O,O,O',O'-tetraoctyl-(branched and linear) S,S'-{methylenebis[imino(3-oxopropane-3,1-diyl)]} bis(phosphorothioate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 November 2014 to 14 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline, with no or minor deviations which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of methylenebis(acrylamide) and (O,O)-diisooctyl dithiophosphoric acid

EC Number:

700-945-5

Molecular formula:

Complex UVCB substance

IUPAC Name:

Reaction product of methylenebis(acrylamide) and (O,O)-diisooctyl dithiophosphoric acid

Details on test material:

- Physical state: Dark yellow, clear viscous liquid
- Analytical purity: 100%
- Expiry data: 18 September 2016
- Storage condition of test material: Room temperature in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Laboratories Inc., Raleigh, NC
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: males 150-228g, females 112-171g
- Housing: Housed in groups of 2-3 per cage by sex in solid bottom cages with ground corncob bedding material (Bed-O'Cobs®). Animals whose cage mate(s) were removed from study (morbidity or unscheduled death) were not re-paired and remained individually housed.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal), ad libitum throughout the study. Except during fasting prior to clinical pathology blood collection when food, but not water, was withheld
- Water (e.g. ad libitum): Reverse osmosis-purified water, delivered by an automatic watering system
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-3 °C
- Humidity (%):50+/-20 %
- Air changes (per hr):at least 10
- Photoperiod (hrs dark / hrs light):12 hours continuous light/dark

IN-LIFE DATES: From: 10 December 2014 To:10-11 March 2015 for non-recovery animals
From: 10 December 2014 To:07-08 April 2015 for recovery animals

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
-Vehicle was dispensed weekly for administration to the control group (Group 1) and for preparation of the test item formulations.
- Aliquots were prepared for daily dispensation to the control group and stored refrigerated, protected from light.
- Vehicle was mixed throughout the sampling and dose administration procedures.
- Aliquots were heated in a water bath at 37 °C ± 5 °C on each dosing day prior to dispensation for dosing.
- Test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated (2°C to 8°C), protected from light.
- Test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis oil was used as the substance formed stable homogenous solutions in this vehicle
- Concentration in vehicle:
Low – 50 mg/mL
Intermediate – 100 mg/mL
High – 150 mg/mL
- Amount of vehicle (if gavage):5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Test item formulations ranging from 10 to 220 mg/mL were previously assessed for homogeneity, resuspension homogeneity, and stability following 7 days of refrigerated storage.
- Samples for homogeneity were collected from the top, middle and bottom strata of the 50-150 mg/mL dosing formulations
- Resuspension homogeneity determinations, a volume of the 50 and 150 mg/mL formulations similar in size to the amount needed for 1 day was stored refrigerated, protected from light for 8 days.
- Samples were collected from the top and bottom of the formulations and analyzed after a minimum remixing of 30 mins using a magnetic stirrer.
Samples for concentrations analysis were collected from the middle stratum of each dosing formulation prepared for study week 0, 3, 7 and 12.
- All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated high performance liquid chromatography method with ultraviolet absorbance detection.
- The analyzed dosing formulations contained 85.2% to 94.9% and were within WIL Research’s SOP range for suspensions (85% to 115%) and were homogeneous. The test item was not detected in the vehicle formulation that was administered to the control group (Group 1).

Duration of treatment / exposure:

Test duration: 90-91 days
Control animals were treated in an identical manner with 5 mL/kg/day of Arachis oil.
Recovery group animals were maintained for a further 28 days treatment-free period following termination of treatment.

Frequency of treatment:

The substance was administered once daily, for 90-91 consecutive days, by gavage.
The volume of test and control material administered to each animal was based on the most recent bodyweight.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
250 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
750 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

15 animals of each sex (m/f) group 1 (control group: vehicle alone) and 4 (high dose, 750 mg/kg bw/day). (5 animals of each sex (m/f) were allocated to these groups to assess recovery persistence.)
10 animals of each sex (m/f) group 2 (low dose, 250 mg/kg bw/day) and 3 (intermediate dose, 500 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

other: recovery control (concurrent vehicle) ... (see attached file)

Details on study design:

- Dose selection rationale: Based on results of a previous 28-day study with no adverse treatment-related effects up to 1000 mg/kg bw/day. Lower dosage levels selected at intervals that were predicted to be narrow enough to reveal any dose-related trends.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Regulatory requirement
- Post-exposure recovery period in satellite groups: 28 days, treatment free

-Animals were then randomized into 5 study replicates to allow for the reasonable conduct of the functional observational battery and motor activity assessments. Each dose group and sex were approximately equally represented within each study replicate.

-Each animal was observed twice daily for mortality and changes in general appearance or Behavior during acclimation period.
- Individual body weights and cage food weights were recorded and detailed physical examinations were performed periodically during acclimation.
- Ophthalmic examination data were also recorded for animals during acclimation.

-First day of dosing was study day 0 and first week of dosing was study week 0.

Positive control:

Not used.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality and signs of overt toxicity. Also, final detailed physical examination prior to euthanasia for moribund animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the time of dose administration and 1-2 hours following dose administration. Once daily during the recovery period. Detailed physical examinations were conducted on all animal within 1 week (+/- 2 days) prior to randomization, on the day of randomization and weekly thereafter, and prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: 1 week (+/- 2 days) prior to randomization, on the day of randomization, prior to dosing on study day 0, weekly thereafter during the study period, and prior to the first day of the scheduled necropsy (non fasted).
The last nonfasted body weights were collected on the second to final day of study weeks 12 and 16, which was the day prior to the first day of the scheduled necropsies.
Final body weights (fasted) were recorded on the day of the scheduled necropsies.


FOOD CONSUMPTION
- Food consumption was recorded for each cage group on the day following randomization and once weekly thereafter throughout the study.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Last food consumption were collected on the second to final day of study weeks 12 and 16, which was the day prior to the first day of the scheduled necropsies.

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all animals were examined pre-treatment (week -2) and before termination of treatment (during Week 12).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Performed for all animals at the end of the study (week 12/13 for non recovery animals and week 16/17 for recovery animals). Blood was collected from the inferior vena cava at the time of necropsy.
- Anaesthetic used for blood collection: Yes. Inhalation of isoflurane.
- Animals fasted: Yes. Overnight prior to blood collection
- How many animals: all
- Parameters examined: Total leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, prothrombin time, activated partial thromboplastin time, reticulocyte count, mean platelet volume, differential leukocyte count (neutrophil, lymphocyte, monocyte, eosinophil, basophil, large unstained cell), red cell distribution width, hemoglobin distribution width, platelet estimate, red cell morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Performed for all animals at the end of the study (week 12/13 for non recovery animals and week 16/17 for recovery animals). Blood was collected from the inferior vena cava at the time of necropsy.
- Animals fasted: Yes. Overnight prior to blood collection
- How many animals: all
- Parameters were examined: Albumin, total protein, globulin, albumin/globulin ratio, total bilirubin, urea nitrogen, creatinine, AP, ALAT, ASAT, gamma GT, glucose, total cholesterol, calcium, chloride, phosphorus, potassium, sodium, triglycerides, sorbitol dehydrogenase, appearance

URINALYSIS: Yes
- Time schedule for collection of urine: Performed for all animals at the end of the study (week 12/13 for non recovery animals and week 16/17 for recovery animals).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes. Overnight prior to blood collection
- Parameters examined: Total volume, Ketones, Specific Gravity, bilirubin, pH, protein, glucose, urobilinogen, occult blood, color, nitrites, clarity, microscopy of sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional Observational battery and motor activity were recorded for all animals on the last day of study week 11 or during study week 12.
- The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB.
Motor activity
- Motor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilizes a series of
infrared photobeams surrounding an amber plastic, rectangular cage to quantify each animal’s motor activity.
- The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB.
- Each animal was tested separately.
- Data were collected in 5-minute epochs (print intervals) and the test session duration was 60 minutes.
- These data were compiled as six, 10-minute subintervals for tabulation.
- Dose groups that were examined: control, low dose, intermediate dose, high dose
- Battery of functions tested: Yes
FOB assessments
- Home Cage Observations: Posture, convulsions/tremors, feces consistency, biting, palpebral (eyelid) fissure
- 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 Filed Observations: mobility, rearing, convulsions/tremors, grooming, bizarre/stereotypic behavior, time to first step, grait, arousal, urination/defecation, grait score, backing
- Sensory Observations: Approach response, startle response, pupil response, forelimb extension, air righting reflex, touch response, tail pinche response, eyeblink response, hindlimb extension, olfactory orientation
- Neuromuscular Observations: Hindlimb extensor strength, hindlimb foot splay, grip strengths-hind and forelimb, rotarod performance
- Physiological Observations: Catalepsy, Body weight, Body temperature

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
DETAILS: Complete necropsy on all animals. Animals were euthanized by carbon dioxide inhalation (in extremis) or isoflurane inhalation (scheduled necropsy) followed by exsanguination.
- Gross lesions were examined from all animals at the primary necropsy for 250 and 500 mg/kg bw/day groups and all animals at the recovery necropsy.
- Necropsies included examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

HISTOPATHOLOGY: Yes
-The following tissues and organs were removed from all animals and preserved in 10% neutral-buffered formalin except where stated.
-Microscopic examination performed on all tissues listed from all animals euthanized in extremis and in the control and 750 mg/kg bw/day groups at primary necropsy.
-The following tissues: Adrenals, aorta, bone and bone marrow, bone marrow smear, brain, caecum, colon, duodenum, esophagus, epididymides, eyes with optic nerve, gross lesions, heart, ileum, jejunum, rectum, kidneys, liver, lungs, lymph nodes, ovaries with oviducts, pancreas, pituitary, prostate, rectum, salivary glands, peripheral nerve, peyer’s patches, seminal vesicles, skeletal muscle, skin with mammary gland, spinal cord, spleen, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, larynx, pharynx, urinary bladder, uterus, vagina

-Organ weight of the following organs: adrenals, brain, epididymis, heart, kidneys, pituitary, prostate with seminal vesicles, liver, ovaries with oviducts, spleen, testes, thymus, thyroid/parathyroid, uterus.

Other examinations:

None

Statistics:

- Statistical analyses were not conducted if the number of animals or cages was 2 or less.

ANALYSIS CONDUCTED BY WIL RESEARCH
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.
Body weight, body weight change, food consumption, continuous FOB, clinical pathology and organ weights data were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test item-treated groups to the control group. FOB parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact test.
In the special case when the statistical analysis included only 2 groups, as in the case during the recovery period, the Student’s t-Test was used to compare the control group to the 750 mg/kg/day group.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One female of the 750 mg/kg/day group euthanized in extremis on Day 9 following evidence of clinical signs (hypoactivity, increased and labored respiration, partial closure of eyes, red material around mouth/nose). Rales noted for other dose level groups on males and females. See details of results below

Mortality:

mortality observed, treatment-related

Description (incidence):

One female of the 750 mg/kg/day group euthanized in extremis on Day 9 following evidence of clinical signs (hypoactivity, increased and labored respiration, partial closure of eyes, red material around mouth/nose). Rales noted for other dose level groups on males and females. See details of results below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean cumulative body weight gains higher or lower than control depending on the study week for females at 500 and 750 mg/kg/ day. Effects attributed to test item but not considered adverse

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Higher mean food consumption values were noted for females at 500 and 750 mg/kg/day. Not dose-related, not correlated to body weight, no considered adverse and thefore, not related to test item

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Higher white blood cell and absolute lymphocyte counts for males at 500 and 750 mg/kg/day and higher absolute neutrophil counts for males at 750 mg/kg/day than concurrent control group. Not observed at recovery period. See details of results below.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Higher ALAT and ASAT for males at 750 mg/kg/day on primary necropsy compared to the concurrent control group. Not observed at recovery period. Other statistically differences not test item-related. See details of results below

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher liver, thyroid/parathyroid and spleen weights for males at 750 mg/kg/day. Also higher thyroid/parathyroid and spleen weights for males at 500 mg/kg/day. Higher thymus weights for females at all dose levels. See details of results below.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

On primary necropsy, stomach edema for females at 500 mg/kg/day and for males and females at 750 mg/kg/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings were noted in liver, thyroid gland and stomach for males and/or females at 500 and 750 mg/kg/day. See details of results below

Details on results:

CLINICAL SIGNS AND MORTALITY:
Mortality
- One female in the 750 mg/kg/day group, no. 9932, was euthanized in extremis on study day 9 following clinical observations of hypoactivity, increased and labored respiration, partial closure of the eyes, and dried red material around the mouth. At necropsy, the kidneys were pale with multiple white areas, dilated pelvis, and reddened corticomedullary junction; the urinary bladder and ureters were distended and the urinary bladder contained a calculus; the adrenal glands were enlarged and dark red; there was an irregularly shaped white area in the right ventricle of the heart; the thyroid glands, spleen, liver, and pancreas were pale; and the mandibular and mesenteric lymph nodes were enlarged.
Microscopically, there was a moderate pyelonephritis of both kidneys characterized by large numbers of neutrophils in the cortex, medulla, and pelvis with associated necrosis (left kidney worse than right). The pelvis of both kidneys was moderately dilated with associated atrophy of the papilla and medulla and hyperplasia of the transitional epithelium lining the pelvis. Hemorrhage was noted in the atrophied medulla. The lumens of the urinary bladder and ureters were moderately dilated with chronic-active inflammation and transitional epithelial hyperplasia. There was mild hemorrhage of the adrenal cortex and mild hypertrophy of the zona fasciculata (cortex). The right ventricle of the heart had mild necrosis characterized by hypereosinophilic cardiomyocytes and associated acute inflammation. Mild increased extramedullary hematopoiesis in the spleen, minimal myeloid hyperplasia in the bone marrow (sternum and femur), and mixed inflammatory cell infiltrate composed of lymphocytes, plasma cells, and neutrophils noted in the sinusoids and portal tracts of the liver were associated with the inflammation of the urinary tract. Mild interstitial edema was observed in the pancreas. Lymphoid necrosis was noted in the thymus (severe), lymph nodes (minimal to mild in mesenteric, mandibular and axillary), and small intestine (duodenum and Peyer’s patches) and was secondary to stress.
The cause of the death was the urinary tract lesions that resulted from the urinary bladder calculus noted at necropsy. The necrosis noted in the heart was most consistent with renal failure (Greaves, 2012a). Gross and microscopic findings and the cause of death were not test item-related.
- All other animals survived to the scheduled necropsies.

Clinical Signs
Remarkable clinical observations noted for female no. 9932 in the 750 mg/kg/day group that was euthanized in extremis included the following: hypoactivity, increased respiration rate, labored respiration, partial closure of the eyes, red and/or clear material around the mouth and nose, and rales. These findings were noted up to 6 days prior to euthanasia.
Test item-related clinical observations noted for the 250, 500, and 750 mg/kg/day group males and 750 mg/kg/day group females included: clear and/or yellow material around the mouth and/or red material around the nose. These findings were noted at the time of dose administration and 1-2 hours following dose administration. In addition, rales was noted on 7(1), 2(2), and 12(8) occurrences (number of males) in the 250, 500, and 750 mg/kg/day groups during the dosing period at the detailed physical examinations and 1-2 hours following dose administration. These findings did not persist to the recovery period and therefore were considered nonadverse.
All other clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.

BODY WEIGHT AND WEIGHT GAIN: Test item-related effects on body weights were noted in the 500 and 750 mg/kg/day group females.
In the 500 and 750 mg/kg/day group females, higher mean cumulative body weight gains were noted when compared to the control group during study weeks 0-3 and continued throughout the dosing period. The differences in cumulative body weight gains in these groups were often statistically significant when compared to the control group. While a statistically significantly lower mean body weight gain was noted for the 750 mg/kg/day group females during study week 3-4, a statistically significantly higher mean body weight gain was noted during study weeks 5-6 when compared to the control group. As a result of the effects on cumulative body weight gains, mean body weights were 9.2% and 5.1% higher (not statistically significant) in the 500 and 750 mg/kg/day group females during study week 13 when compared to the control group. During the recovery period, cumulative mean body weight loss or lower cumulative mean body weight gains (often statistically significant) were noted for the 750 mg/kg/day group females and resulted in
mean body weights that were up to 4.4% lower (not statistically significant) when compared to the control group. These effects on body weights noted in the 500 and 750 mg/kg/day group females were attributed to test item administration but were not considered adverse.
There were no test item-related effects on body weight noted for the 250 mg/kg/day roup females and 250, 500, and 750 mg/kg/day group males. However, some statistically significant differences were observed when the control and test item-treated groups were compared. In the 250 mg/kg/day group females, a statistically significantly lower mean body weight gain was noted during study week 3-4 and statistically significantly higher mean body weight gains were noted during study weeks 5-6 and 9-10 when compared to the control group. These transient changes in body weight gains for the 250 mg/kg/day group females were not attributed to test item administration as there were no effects on body weights and cumulative body weight gains noted in this group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Test item-related effects on food consumption were noted for the 500 and 750 mg/kg/day group females.
Higher mean food consumption values were noted in the 500 and 750 mg/kg/day group females throughout the dosing period. The differences from the control group were statistically significant during study weeks 6-7 and 8-9 (750 mg/kg/day group only). The effects on food consumption noted for females in these groups correlated with the higher body weight gains noted during the dosing period but were not considered adverse.
During the recovery period, mean food consumption for the 750 mg/kg/day group females was similar to the control group.
There were no test item-related effects on food consumption noted for the 250 mg/kg/day group females and 250, 500, and 750 mg/kg/day group males. However, some statistically significant differences were observed when the control and test item-treated groups were compared. Statistically significantly lower mean food consumption was noted during study weeks 6-7 and 9-10 for the 250 mg/kg/day group males, study weeks 7-10 for the 500 mg/kg/day group males, and study weeks 0-1 for the 250 mg/kg/day group females during. These effects on food consumption did not occur in a dose-related manner, were transient in nature, and/or did not have any correlating effect on body weights, and therefore, were not attributed to the test item.

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

HAEMATOLOGY: Test item-related higher white blood cell (WBC) and absolute lymphocyte counts were noted in the 500 and 750 mg/kg/day group males and higher absolute neutrophil counts
were noted in the 750 mg/kg/day group males at the primary necropsy (study week 12/13).
Higher mean white blood cell counts resulted from higher mean absolute neutrophil and lymphocyte counts. Higher mean WBC values noted in the 500 and 750 mg/kg/day group males resulted from individual values that were above the concurrent control group range in the mid- (4 of 10 rats) and high-dose (5 of 10 rats) group males. Higher mean absolute neutrophil counts noted in the 750 mg/kg/day group males resulted from individual values in 6 of 10 rats that were above the concurrent control group range.
Higher mean absolute lymphocyte values resulted from individual values that were above the concurrent control group range in the mid- (4 of 10 rats) and high-dose (5 of 10 rats) group males.
White blood cell and absolute neutrophil and lymphocyte count elevations noted at the primary necropsy in the 750 mg/kg/day group males were not observed at the recovery necropsy (study week 16/17).
There were no other test item-related effects on hematology or coagulation parameters.
Statistically significant findings that involved percentage leukocyte differential counts were not itemized above, and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.

CLINICAL CHEMISTRY: Test item-related higher alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were noted in the 750 mg/kg/day group males at the primary necropsy (study week 12/13).
Higher ALT and AST mean values in the 750 mg/kg/day group males resulted from elevated individual values in 2 of 10 males (nos. 9855 and 9866) and 4 of 10 males (nos. 9841, 9844, 9855, and 9866), respectively. The highest individual values were noted in male no. 9855 and this rat also had multifocal, mild hepatocellular necrosis, but hepatocellular necrosis was not observed in any other rat.
ALT and AST elevations noted at study week 12/13 in the 750 mg/kg/day group males were not observed at the recovery period.
There were no other test item-related effects on serum chemistry parameters. However, some statistically significant differences were observed when the control and test item-treated groups were compared. Lower mean globulin and calcium values in the 750 mg/kg/day group males at study week 16/17 were not test item-related as the values were the same as the study week 12/13 values. The lower mean globulin level in the 750 mg/kg/day group males (study week 16/17) resulted in a higher A/G ratio that was not considered to be test item-related. Minimally higher mean creatinine value in the 750 mg/kg/day group females at study week 16/17 was not test item-related, as it was most consistent with biological variation. The lower mean chloride value in the 500 mg/kg/day group females at study week 12/13 was not test item-related due to the lack of a dose-related response. The higher mean potassium value in the 750 mg/kg/day group females at study week 16/17 was not test item-related as it resulted from a single
high individual animal value.

URINALYSIS: There were no test item-related alterations in urinalysis parameters at the primary or recovery necropsies at any dosage level.

NEUROBEHAVIOUR:
Functional Observational Battery (FOB)
- Home cage observations, handling observations, open field observations, sensory observations, neuromuscular observations, and physiological observations were unaffected by test item administration. There were no statistically significant differences when the test item-treated males and females were compared to the control group at the study week 12 evaluation.
- Motor activity: Within-session repeated measures analyses of variance were conducted across the subintervals of each test session for total and ambulatory counts and for overall interval means (representing the entire 60-minute session activity) during each test session.
Motor activity patterns (total and ambulatory activity counts) were unaffected by test item administration. There were no statistically significant changes for the test item-treated males and females at any dosage level when compared to the control group at the study week 12 evaluation. 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 item-treated groups when the animals were evaluated on study week 12.

ORGAN WEIGHTS: At the primary necropsy (study week 12/13), test item-related higher liver weights were noted in the 750 mg/kg/day group males. Higher thyroid/parathyroid and spleen weights were noted in the 500 and 750 mg/kg/day group males. Higher thymus weights were noted in the 250, 500, and 750 mg/kg/day group females.
Higher liver weights in males were most evident when liver weights relative to body weight were compared to the control group. Mean absolute liver weights were minimally higher than the control group mean (with slightly lower mean final body weight in the high dose males) but weights relative to body weight were statistically significantly higher. Individual weights relative to final body weight were above the concurrent control group range in 7 of 10 males in the 750 mg/kg/day group. Higher liver weight parameters were associated with minimal centrilobular hepatocellular hypertrophy.
Higher mean thyroid/parathyroid weights in the 500 and 750 mg/kg/day group males (absolute and relative to body and brain weight) were generally statistically significantly different from control group means and were associated with minimal follicular cell hyperplasia.
Higher spleen weights in the 500 and 750 mg/kg/day group males (absolute and relative to body and brain weight) and higher thymus weights in the 250, 500, and 750 mg/kg/day group females (absolute and relative to body and brain weight) were not associated with relevant microscopic changes.
Some organ weight differences were statistically significant when compared to the control group but were considered to be a result of a higher final body weight. Higher kidney weight relative to brain weight in the 750 mg/kg/day group females and lower brain weights relative to body weight were noted in the 500 and 750 mg/kg/day group females. Higher liver weights (absolute and/or relative to brain weight) were noted in the 500 and 750 mg/kg/day group females.
There were no other test item-related effects on organ weights. However, some statistically significant differences were observed when the control and test item-treated groups were compared. Lower pituitary weights (absolute, relative to brain weight) and a higher testes weight relative to body weight in the 500 mg/kg/day group males were not test item-related given the lack of a dose-related response.
Higher thyroid/parathyroid weights persisted in the 750 mg/kg/day group males at the study week 16/17 recovery necropsy.

GROSS PATHOLOGY: At the primary necropsy (study week 12/13), test item-related stomach edema was noted in the 500 mg/kg/day group females and 750 mg/kg/day group males and females and correlated with microscopically visible edema in the glandular and/or nonglandular
stomach.
There were no test item-related gross observations noted at the recovery necropsy (study week 16/17).

HISTOPATHOLOGY: NON-NEOPLASTIC
At the primary necropsy (study week 12), test item-related microscopic findings were noted in the liver, thyroid gland, and stomach (glandular and nonglandular) of the 500 and 750 mg/kg/day group males and/or females.
Minimal hypertrophy of centrilobular hepatocytes of the liver, characterized by expansion of eosinophilic, glassy cytoplasm, was observed in a single male in the 500 mg/kg/day group and 6 males and 1 female in the 750 mg/kg/day group and was not considered adverse. A single male in the 750 mg/kg/day group (no. 9855) had adverse mild hepatocellular necrosis characterized by multiple, randomly scattered foci of hypereosinophilic, lysed hepatocytes with hemorrhage, and peripheral inflammation. Minimal follicular cell hyperplasia of the thyroid, characterized by increased size and number of follicular cells, was observed in 4 males and 1 female in the 500 mg/kg/day group and 5 males and 2 females in the 750 mg/kg/day group. Thyroid follicular changes were not considered adverse.
The majority of findings in the stomach were noted in the nonglandular region of 2 males and 1 female in the 500 mg/kg/day group and 3 males and 2 females in the 750 mg/kg/day group and were considered adverse. Nonglandular stomach findings included edema in the lamina propria and submucosa (males and females), epithelial degeneration with or without ulceration (females), or epithelial hyperplasia (males and females) of the squamous epithelium and mixed inflammatory cell infiltrate (males and females). Submucosal edema of the glandular stomach was noted in 1 female in the 500 mg/kg/day group and 1 male and 1 female in the 750 mg/kg/day group.
Hepatocellular hypertrophy and/or necrosis, stomach findings (edema, inflammation, squamous epithelial changes), and thyroid follicular cell hyperplasia were not observed at the study week 16/17 recovery necropsy.
There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test item. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The no-observed-adverse-effect level (NOAEL) specific to the rat was considered to be 250 mg/kg/day for males and females due to adverse effects for the males and females at 500 mg/kg/day and for the males at 750 mg/kg/day.

2

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Nonglandular stomach findings were considered specific to the rat and not relevant to humans

3

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: Nonglandular stomach findings were considered specific to the rat and not relevant to humans

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

DISCUSSION

 

The objectives of the study were to evaluate the potential toxic effects of the test item, when administered via gavage to rats for a minimum of 90 consecutive days.

This included evaluation of potential neurotoxicity by functional observational battery (FOB) and motor activity (MA) assessment.

Test item-related, nonadverse clinical observations of clear and/or yellow material around the mouth and/or red material around the nose were noted for the 250, 500, and 750 mg/kg/day group males and 750 mg/kg/day group females at the time of dose administration and 1-2 hours following dose administration. In addition, increased incidences of rales were noted in the 250, 500, and 750 mg/kg/day group males during the dosing period at the detailed physical examinations and 1-2 hours following dose administration but was considered nonadverse.

Test item-related higher body weights and correlating higher food consumption were noted for the 500 and 750 mg/kg/day group females throughout the dosing period.

However, as these findings did not persist during the recovery period, they were not considered adverse.

Liver hypertrophy and thyroid hyperplasia with associated weight elevations were most consistent with an adaptive response secondary to liver microsomal enzyme induction(Hoodet al., 1999; Maronpotet al., 2010). Minimal liver hypertrophy and liver weight elevations were associated with 1.5-fold elevations in ALT and AST levels and, along with thyroid hyperplasia, were not considered adverse(Hallet al., 2012). In addition to hepatocellular hypertrophy, a single male (no. 9855) in the 750 mg/kg/day group had multifocal hepatocellular necrosis that was randomly scattered and associated with the highest individual ALT and AST levels. Liver necrosis was considered adverse.

Adverse changes in the nonglandular stomach in rodent toxicology studies limited to the mid- and high-dose groups were most consistent with direct local irritating effects to the nonglandular stomach mucosa(Greaves, 2012b).The rodent stomach is divided into a proximal, nonglandular forestomach that is lined by stratified squamous epithelium that is devoid of glands and protective mucous, and a distal, glandular stomach(Proctoret al.,2007). The forestomach constitutes approximately three-fifths of the rodent stomach area and functions as a food reservoir(Proctoret al., 2007).Therefore, the squamous epithelium in the forestomach may be exposed to the test item within undigested food for longer periods than the remaining digestive tract. The potential prolonged exposure to test item and the lack of a protective lining along with higher pH levels in the rodent nonglandular stomach may contribute to a greater vulnerability to local toxicity(Proctoret al., 2007).The nonglandular stomach findings were considered adverse but were considered specific to the rat.

Applicant's summary and conclusion

Conclusions:

Oral administration of the test material to Crl:CD(SD) rats at dosage levels of 250, 500, and 750 mg/kg/day for a minimum of 90 consecutive days resulted in effects considered to be nonadverse with the exception of adverse edema, squamous epithelial hyperplasia, and/or degeneration (females), mixed inflammatory infiltrate and ulcerate (females) in the nonglandular stomach at ≥500 mg/kg/day for the males and females and liver necrosis noted in the 750 mg/kg/day group males. Therefore, the no-observed-adverse-effect level (NOAEL) specific to the rat was considered to be 250 mg/kg/day for males and females.
However, given that the nonglandular stomach findings were considered specific to the rat and not relevant to humans, the NOAEL for findings relevant to humans would be considered to be 500 mg/kg/day for males and 750 mg/kg/day for females.

Executive summary:

OBJECTIVE

The objectives of the study were to evaluate the potential toxic effects of the test item when administered via gavage to rats for a minimum of 90 consecutive days.

This included evaluation of potential neurotoxicity by functional observational battery (FOB) and motor activity (MA) assessment.

STUDY DESIGN

The test item in the vehicle (peanut oil) was administered orally by gavage once daily for a minimum of 90 consecutive days to 3 groups (Groups 2-4) of Crl:CD(SD) rats. Dosage levels were 250, 500, and 750 mg/kg/day for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Groups 1 and 4 each consisted of 15 animals/sex and Groups 2-3 each consisted of 10 animals/sex. Following at least 90 days of dose administration, 10 animals/sex/group were euthanized; the remaining ≤5 animals/sex in the control and high-dose groups were euthanized following a minimum 28-day nondosing (recovery) period.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body weights and cage food weights were recorded weekly (± 2 days). FOB and MA data were recorded for all animals during study week 11/12 (hereafter referred to as study week 12). Ophthalmic examinations were performed during study weeks -2 and 12. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals assigned to the primary (study week 12/13) and recovery (study week 16/17) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies.

Selected tissues were examined microscopically from all animals.

 

RESULTS

There were no test item-related effects on survival, ophthalmic findings, FOB or MA effects, or urinalysis alterations. One female in the 750 mg/kg/day group was euthanized in extremison study day 9 following clinical observations of hypoactivity, increased and labored respiration, partial closure of the eyes, and dried red material around the mouth.

The cause of death for this animal (microscopic urinary tract lesions resulting from a grossly observed urinary bladder calculus) was not test item-related. All other animals survived to the scheduled necropsies.

Test item-related clinical observations of clear and/or yellow material around the mouth and/or red material around the nose were noted for the 250, 500, and 750 mg/kg/day group males and 750 mg/kg/day group females at the time of dose administration and 1-2 hours following dose administration. In addition, increased incidences of rales were noted in the 250, 500, and 750 mg/kg/day group males during the dosing period at the detailed physical examinations and 1-2 hours following dose administration. These findings did not persist to the recovery period and therefore were considered nonadverse. There were no test item-related clinical findings in the 250 and 500 mg/kg/day group females.

Test item-related, nonadverse higher body weights and correlating higher food consumption were noted for the 500 and 750 mg/kg/day group females throughout the dosing period. The effects on body weights and food consumption did not persist during the recovery period for the 750 mg/kg/day group females. There were no test item-related effects on body weights or food consumption for the 250 mg/kg/day group females or the 250, 500, and 750 mg/kg/day group males.

Test item-related clinical pathology changes at the primary necropsy included higher nonadverse white blood cell (WBC) and absolute lymphocyte counts in the 500 and 750 mg/kg/day group males and higher absolute neutrophil counts and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the 750 mg/kg/day group males. These changes were not observed at the recovery evaluation.

Test item-related pathology findings and/or organ weight changes were noted in the liver, thyroid, stomach, spleen, and thymus at the primary necropsy (study week 12/13). There was nonadverse centrilobular hepatocellular hypertrophy at ≥500 mg/kg/day for the males and at 750 mg/kg/day for the females and thyroid follicular cell hyperplasia in the 500 and 750 mg/kg/day group males and females and adverse edema, squamous epithelial hyperplasia, and/or degeneration (females), mixed inflammatory infiltrate and ulceration (females) in the nonglandular stomach at ≥500 mg/kg/day for the males and females, and edema in the glandular stomach of the single animals in the 500 and 750 mg/kg/day groups. Liver hypertrophy and/or liver necrosis (necrosis noted in 1 male and was adverse) were associated with ALT, AST, and liver weight elevations in the 750 mg/kg/day group males. Thyroid hyperplasia was associated with thyroid weight elevations in ≥500 mg/kg/day group males at study week 12/13. Higher thyroid weights persisted at study week 16/17 in 750 mg/kg/day group males). Edema (males and females), squamous epithelial hyperplasia (males and females, and/or degeneration (females); mixed inflammatory infiltrate (males and females) and ulceration (females) were noted in the nonglandular stomach of ≥500 mg/kg/day group males and females and were considered adverse. Edema was noted in the glandular stomach of a single 500 mg/kg/day group female and a single male and female in the 750 mg/kg/day group.

Nonglandular and glandular stomach findings were associated with gross observations of stomach edema and were considered rat specific findings. Nonadverse spleen weight elevations in ≥500 mg/kg/day group males and thymus weight elevations in ≥250 mg/kg/day group females did not have a microscopic correlate and were only observed at study week 12/13. Nonadverse higher white blood cell and absolute lymphocyte counts were noted in ≥500 mg/kg/day group males and higher absolute neutrophil counts were noted in the 750 mg/kg/day group males at study week 12/13 only. There were no test item-related liver, thyroid, and stomach findings observed at study week 16/17.

 

CONCLUSIONS

Oral administration of the test item to Crl:CD(SD) rats at dosage levels of 250, 500, and 750 mg/kg/day for a minimum of 90 consecutive days resulted in effects considered to be nonadverse with the exception of adverse edema, squamous epithelial hyperplasia, and/or degeneration (females), mixed inflammatory infiltrate and ulcerate (females) in the nonglandular stomach at ≥500 mg/kg/day for the males and females and liver necrosis noted in the 750 mg/kg/day group males. Therefore, the no-observed-adverse-effect level (NOAEL) specific to the rat was considered to be 250 mg/kg/day for males and females. However, given that the nonglandular stomach findings were considered specific to the rat and not relevant to humans, the NOAEL for findings relevant to humans would be considered to be 500 mg/kg/day for males and 750 mg/kg/day for females.