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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:
30 May - 29 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: A022I34007
- Purity: 97.84 wt%
- Water content: 0.06 wt%
- Manufacturing date: 2018-03-04

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (room temperature)
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Solubility and stability of the test substance in the solvent/vehicle: Solubility analyses performed previously demonstrated that the formulation was soluble in the vehicle at a concentration of 12.0 mg/mL. Stability analyses performed previously demonstrated that the test substance is stable in the vehicle following at least 49 hours of room temperature storage, and following at least 8 days of refrigerated storage in formulations at concentrations bracketing those used in the present study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dilution in vehicle

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species and strain for toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: no information available
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: 139 - 270 g
- Housing: in groups of 3 animals of the same sex until randomization; in groups of 2 animals of the same sex and dosing group following randomization
- Diet: ad libitum; PMI Nutrition International, LLC Certified Rodent LabDiet® 5CR4 meal
- Water: ad libitum; municipal tap water after treatment by reverse osmosis
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water was performed. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 17 May 2018 To: 29 Aug 2018

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since oral ingestion is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
Vehicle:
water
Remarks:
deionized
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test substance dosing formulations were prepared based on Sponsor instructions at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily during Week 1 and approximately weekly thereafter. An adequate amount of each formulation was dispensed into daily aliquots, which were stored in a refrigerator set to maintain a target of 5°C, until use. The dosing formulations were stirred continuously during dosing.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis:
Day -1: Group 1
Day 1: Groups 2 - 4
Day 4: Groups 2 - 4
Day 28: All groups
Day 56: All groups
Day 84: All groups
Analyses were performed by a gas chromatography method with flame ionization detection using a validated analytical procedure.

The analyzed dosing formulations contained 90.2% to 110% of the test substance which was within the protocol-specified range of target concentrations for solutions (90% to 110%) with the following exceptions. The mean analyzed concentrations of the 30 May 2018 Group 2, Group 3, and Group 4 formulations were 118%, 87.3%, and 84.9% of the target concentrations, respectively. The back-up samples were processed and analyzed on 31 May 2018. The mean
analyzed concentration of the Group 2 formulation met the previously stated protocol-specified acceptance criteria for concentration. The results of the analyzed Group 3 and Group 4 back-up samples confirmed the initial results. This is not expected to have an impact on the study since the formulations were being prepared daily at the time, and the samples collected on Day 4 were within the acceptable range. In addition, the mean analyzed concentrations of the 26 Jun 2018 Group 2 and Group 3 formulations were 89.1% and 84.0% of the target concentration, respectively. Although formulations were being prepared approximately weekly at this sampling interval, this is not expected to impact the study since the analyzed values for Groups 2 and 3 were within 20% of the targeted concentration and Group 4 samples met acceptance criteria.
No test substance was detected in the analyzed vehicle administered to the control group (Group 1).
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 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: The dose selection was based on the results of an OECD TG 422 in Wistar rats, in which strong irritation including erosion and ulcer in the forestomach, as well as inflammatory changes in the duodenum were detected in the parental animals of the high dose (150 mg/kg). The effects in the forestomach and duodenum were still observed in the mid dose (50 mg/kg), but less pronounced. It was anticipated that the high-dosage level would show substance-specific effects but not produce an incidence of fatalities that would prevent a meaningful evaluation. The lower dosage levels were selected at intervals that were predicted to be narrow enough to reveal any dose-related trends. Though priority was given to detecting a dose-related trend, it was expected that the low-dosage level would be a no-observed-adverse-effect level (NOAEL).

- Fasting period before blood sampling for clinical biochemistry: overnight
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (for general health/mortality and moribundity)
- Time schedule: twice daily, once in the morning and once in the afternoon
- Cage side observations were performed daily, beginning on Day 1 and lasting throughout the dosing period. During the dosing period, these observations were performed 1 to 3 hours postdose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: within 4 days of receipt, on the day of randomization, on Day 1 (prior to dosing), weekly (± 2 days) during the study period, and on the day of the scheduled necropsy
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size (further parameters as required)

BODY WEIGHT: Yes
- Time schedule for examinations: within 4 days of receipt, on the day of randomization, on Day 1 (prior to dosing), weekly (± 2 days) during the study period, on the day prior to the first day of scheduled necropsy, and on the day of the scheduled necropsy. A fasted weight was recorded on the day of the scheduled necropsy.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: weekly (± 2 days) starting on Day 1 and continuing weekly throughout the study

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during the acclimation period (Day -9) and near the end of the dosing period (Day 87)
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13 (Day 91 or 92)
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (overnight)
- How many animals: all
- 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); Reticulocyte count; Percent (RETIC); Absolute (RETIC Absolute); Differential leukocyte count - Percent and absolute; -Neutrophil (NEU); -Lymphocyte (LYMPH); -Monocyte (MONO); -Eosinophil (EOS); -Basophil (BASO); -Large unstained cell (LUC); Red cell distribution width (RDW); Platelet estimate; Red cell morphology (RBC Morphology)
Activated partial thromboplastin time; Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 (Day 91 or 92)
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters checked: Alanine aminotransferase (ALT); Albumin; Albumin/globulin (A/G) ratio (calculated); Alkaline phosphatase (ALP); Aspartate aminotransferase (AST); Calcium; Chloride; Creatinine; Gamma glutamyltransferase (GGT); Globulin (calculated); Glucose; Phosphorus; Potassium; Sodium; Sorbitol dehydrogenase (SDH); Total bilirubin; Total cholesterol; Total protein; Triglycerides; Urea nitrogen; Appearance;

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13 (Day 91 or 92)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (during stay in the metabolism cages)
- Parameters checked: Bilirubin; Color and clarity; Glucose; Ketones; Occult blood; pH; Protein; Specific gravity; Total volume

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:
Functional observational battery (FOB): for all animals/sex/group during Week 13 prior to exposure
- The functional observational battery was carried out in 10 animals/sex/group during Study Week 12.
- Examined parameters:
• Home cage observations: Biting, Convulsions/tremors, Feces consistency, Palpebral (eyelid) closure, Posture
• Handling observations: Ease of removal from cage, Lacrimation/chromodacryorrhea, Piloerection, Palpebral closure, Eye prominence, Red/crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character,
Mucous membranes/eye/skin color, Muscle tone
• Open field observations (evaluated over a 2-minute observation period): Mobility, Rearing, Convulsions/tremors, Grooming, Bizarre/stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/defecation, Gait score, Backing
• Sensory observations: Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eyeblink response, Hindlimb extension, Olfactory orientation
• Neuromuscular observations: Grip strength-hind and forelimb, Hind limb extensor strength, Hind limb foot splay, Rotarod performance
• Physiological observations: Body temperature, Body weight, Catalepsy

Motor activity assessment:
- assessed for all animals/sex/group during Week 13
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities, including viscera.

HISTOPATHOLOGY: Yes
- Microscopic examination of hematoxylin-eosin stained paraffin sections was performed from animals in the control and high-dose groups at the scheduled necropsy.

Organs weighed: Adrenal glands; Brain; Epididymides (total and caudal); Heart; Kidneys; Liver; Ovaries; Pituitary; Prostate with seminal vesicles; Spleen; Testes; Thymus; Thyroid with parathyroid; Uterus

Tissue collection and preservation: Adrenal glands; Aorta; Bone with marrow; Sternum; Femur; Bone marrow smear (from femur); Brain; Cervix; Epididymides; Eyes with optic nerves; Gastrointestinal tract; Esophagus; Stomach; Duodenum; Jejunum; Ileum; Cecum; Colon; Rectum; Harderian glands; Heart; Kidneys; Larynx; Liver (sections of 2 lobes); Lungs (including bronchi, fixed by inflation with fixative); Lymph nodes; Axillary; Mesenteric; Mandibular; Ovariesf with oviductsd; Pancreas; Peripheral nerve (sciatic); Peyer's patches; Pharynx; Pituitary; Prostate; Salivary glands (mandibular); Seminal vesicles; Skeletal muscle (rectus femoris); Skin with mammary glande; Spinal cord (cervical, thoracic, lumbar); Spleen; Testes; Thymus;Thyroid (with parathyroids; Tongue; Trachea; Uterus; Urinary bladder; Vagina; Gross lesions (when possible)
Other examinations:
Coagulation parameters: at the time of euthanasia from animals euthanized via carbon dioxide inhalation

Spermatogenic evaluations: The following quantitative assessment of the process of spermatogenesis was performed on all surviving males at the scheduled necropsy.
- Motility/viability assessment
- Morphology assessment
- Enumeration of epididymal and testicular sperm and sperm production rate (SPR)

Stage-dependent qualitative light microscopic evaluation of spermatogenesis was conducted on sections of testicular tissues with special attention given to the normal progression of stages of the spermatogenesis cycle, cell associations, and cell proportions expected to be present during spermatogenesis.

Thyroid Hormone Assessments:
- Blood samples were collected from all study animals for analysis of T3, T4, and TSH levels on the day of the terminal necropsy.

On the day of scheduled necropsy, all females received a vaginal lavage to determine the stage of estrus.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and are reported at the 1% and 5% levels.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Datasets were compared using an overall one-way ANOVA F-test.12 If the overall F-test was found to be significant, then the above pairwise comparisons were conducted using Dunnett’s test.
Further details are given in table 1.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- body weights/body weight gains were lower than expected from Days 85–90 in both sexes and all groups, which could be due to a brief period of fasting for the functional observational battery evaluations.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- test substance-related higher mean food consumption in the 100 mg/kg/day group males and females throughout the dosing period (Day 1–90) and in the 30 mg/kg/day group males from Day 29 through Day 90 (frequently statistically significant)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- statistically significantly higher mean cholesterol and potassium, and lower mean chloride levels in the 100 mg/kg/day group males on Week 13
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Home cage observations were unaffected by test substance administration. The only statistical significant difference from the control group was a higher number of females in the 10 m/kg/day group without fecal pellets. Based on a lack of dose response, this finding was not considered to be test substance-related.

- Handling observations, open field observations, sensory observations, neuromuscular observations and physiological observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the Week 13 evaluation.
- Motor activity patterns (total and ambulatory activity counts) were unaffected by test substance administration. Values obtained from the 6 epochs evaluated (0-10 minutes, 11-20 minutes, 21-30 minutes, 31-40 minutes, 41-50 minutes, and 51-60 minutes) and the overall 60-minute test session were comparable to the concurrent control values. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the animals were evaluated on Week 13.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- statistically significantly higher mean liver weights (absolute and relative to body and brain weights) and higher mean absolute and relative thyroid gland weights (not statistically significant) in the 100 mg/kg/day group males. There were no corresponding microscopic alterations in the liver or thyroid gland, thus, the weight changes were considered test substance-related and non-adverse.
- higher mean organ to body weight ratio values for the liver and thyroid/parathyroid in the 100 mg/kg/day group males, compatible with a test substance-related effect.
- higher mean kidney weights in the 100 mg/kg/day group males and lower mean thymus weights in the >10 mg/kg/day group females, but relationship of these to the test substance was considered uncertain (absence of microscopic changes)
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test substance-related changes in mean T3, T4, or TSH levels were detected on this study.
No test substance-related changes in sperm morphology or differential counts were detected on this study
There was no test substance-related effect on ovarian follicle counts in the 100 mg/kg/day group compared to the control group

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to and including the highest dose group

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

For summary tables of body weight, body weight changes, hematology and coagulation values, serum chemistry values, other chemistry values, sperm motility and concentrations, sperm morphology differential counts, gross pathology findings, organ weights absolute and relative, and histopathology findings see "Attached background material".

 

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, oral administration of the test substance to Wistar Han rats at dosage levels of 10, 30, and 100 mg/kg/day for a minimum of 90 days was well tolerated at all dosages. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 100 mg/kg/day.
Executive summary:

The test substance was administered daily by oral gavage to 10 male and 10 female Wistar Han rats at doses of 0 mg/kg bw/day (test group 1), 10 mg/kg bw/day (test group 2), 30 mg/kg bw/day (test group 3) and 100 mg/kg bw/day (test group 4) for at least 90 consecutive days.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, functional observational battery, motor activity, ophthalmology, thyroid hormone assessment, clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis), spermatogenesis evaluation, gross necropsy findings, organ weights, and histopathologic examinations.

All animals survived to the scheduled necropsy. There were no test substance-related clinical observations or effects on body weight, hematology, coagulation, or urinalysis. There were no test substance-related ophthalmic, macroscopic, microscopic findings, or effects on ovarian follicle counts, thyriod hormones (T3, T4 or TSH levels), sperm morphology or differential counts.

Test substance-related higher food consumption was noted in the 100 mg/kg/day group males and females (Days 1–90), and in the 30 mg/kg/day group males (Days 29–90), compared to the control group.

Test substance-related higher cholesterol and potassium, and lower mean chloride levels were noted in the 100 mg/kg/day group males on Week 13, compared to the control group.

Test substance-related higher mean organ weights were noted in the liver and thyroid/parathyroid gland of the100 mg/kg/day group males. Higher mean kidney weights were present in the 100 mg/kg/day group males, and lower mean thymus weights were seen in the >10 mg/kg/day group females, but relationship of these to the test substance was considered uncertain.

Based on the results of this study, oral administration of the test substance to Wistar Han rats at dosage levels of 10, 30, and 100 mg/kg/day for a minimum of 90 days was well tolerated at all dosages. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 100 mg/kg/day.