27. There were no dosing incidents reported and no abnormal clinical signs noted prior to death. The animal was eating well and gaining weight normally prior to death. Microscopically, Neononylacetate-related changes consisted of moderate renal tubular hyaline cast consistent with a possible protein losing nephropathy and minimal tubular vacuolation in the kidneys, as well as up to moderate lipid deposits in the liver and minimal hepatocellular centrilobular hypertrophy. A definitive cause of death could not be ascertained but was likely related to the test item. The control female (No. 1507) was presenting with a fracture to the right hindlimb (of unknown cause) and was therefore euthanized for humane reason. The clinical pathology parameters measured at euthanasia of this animal were overall normal (unfasted).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on body weights and body weight changes. Minor though significant variations in body weight changes were observed in groups of animals treated at 300 mg/kg bw/day when compared to concurrent controls (Weeks 9 and 15 in males, and Week 13 in females). However, as it was not reflected in the mean body weights which remained comparable to control throughout the dosing and recovery phases, these variations in body weight changes were considered incidental; furthermore most individual values were generally within the range of control values. Other changes in body weights or body weight change were considered incidental since similar body weights or body weight changes were noted in some individual control animals, and/or reflected the normal inter-animal variation in this species.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significant increases in mean food consumption were observed at 300 mg/kg bw/day (both sexes); however, these increases had no impact on mean body weights.
In males at 300 mg/kg bw/day, the mean food consumption increase compared to control, first observed at Week 7 and persisting during the recovery period, reached significance at Weeks 10 to 12 (+16 to 24 % from control mean). It should be noted that one high dose recovery male (No. 4015) persistently exhibited a weekly food intake considerably higher (except at Week 1) compared to any other males on the study, which could have contributed to the mean differences, especially during the recovery period where there were less animals per group. In females at 300 mg/kg bw/day, the increase in mean food consumption compared to control reached significance from Week 8 and for the remainder of the dosing phase (+11 to 26 % from control mean), and persisted during the recovery period however only showing significance during the last week of recovery (+12 % from control). Other variations in food consumption were considered incidental since similar food consumption was noted in some individual control animals, and/or reflected the normal interanimal variation in this species.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There was no adverse ocular effect caused by the administration of test item. Any findings noted at Week 13 examination were observed at the pretreatment evaluation or are spontaneous or considered normal for a rat population.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on hematology parameters. There was a minimal decrease in hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) in males, reaching statistical significance at the highest dose level only (-5 to -9% from mean control), and without a significant impact on the total red blood cell count or red cell distribution width (RDW). The mean hemoglobin distribution width (HDW) was slightly increased in high dose males when compared to controls (+11% from controls). In females, variations on red cell parameters were limited to minimal, though statistically significant, decreases in MCV and MCH at all dose levels, while the RDW and HDW were increased at the mid- and high-dose levels, though without being associated with decreased red blood cell counts, HGB and HCT, nor variations in levels of reticulocytes. These minimal variations in females (all less than 7% different from controls) did not follow a dose-dependent trend, and all individual values for these parameters were within normal limits for this species. The toxicological significance of these changes in red cell parameters, if any, is uncertain, and not considered adverse based on their low magnitude and in the absence of correlating histopathological changes.
Any other changes in hematology values, including some that resulted in statistically
significant differences between groups (i.e. at 300 mg/kg/day, decreased absolute and relative
eosinophils in Main females and increased absolute neutrophils in Recovery males), were
considered incidental since they resulted in values that were within the range of values
observed across control group, and/or reflected the normal inter-animal variation in this
species.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The use of corn oil, an oil containing high levels of polyunsaturated and monounsaturated fats, as the vehicle may have had a confounding influence on interpretation of test item-related effects on some clinical chemistry parameters. At main study termination, significant changes in clinical chemistry parameters were observed in females only and were signs of altered liver function, correlating with histopathological findings of hepatocellular vacuolation and/or necrosis. In males, significant test item-related effects on clinical chemistry parameters were only noted after completion of the 28-day recovery period and correlated with histological evidence of renal injury in affected animals. At the end of the Main phase, mean serum AST, ALT and ALP activities in females were increased compared to controls, and generally followed a dose-dependent trend, reaching statistical significance at the mid and/or high dose level (+55 to +226% compared to controls). The individuals showing the most marked elevations in liver enzyme activities (i.e., Nos. 2504, 2505, 3506, 3507, 3508, 4501, 4506, 4507, 4509 and 4510) were also generally those with the most prominent microscopic findings in the liver (hepatocellular vacuolation and/or hepatocellular necrosis). Similarly, significant increases in globulin and decreases in albumin, with correlating significantly decreased albumin/globulin ratio (though without effect on the mean total protein level), were noted in females at > 80 mg/kg bw/day when compared to controls. These changes were likely associated with the liver changes observed microscopically. In addition, there was a slight dose-dependent decrease in serum glucose in females, reaching statistical significance at the high dose only (-28% from controls). However, nearly all individual glucose values for treated females were within the normal range for this species, while most of the control values were outside the normal range. This reduction in serum glucose level was of uncertain relationship to treatment.

A minor though statistically significant decrease in mean calcium was observed in females at all dose levels compared to control groups (-3 to -4%). This decrease in calcium which was not dose-dependent, was possibly linked with the decreased albumin (this protein being the major carrier of blood calcium); however, there was no clear association at the individual level (i.e. animals with decreased calcium were not necessarily those presenting decreased albumin). Therefore, this decrease was not considered adverse or toxicologically significant, as it likely reflected subtle homeostatic effects without impact on animals’ health (i.e. no signs of gastro-intestinal fluid loss, dehydration, or renal failure in females).

Dose-dependent increases in mean total bile acids (TBA) were noted in all groups of treated females when compared to controls, though not reaching statistical significance at any dose levels. The mean TBA values in each treated group was generally attributable to one or a few individual animals presenting with slightly to markedly elevated levels compared to control range (e.g. Nos. 4506 and 4508 with TBA above 130 μmol/L). These changes were of limited toxicological relevance since the animals exhibiting the highest TBA values were not necessarily those presenting the most severe histopathological findings.

At the end of the Main phase, there was a slight dose-dependent decrease in triglycerides in males, reaching statistical significance at the highest dose level only (-60% from controls). Although, all individual triglyceride values for treated males were within the normal range for this species, all individual Group 4 values were below the range of control values. Since there were no significant increases in thyroid hormones in this study, these reductions in triglyceride levels, which were not associated with signs of fat malabsorption (although there was hepatocellular hypertrophy – enzymatic induction - in many treated males) and were only noted in males (although the adverse liver findings were noted in females), were not considered toxicologically significant. Given the histopathological changes in the kidneys observed in males, statistical changes in mean urea and/or creatinine were expected; however, only a few individual males (furthermore, not the ones presenting the most prominent renal histological changes) showed elevations in these clinical chemistry parameters.

At the end of the recovery period, test item-treated high dose males were observed with increased serum triglycerides, cholesterol, urea and/or glucose, and decreased potassium. These changes were most notable in animals with more significant kidney histological findings (e.g., moderate tubular degeneration/necrosis – Nos. 4011 and 4012) reaching statistical significance for the triglycerides and potassium only. Other changes in clinical chemistry values, (i.e., at 80 mg/kg bw/day, increased urea in Main females), were considered incidental since they resulted in values that were within the range of values observed across control group, and/or reflected the normal inter-animal variation in this species.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes in urinalysis parameters were limited to an increased volume of urine, correlating with the renal changes observed microscopically. The mean volume of urine samples was greater in Groups 3 and 4 (80 and 300 mg/kg bw/day – both sexes), in the range of +32 to 219% compared to the concurrent control mean; however, the urine specific gravity was generally comparable between treated groups and to the control group. Similarly, at the end of the recovery period, the mean volume of urine samples in Group 4 males (300 mg/kg bw/day) was 63% greater than the concurrent control mean; without significant impact on the urine specific gravity. These increased urine output occurred in some individual Main animals which had test item-related microscopic kidney changes (tubular degeneration/necrosis, granular cast, cellular debris, interstitial inflammation and increased amount of hyaline droplets in males, and tubular vacuolation in females) and overall higher mean kidney weights (in males). Other variations in urinalysis values (including animal No. 4012 presenting protein graded above or equal to 3.0 g/L at the end of the recovery) were considered incidental and/or reflected the normal inter-animal variation in this species.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional Observation Battery (FOB):
The general behavior of animals was analyzed and compared to the observations before dosing (pre-dosing occasion) and to the concurrent control (Group 1). There were no test item-related quantitative or qualitative FOB changes. Differences noted in a few parameters of the animals treated with test item, including those reaching statistical significance [i.e. in males at 2 to 3 hours post-dose at Weeks 11 to 13: number of rears in Group 4 (300 mg/kg bw/day), arousal in Group 3 and 4 (≥ 80 mg/kg bw/day), tail pinch response in Group 2 (20 mg/kg bw/day) and forelimb grip strength trial #1 in Group 3 (80 mg/kg bw/day)], were minor, comparable to pretreatment values, and/or did not exhibit a dose-response, and were considered to reflect normal biological variation of normal laboratory rats.

Motor Activity:
There were no treatment-related effects on motor activity. There were no significant differences between treated and control groups for the motor activity counts. An overall difference was detected for females Linear Time Contrast at the pre-treatment occasion, but the pairwise comparisons only revealed a significant difference from control for the lowest dose level (Group 2).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Main Phase: Organ weight changes (absolute and relative to body and brain weights) considered related to test item during the main phase included dose proportional increase in liver weights in both genders at 80 and 300 mg/kg bw/day and dose proportional increase in kidney weights in males at all dose levels, when compared to controls. The liver and kidney weight increases were often statistically significant. All other organ weight differences noted during the main period were considered to be within the range of normal biological variation in the rat.

Recovery: Following the 28-day recovery period, increased liver weight was generally reversed at 300 mg/kg bw/day (both sexes). However, increased kidney weight persisted in males at 300 mg/kg bw/day, but at a reduced magnitude compared to that observed at the end of the main phase, which was indicative of partial reversal. All other organ weight differences noted during the recovery period were considered to be within the range of normal biological variation in the rat.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Main Period:
Macroscopic changes considered Neononylacetate-related were observed in a dosed proportional manner in the liver of both sexes and kidney of males. In the liver, the changes consisted of prominent lobular architecture or pale discoloration in males and females at > 20 mg/kg bw/day, and were generally correlated microscopically with hepatocellular vacuolation. In the kidney, irregular surface, pale discoloration and/or mottled appearance were observed only in males dosed at 80 and 300 mg/kg bw/day, and were generally correlated microscopically with tubular degeneration/necrosis. A single female dosed with 80 mg/kg bw/day (No. 3508) had pale discoloration in the kidney which was interpreted to be incidental in nature and not related to test item. All other macroscopic findings noted during the main period in various organs and tissues distributed randomly among groups, including controls lacked any dose relationship to the test item were interpreted to be incidental, spontaneous or agonal and not test-item related.

Recovery:
Following a 28-Day recovery period, macroscopic changes were not observed in the liver of males and females dosed at 300 mg/kg/day, which was indicative of reversal. However, macroscopic changes of irregular surface and/or raised area persisted in the kidney of 2/5 male animals dosed at 300 mg/kg/day and were correlated microscopically with renal tubular degeneration/necrosis. All other macroscopic findings noted during the recovery period in various organs and tissues distributed randomly among groups, including controls lacked any dose relationship to the test item were interpreted to be incidental, spontaneous or agonal and not test-item related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Main Period:
In the liver, minimal to mild or moderate hepatocellular hypertrophy was observed in both sexes at 80 or 300 mg/kg bw/day, as well as in 2/10 males at 20 mg/kg bw/day. Hepatocellular hypertrophy was generally more pronounced in males and was interpreted to be likely adaptive in nature due to hepatic microsomal enzyme induction noted primarily in males at all dose levels, and in females at the high dose. Minimal to marked hepatocellular vacuolation was observed in the liver of both sexes at all dose levels including controls. The vacuoles stained positive with Oil-red-O which is consistent with fat vacuoles. Although fat vacuoles were present in the liver of all groups including controls, there appeared to be an increase in incidence and/or severity of fat vacuoles in the liver of males at 80 and 300 mg/kg bw/day and females at ≥ 20 mg/kg bw/day, based on a combination of both H&E and Oil-red-O staining patterns, where the higher severity grades of moderate to marked were mostly recorded in the test item-treated animals. Minimal to mild hepatocellular vacuolation could be within background levels in rats. However, hepatocellular vacuolation of moderate to marked severity was interpreted to be likely due to the vehicle control item, with exacerbation by the test item.
Both hepatocellular hypertrophy and hepatocellular vacuolation in the liver of both genders were likely responsible for the higher liver weights seen at 80 and 300 mg/kg bw/day, and accounted for the prominent lobular architecture/pale discoloration observed macroscopically in the liver. Also, 2/10 females (Nos. 4501 and 4506) at 300 mg/kg bw/day had minimal or mild hepatocellular necrosis associated with areas of hepatocellular vacuolation.
Overall, hepatocellular vacuolation was generally more pronounced in the liver of test item treated females compared to males. This along with the hepatocellular necrosis could be responsible for the slight elevation in serum liver enzymes noted in females at Day 92/93.

In the kidney, minimal to marked renal tubular degeneration/necrosis was observed in males at ≥ 20 mg/kg bw/day and accounted for the increased kidney weights and the macroscopic changes of irregular surface/mottled/pale discoloration noted in the kidneys. The renal tubular degeneration/necrosis in the kidneys of males at ≥ 20 mg/kg bw/day was accompanied by minimal to moderate granular cast consistent with cellular debris, minimal to moderate interstitial inflammation and minimal to marked increased amounts of hyaline droplets in tubular epithelium. All of the aforementioned changes were generally dose proportional based on incidence and/or severity, and were not observed in females, except for minimal interstitial inflammation in a single female at 20 mg/kg bw/day, which was interpreted to be likely spontaneous. In females, test item-related changes in the kidneys consisted of dose-related, minimal increase of renal tubular vacuolation at ≥ 20 mg/kg bw/day, compared to controls. This change was subtle in all affected animals.

Increased α2μ-globulin-positive hyaline droplets were observed in the tubular epithelium of the kidneys of males administered the test item when compared to males given the reference item/vehicle (corn oil) at the end of the 13-week treatment-period. Using Chromotrop Anilin Blue (CAB) staining, there were clear evidences of increased numbers of hyaline droplets in the test item-treated animals when compared to controls. This finding was observed only in the males and was highly suggestive of α2μ-globulin nephropathy, and was confirmed by automated IHC techniques using a monoclonal antibody from RD System, raised in the mouse species against rat urine-derived α2μ-globulin. The hyaline droplet nephropathy observed in males was considered to represent an adverse change for the rat only and to have limited relevance for other species including man.

All other microscopic changes recorded in various organs during the main period were considered incidental or spontaneous and not due to the test item.

Recovery:
Following the 28-Day recovery period, microscopic changes in the liver were generally reversed to minimal to mild severity that was interpreted to be within background levels. In the kidneys, increased hyaline droplet was not observed in renal tubular epithelium. Renal tubular degeneration/necrosis persisted at minimal to moderate severity in the kidney of all 5/5 males given 300 mg/kg bw/day, accompanied with minimal to mild granular casts and minimal to moderate bilateral interstitial inflammation. The renal tubular degeneration accounted for the increased kidney weights in recovery males as well as the irregular surface/raised area seen macroscopically in 2/5 animals given 300 mg/kg bw/day. One control and one high dose female animals also had minimal unilateral interstitial inflammation in the kidney which was interpreted to be spontaneous in nature.
All other microscopic changes recorded in various organs during the recovery period were considered incidental or spontaneous and not due to the test item.

Overall, renal tubular degeneration/necrosis of moderate to marked severity was considered adverse based on the magnitude of the change, a lack of reversibility following the 28-day recovery period and altered renal function. However, the hyaline droplet nephropathy
observed in males was considered to represent an adverse change for the rat only and to have limited relevance for other species including man. The hepatocellular vacuolation/necrosis was considered adverse in females based on elevated liver enzymes indicative of altered liver function.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Estrous Cycle:
There was an apparent dose-related trend in estrous prolongation with test item treatment when evaluated during the final two weeks of this study; this was suggestive of a test item-related effect most notably at the high dose. In the Main control group, every cycle in control females included only 1 estrous day except for one animal (No. 1509) which exhibited one sequence of 4 consecutive days of estrous. In groups of test item-treated Main animals, there was a trend for increased number of animals with prolonged estrous, as well as an increased total number of sequences of consecutive estrous days (2 to 4 days) per group. In the Recovery control group, every cycle in control females included only 1 estrous day. In the group of test item-treated Recovery females (300 mg/kg bw/day), there was 1 animal (No. 4512) which had a total of 12 estrous days over the 15 days of examination, including a sequence of 9 consecutive estrous days. None of the Main and Recovery Groups 1 and 4 animals with persistent estrous had histopathological findings in the ovaries or reproductive tract adding uncertainty as to its direct relationship to the test item. It is also unknown whether the liver effects noted in this study including liver hypertrophy, microsomal enzyme induction, and/or histological vacuolation/necrosis, may have indirectly contributed to hormonal changes potentially affecting estrus. In the absence of quantification of circulating gonadotropins and/or estrogen levels, it is unclear how test item treatment may be responsible for the apparent prolongation of estrus at the high dose. Any other variations in estrous cycle duration and/or sequence were considered incidental and/or reflected the normal inter-animal variation in female rats of this age (i.e. 17 to 19 weeks old or 22 to 24 weeks old for Main and Recovery females, respectively).
Details on results:
FURTHER EXAMINATIONS:

Thyroid Hormone Determination:
There were no test item-related effects on levels of thyroid hormones. At Week 12, T3 levels were slightly decreased in females, reaching statistical significance at 300 mg/kg bw/day when compared to control mean. At the end of the recovery period, the levels of T3 were slightly elevated compared to concurrent controls in 4 of 5 high dose females. Since most individual T3 values of treated females were comparable to individual concurrent control values or to predose values observed across groups, and/or were within the normal range for this species, this change was considered incidental. Other variations in thyroid hormones levels were considered incidental since they were noted in some control animals, resulted in values that were within the range of values observed across groups predose, and/or reflected the normal inter-animal variation in this species.

Male Reproduction Assessment:
There were no test item-related effects on spermatozoa motility, count and morphology. Any variations in male reproductive parameters were considered incidental and/or reflected the normal inter-animal variation in this species.

Hepatic Microsomal Enzyme Activity:
Test item-related effects on hepatic microsomal enzyme activity were limited to increased hepatic CYP1A1/2, CYP2B1/2, CYP3A1/2 and UDPGT activities in males, while in females, the effects were more related to the Phase II conjugation UDP-glucuronosyltransferase enzymes, and were negligible for the Phase I oxidative enzymes. This enzymatic induction correlated with the hepatocellular hypertrophy (minimal to moderate) observed in both sexes at 80 or 300 mg/kg bw/day, as well as in 2/10 males at 20 mg/kg bw/day, but generally more pronounced in males. Administration of test item for 13 weeks at dose levels up to 300 mg/kg bw/day predominantly affected the male inducible cytochrome P450 system, as well as the Phase II UDPGT metabolizing enzymes. The cytochrome P450 enzyme activities evaluated in this study were not significantly affected in females, and only at 300 mg/kg bw/day was there a small increase of the UDPGT family noted. The results of the enzyme activity analyses demonstrated that administration of test item at 20, 80 and 300 mg/kg bw/day for 13 weeks caused a consistent increase of hepatic microsomal enzyme activity in males, either the Phase I oxidative enzymes (CYP1A1/2 and CYP3A1/2) or the conjugative Phase II glucuronidation isoforms. However, only the highest dose showed a statistically significant difference with the control group for the four (4) tested hepatic enzymes. The effects of test item in the female groups were less noticeable. Indeed, only UDP-glucuronosyltransferase was increased in a statistically significant manner for the highest dose group, while a small but significant decrease of PROD activity was noted at 80 mg/kg bw/day. EROD activity was not affected by the test item while the testosterone 6B-hydroxylase activity was increased slightly, reaching approximately 1.7-fold for the highest dose, but not in a statistically-significant manner.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Once daily oral gavage administration of test item to male and female Sprague-Dawley rats at dose levels of 20, 80 and 300 mg/kg bw/day for at least 13 weeks resulted in mortality at the high dose in a single animal after roughly 1 month of dosing.
Salient test item-related effects were noted primarily in the liver and kidney and included hepatocellular hypertrophy (related to microsomal enzyme induction at all dose levels), hepatocellular vacuolation/necrosis at the high dose with increased serum liver enzymes, renal tubular degeneration/necrosis, casts, and inflammation (males) with increased urine volume and serum analyte changes in recovery males. The liver changes demonstrated complete or partial reversibility, while the kidney changes in males were not reversed within the 28-day observation period. The hyaline droplet (α2μ-globulin) nephropathy observed in males was considered to represent an adverse change for the rat only and to have limited relevance for other species including man.
Based on the results of this study and the observations of non-reversible microscopic kidney changes of minimal to marked severity in males at all dose levels, including up to moderate severity in low dose Main males, it was not possible to determine a No Observed Adverse Effect Level (NOAEL) for daily oral administration of test item in Sprague-Dawley rats for 13 weeks. In female rats, however, all test item-related effects were reversible at all dose levels, and since the liver changes were more severe at the high dose, and given that the mortality of one high dose animal was likely test item-related, the NOAEL was considered the mid dose of 80 mg/kg bw/day. Effects observed in male rats are probably caused by alpha2 -microglobin-associated nephropathy and are therefore considedered to be sex and species specific and thus not of relevance for humans. Therefore, these effects were not taken into account for risk assessment (DNEL derivation) and classification and labelling.
Executive summary:

The objective of the study was to determine the toxicity of the test item following daily oral gavage administration to the Sprague-Dawley rat for at least 13 weeks, and to assess the reversibility of any changes following a 28-day recovery period. The test item and reference item/vehicle were administered daily, during the dosing period, as shown in table 1.

Table 1. Study Design

Treatment

Group

Dose Level

(mg/kg/day)

Dose Conc.

(mg/mL)

Dose Volume

(mL/kg)

Number of Animals

Main

Recovery

M

F

M

F

1. Control*

0

0

4

10

10

5

5

2. Test Item-Low Dose

20

5

10

10

-

-

3. Test Item-Mid Dose

80

20

10

10

-

-

4. Test Item-High Dose

300

75

10

10

5

5

  *Group 1 animals received the reference item/vehicle (corn oil). M: male; F: female

The control group received the reference item (vehicle), corn oil. Animals were received on October 22, 2015, were 6 weeks old and weighed 137 to 237 grams at the start of treatment (November 05, 2015). During this study, assessments included mortality checks, clinical observations, body weight, food consumption, ophthalmology, functional observation battery, motor activity, and estrous cycle monitoring. Blood samples were collected for thyroid hormone determinations once pretreatment and at Weeks 12 (Main) and 16 (Recovery). Blood and urine samples were collected for clinical pathology (hematology, coagulation, clinical chemistry and urinalysis) from all surviving animals prior to their scheduled necropsies. Organ weight evaluation and complete necropsy with macroscopic examination of a comprehensive list of organs and tissues were performed on all Main and Recovery animals (euthanized on Days 92 or 93, and Day 120, respectively. Histopathological examination was performed in all Main and Recovery animals of Groups 1 (Control) and 4 (High Dose), target organs (liver and kidneys) from Main animals of Groups 2 (Low Dose) and 3 (Mid Dose), as well as any gross abnormalities from animals of all groups. Male reproductive assessments (sperm count, motility and morphology) were performed for Main males, using the right epididymis. Kidney samples from Main control and high dose males were collected for immunohistochemistry evaluation of alpha-2u-globulin, while liver samples from 4 Main animals/sex/group were used for determination of hepatic microsomal enzyme (cytochrome p450) activity.

Once daily oral gavage administration of test item to male and female Sprague-Dawley rats at dose levels of 20, 80, and 300 mg/kg bw/day for at least 13 weeks was well tolerated at all dose levels with the exception of a single high-dose animal death on Day 27, with no premonitory clinical signs or indication of intolerability. There were no test item-related effects on body weights, body weight changes, general behavior and motor activity, ophthalmology, hematology and coagulation, on levels of thyroid hormones, and on male reproductive assessments at any dose levels. There were no adverse test item-related clinical signs (limited to sporadic/transient salivation and wet/stained fur/skin at the tail and urogenital area) or non-adverse effects on food consumption (generally increased in females) mainly at the high dose (300 mg/kg bw/day). In test item-treated Main animals, there was a dose-related trend in the number of animals with prolonged estrous, as well as an increased total number of sequences of consecutive estrous days per group, most notably in the high dose group and which was also observed in one (high dose) recovery female. Although none of these animals had histopathological findings in the ovaries or reproductive tract; either a direct or indirect effect of the test item could not be ruled out.

Test item-related effects on hepatic microsomal enzyme activity were limited to increased hepatic CYP1A1/2, CYP2B1/2, CYP3A1/2 and UDP-glucuronosyl transferase (UDPGT) activities at all dose levels in males, while in females, UDPGT was most notably increased at the high dose. Liver enzyme induction correlated with microscopic hepatocellular hypertrophy (minimal to moderate) observed in both sexes, but generally more pronounced in males, at 80 or 300 mg/kg bw/day, as well as in males at 20 mg/kg/day.

Serum chemistry changes reflective of liver injury (dose-related increases in ALT, AST and alkaline phosphatase) were noted in test item-treated females only, and correlated with liver degenerative changes noted microscopically in individual animals. Reduced serum albumin, increased globulin, and reduced A/G ratios in mid- and high-dose females were considered likely related to liver injury in affected animals. The serum changes in females were no longer present in recovery animals. In contrast, serum chemistry changes in males were only noted in recovery animals (high dose) and included increased serum cholesterol, triglycerides, urea and/or glucose, with decreased potassium. Affected recovery animals were noted with histological renal tubule degeneration, casts, and inflammation. At main study termination at the mid and high dose levels, urinary output was increased in both sexes and persisted in recovery males (high dose). Microscopic changes considered related to the test item were present in the liver and kidneys.

Changes in the liver included hepatocellular hypertrophy at 80 and 300 mg/kg bw/day (both sexes) as well as in males at 20 mg/kg bw/day; hepatocellular necrosis in two females at 300 mg/kg bw/day; and hepatocellular vacuolation consistent with fat vacuoles which stained positive with Oil-red-O and considered to be largely due to the vehicle with exacerbation by the test item, particularly at moderate to marked severity. Hepatocellular vacuolation and hepatocellular hypertrophy accounted for the increased liver weights at 80 or 300 mg/kg bw/day (both sexes) and correlated with the macroscopic changes of prominent lobular architecture/pale discoloration. Hepatocellular hypertrophy was likely due to microsomal enzyme induction, and was more pronounced in males. In contrast, hepatocellular vacuolation was more pronounced in females. Changes in the liver were generally reversible following the 28-day recovery period.

Renal tubular degeneration/necrosis was noted in males at all dose levels accompanied with increased hyaline droplets, granular casts and interstitial inflammation. These changes accounted for the increased kidney weights in males as well as the macroscopic changes of irregular surface/mottled or pale appearance. Renal tubular degeneration/necrosis persisted in recovery animals, along with interstitial inflammation and granular casts, and accounted for the increased kidney weight and macroscopic changes of irregular surface/raised areas. Overall, moderate to marked histologic renal tubular degeneration/necrosis noted across all dose levels in males was considered adverse based on the magnitude of the change, lack of reversibility following the 28-day recovery period and serum analyte changes indicative of altered renal function.Immunohistochemistry in renal tubular epithelium in males indicated an increased presence of α2μ-globulin-positive hyaline droplets in test item-treated animals when compared to controls. In addition, hepatocellular vacuolation/necrosis was considered adverse in (high-dose) females based on severity, absence of full recovery, and magnitude of serum liver enzyme increases at study termination. Minimal renal tubular vacuolation was observed in females at ≥ 20 mg/kg bw/day, but not tubular degeneration/necrosis.

In summary, once daily oral gavage administration of test item to male and female Sprague-Dawley rats at dose levels of 20, 80 and 300 mg/kg bw/day for at least 13 weeks resulted in mortality at the high dose in a single animal after roughly 1 month of dosing.

Salient test item-related effects were noted primarily in the liver and kidney and included hepatocellular hypertrophy (related to microsomal enzyme induction at all dose levels), hepatocellular vacuolation/necrosis at the high dose with increased serum liver enzymes, renal tubular degeneration/necrosis, casts, and inflammation (males) with increased urine volume and serum analyte changes in recovery males. The liver changes demonstrated complete or partial reversibility, while the kidney changes in males were not reversed within the 28-day observation period. The renal changes noted in males were considered representative of a hyaline droplet (α2μ-globulin) nephropathy, an adverse change known to have limited relevance for other species including man.

Based on the results of this study and the observations of non-reversible microscopic kidney changes of minimal to marked severity in males at all dose levels, including up to moderate severity in low dose Main males, it was not possible to determine a No Observed Adverse Effect Level (NOAEL) for daily oral administration of test item in Sprague-Dawley rats for 13 weeks. In female rats, however, all test item-related effects were reversible at all dose levels, and since the liver changes were more severe at the high dose, and given that the mortality of one high dose animal was likely test item-related, the NOAEL was considered the mid dose of 80 mg/kg bw/day. Effects observed in male rats are probably caused by alpha2 -microglobin-associated nephropathy and are therefore considedered to be sex and species specific and thus not of relevance for humans.