Reaction mass of 2-[[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]methyl]oxirane and 1,3-bis[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]propan-2-ol and 1-[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]-3-[4-[[4-[3-[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]-2-hydroxy-propoxy]-3,5-dimethyl-phenyl]methyl]-2,6-dimethyl-phenoxy]propan-2-ol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]propan-2-ol; 1-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-3-{4-[(4-{3-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-2-hydroxypropoxy}-3,5-dimethylphenyl)methyl]-2,6-dimethylphenoxy}propan-2-ol; 2-{[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]methyl}oxirane

EC Number:

941-357-0

Molecular formula:

Not applicable for multi-constituent substances

IUPAC Name:

1,3-bis[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]propan-2-ol; 1-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-3-{4-[(4-{3-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-2-hydroxypropoxy}-3,5-dimethylphenyl)methyl]-2,6-dimethylphenoxy}propan-2-ol; 2-{[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]methyl}oxirane

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The rat was selected because it is a rodent species known to be accepted by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Margate, United Kingdom
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Between 5 and 6 weeks.
- Weight at study initiation: Males 124.1 and 176.8g and females 103.7 and 160.8g
- Fasting period before study: No
- Housing: Animals were housed in a single, exclusive room. Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes (Home Office, 2014). Animals were housed in groups of up to three.
- Diet (e.g. ad libitum): Animals had ad libitum access to 5LF2 EU Rodent Diet (International Product Supplied Ltd., London, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. On occasion, selected animals were offered a few pellets of moistened diet to aid consumption.
- Water (e.g. ad libitum): Main supply water was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants.
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY: No contaminants were present in the water or food at levels that might have interfered with achieving the objective of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 to 70%
- Air changes (per hr): At least 15
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle except when otherwise dictated by experimental procedures.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral (gavage) route of administration was chosen because it is considered an acceptable and commonly used exposure route for regulatory studies of this type.

Vehicle:

polyethylene glycol

Remarks:

PEG400

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: Formulations were prepared daily on Days 1 through 11 of the dosing phase and administered to the animals within 6 hours of preparation, and then weekly from Day 12 of the dosing phase onwards.

The test article was formulated as a suspension in Polyethylene Glycol 400 (PEG 400) following dispensary SOPs and a suitable formulation method. Formulations were stored at 15 to 25°C, in a sealed container.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): standard vehicle deemed suitable for use with the substance.
- Concentration in vehicle: 0, 20, 60 and 120 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:

Formulations prepared for use during Weeks 1 and 13 of the dosing phase were analyzed to determine the achieved concentration. Triplicate samples were removed from the middle of the test article formulations and were analyzed. A single sample was taken from the middle of control formulations, and analyzed. Suspensions of 50 and 200 mg/g were previously found to be homogeneous. Suspensions of 16.5 and 162 mg/g were found to be stable for 6 hours at room temperature. The trial work established that dose formulations prepared at 1 and 200 mg/mL were stable for 12 days at room temperature.

Duration of treatment / exposure:

Males: 91 days
Females: 92 days

Frequency of treatment:

Daily dosing

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

High

No. of animals per sex per dose:

10 males and 10 females per sex per group with an additional 5 males and 5 females in the control and high dose groups subject to a 4 week recovery period.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses based on the results of a previously conducted OECD 422 study.
- Rationale for animal assignment (if not random): Animals were assigned to dose groups during acclimation using a total randomization procedure. Animals were individually identified by electronic implant and by tail mark during FOB tests and on one occasion when the computer system for reading the electronic implants was not operational. Following the first full weighing (Day 1 of the predose phase), group mean body weights and standard deviations were calculated and inspected to ensure no unacceptable differences occurred between groups.
- Rationale for selecting satellite groups: Randomly assigned
- Post-exposure recovery period in satellite groups: 4 weeks

Positive control:

Not a guideline requiement

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at the beginning and end of each working day for signs of ill health or overt toxicity. Each animal was given a detailed physical examination once weekly and on the day of terminal necropsy. An individual record of the clinical condition of each animal was maintained. Additional observations were recorded, when deemed necessary. During Week 1 of the dosing phase, animals were observed daily at 1 hour postdose; then, from Week 2 until the end of the dosing phase, animals were observed once weekly at 1 hour postdose. Additional observations were recorded when deemed necessary.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed during the predose phase, during Week 13 of the dosing phase, and during Week 3 of the recovery phase.
- Parameters checked: Animals were observed within the home cage, in hand and in the arena, for 2 minutes. Animals were observed for the potential effects on behaviour, gait, posture, respiration, secretion, excretion, involuntary movements, skin, tail, eyes, pelage and activity.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of all animals were recorded on Day 1 of the predose phase, at least once weekly from Day 1 of the dosing phase, on the same day each week, and before each necropsy. Additional bodyweights were taken when deemed necessary.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: The amount of food consumed by each cage of toxicity was determined at least once weekly from Day 1 of the dosing phase. Consumption was calculated as g/animal/day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule and dose groups for examinations: Investigations were performed on all animals during the predose phase and on Group 1 and 4 animals during Week 12 of the dosing phase.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13 of the dosing phase and week 4 of the recovery phase. Coagulation samples were taken from females at terminal and recovery necropsies via the abdominal aorta instead, as they did not meet the necessary weight requirement during the other sampling occasions.
- Anaesthetic used for blood collection: Yes (isoflurane anesthesia).
- Animals fasted: Animals had access to water, but food was removed overnight prior to clinical pathology blood sampling taken at the scheduled terminal necropsies.
- How many animals: All animals.
- Parameters checked: Haemoglobin, mean cell haemoglobin concentration, red blood cell count, reticulocyte count, packed cell volume, red cell distribution width, mean cell volume, total and differential white cell count, mean cell haemoglobin and platelet count (including clump assessment). Coagulation tests included prothrombin time, activated partial thromboplastin time and fibrinogen.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 of the dosing phase and week 4 of the recovery phase.
- Animals fasted: Animals had access to water, but food was removed overnight prior to clinical pathology blood sampling taken at the scheduled terminal necropsies.
- How many animals: All animals
- Parameters checked: Aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, alkaline phosphatase, total cholesterol, triglycerides, total bilirubin, total protein, albumin, globulin, A/G ratio, sodium, potassium, chloride, calcium, inorganic phosphate, creatinine, urea, glucose and creatinine kinase.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected from all toxicity animals overnight during Week 12 of the dosing phase and from all recovery animals overnight during Week 3 of the recovery phase.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Food was removed during collection, but water remained available.
- Parameters checked: Volume, colour, turbidity, specific gravity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood and microscopy of sediment. Some of the procedures were assessed semi-quantitatively.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Animals were observed during the predose phase, during Week 13 of the dosing phase, and during Week 3 of the recovery phase. Where possible, observations were performed at the same time on each occasion. At the time of testing, the observer was unaware of the dose levels for each animal.
- Dose groups that were examined: All animals
- Battery of functions tested: Motor activity (total activity counts, total mobile counts and total rears). Measurements of quantitative forelimb and hind limb grip strength and hind limb foot splay were made.

IMMUNOLOGY: No

OTHER:
Bone marrow smear evaluation: Bone marrow smears were prepared at necropsy. Tissues were smear-prepared, air-dried, and fixed in methanol, but not examined.

Sacrifice and pathology:

TIMEFRAME: With the exception of fasting, these procedures were also followed for unscheduled sacrifices and deaths. All toxicity and recovery animals, including unscheduled deaths/sacrifices, were subjected to necropsy. The scheduled necropsies were performed on Day 92 or 93 of the dosing phase and on Day 30 or 31 of the recovery phase, after an overnight period without food. When possible, necropsies were carried out via controlled randomization (i.e., one cage of animals from Groups 1, 4, 2, and then 3 and repeated in the same sequence until all animals were terminated).
Each animal was administered isoflurane anesthesia. Once a suitable deep plane of anesthesia was established, the animal was exsanguinated by severing its major blood vessels. Animals were weighed before necropsy.

GROSS PATHOLOGY: A full macroscopic examination was performed under the general supervision of a Pathologist, and all lesions were recorded.

ORGAN WEIGHTS: Organs weighed from all animals excluding decedents and were dissected free from fat and other contiguous tissue and weighed before fixation. Left and right organs were weighed together.
Organs weighed: adrenal, brain, epididymis, heart, kidney, liver, lungs with main stem bronchi and bronchioles, ovary and oviducts, pituitary, prostate, spleen, testis, thymus, thyroid with parathyroid and the uterus with cervix.

HISTOPATHOLOGY: Tissues were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 μm, and stained with hematoxylin and eosin.

Organs/tissues observed microscopically (Toxicity animals):
Group 1: Adrenal, aorta, brain, caecum, colon, duodenum, epididmyis, aesophagus, eye, femur with bone marrow and femorotibial joint gut-associated lymphoid tissue (GALT)/Peyer's patch, gross lesions, heart, ileum, jujunum, kidney, liver, lungs with main stem bronchi and bronchioles, lymph node (mesenteric and mandibular), mammary, mandibular salivary gland, muscle (biceps femoris), optic nerve, sciatic nerve, ovary, oviduct, pancreas, pituitary, prostate, rectum, seminal vesicles, skin and subcutis, spinal cord (cervical, lumbar, thoracic), spleen, sternum with bone marrow, stomach, sublingual salivary gland, testis, thyroid with parathyroid, tongue, trachea, urinary bladder, uterus with cervix, vagina and vertebrae (cervical, lumbar and thoracic).
Group 2: Liver, kidney, colon, rectum, lung, mandibular salivary gland, mesenteric lymph node, ovary/uterus, pancreas thyroid and gross lesions.
Group 3: Same as Group 2.
Group 4: Same as Group 1.
Unscheduled deaths/sacrifices: Same as groups 1 and 4.

Organs/tissues observed microscopically (Recovery animals):
Group 1: Liver, kidney, colon, rectum, lung, mandibular salivary gland, mesenteric lymph node, ovary/uterus, pancreas thyroid and gross lesions.
Group 4: Same as Group 1.
Unscheduled deaths/sacrifices: Same as for unscheduled deaths/sacrifices made in the toxicity animals

Other examinations:

None

Statistics:

When tables were computer-generated, rounding of individual values may have occurred during the calculation of derived values. Therefore, recalculation of derived values from the individual data, as presented in this report, may have, in some instances, yielded minor variations.

Data from test article-treated animals were compared with control data. Statistical analyses were performed, when appropriate. The comparisons of interest were Group 1 versus Groups 2, 3, and 4.

The following data were analyzed statistically.
- Functional observational battery - quantitative assessment
- Body weight changes
- Food consumption over selected intervals
- Clinical pathology parameters
- Absolute organ weights, organ:body weight percentages, and organ:brain weight percentages

Data for each sex were analyzed separately; only data collected on or after the first day of dosing were analyzed statistically.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance related clinical observations included minimal to moderate salivation on multiple days for animals administered 100, 300, or 600 mg/kg/day. Occasionally, thin appearance, raised hair, and staining of the fur and skin were noted for animals administered 600 mg/kg/day; these were non-specific indicators of stress, poor appetite, and/or general debilitation and were attributed to test article administration.

During 1-hour postdose observations, the only observation recorded was salivation on Day 36 or 43 of the dosing phase for males administered 300 or 600 mg/kg/day.

All other clinical observations noted for control or test article-treated animals were considered incidental, as they were infrequent, showed no relationship to dose, were observations typically observed in this species, or were comparable with observations routinely noted at this laboratory.

See attached summary tables (attachment [1]) for more information.

Mortality:

mortality observed, treatment-related

Description (incidence):

All animals survived to the scheduled sacrifice on Day 92/93 of the dosing phase or Day 30/31 of the recovery sacrifice, except three males administered 600 mg/kg/day and one female administered 100 mg/kg/day.

One toxicity male administered 600 mg/kg/day was humanely sacrificed on Day 57 of the dosing phase due to the severity of clinical observations, including body weight loss, hunched posture, being thin, raised fur, and staining around the mouth and genital area. This animal had microscopic, slight degeneration, with inflammation within the myocardium of the heart; this finding was considered related to events just prior to death. No microscopic findings were considered the primary cause of demise, but the cause of demise was considered related to the test substance.
Other microscopic findings in this animal were similar to those found in terminal sacrifice males administered the same dose.

One recovery cohort male administered 600 mg/kg/day was humanely sacrificed on Day 79 of the dosing phase due to the severity of clinical observations, which included body weight loss and thin. Microscopic findings in this animal were similar to those found in terminal sacrifice males administered the same dose, with the addition of slightly decreased cellularity within the thymus, which was consistent with the effects of stress. The cause of demise remained undetermined but was considered related to the test substance.

One toxicity cohort male administered 600 mg/kg/day was found dead on Day 46 of the dosing phase. Prior to death, this animal was cold to touch and had pale extremities. At necropsy, this animal had macroscopic rupture of the esophagus, with abnormal fluid within the thoracic cavity. These macroscopic findings were consistent with mis-dosing of the test article. The cause of demise was, therefore, considered not related to the test substance. Other microscopic findings in this animal were similar to those found in terminal sacrifice males administered the same dose, with the addition of slightly decreased cellularity within the thymus, which was consistent with the effects of stress.

One toxicity cohort female administered 100 mg/kg/day (Animal R0504) was found dead on Day 32 of the dosing phase. Prior to death, opaque eyes were noted for this animal. At necropsy, this animal had macroscopic rupture of the esophagus, with red fluid/contents within the thoracic cavity. These macroscopic findings were consistent with mis-dosing of the test article. The cause of demise was, therefore, considered not related to the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Compared with controls, test substance related decreased body weight gain was noted in males administered ≥ 100 mg/kg/day. At the end of the dosing phase, overall bodyweight gain was lower in males administered 100, 300, or 600 mg/kg/day (12%, 7%, and 24%, respectively) and reached statistical significance for males administered 100 or 600 mg/kg/day. In males administered 600 mg/kg/day, decreased body weight gain was evident beginning the first week of dosing and generally persisted throughout the dosing phase, with occasional body weight loss (e.g., Weeks 8 to 9, 10 to 11, and 12 to 13); this finding was considered adverse given the high magnitude of change. While a clear dose response was not evident for the effect on body weight gain in males administered 100 or 300 mg/kg/day, these decreases were considered related to test article administration, but were not adverse. Lower body weight gain was limited to first few weeks of the dosing phase for males administered 300 mg/kg/day and was subsequently comparable with controls.

During the recovery phase, body weight gain was greater in males previously administered 600 mg/kg/day, which confirmed complete reversibility of the finding during the dosing phase. Compared with controls, test substance related increased body weight gain was noted in females administered 300 or 600 mg/kg/day, which correlated with increased food consumption for these animals. Overall, mean body weight gain was 15 or 16% higher in females administered 300 or 600 mg/kg/day, compared with control. Given the small magnitude of changes, the effect on body weight gain was considered not adverse.

Overall body weight gain was unaffected in females administered 100 mg/kg/day. During the recovery phase, body weight gain was significantly lower in females previously administered 600 mg/kg/day, reflecting reversibility of the effect on body weight gain.

See attached summary tables (attachment [1]) for more information.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For males administered 100 or 600 mg/kg/day, overall mean food consumption was minimally lower (9 or 4%, respectively), compared with controls. These decreases lacked dose response and did not achieve statistical significance; nonetheless, they correlated with decreased mean body weight gain in these animals and, therefore, were considered TMBPF-DGE related. During the recovery phase, mean food consumption for males previously administered 600 mg/kg/day was generally comparable with controls.

For females administered 300 or 600 mg/kg/day, slightly higher mean food consumption was noted over the course of the dosing phase. Overall food consumption was 6 and 14% higher for females administered 300 or 600 mg/kg/day, respectively, and reached statistical significance for females administered 600 mg/kg/day. During the recovery phase, food consumption for females previously
administered 600 mg/kg/day was generally comparable with controls, which confirmed reversibility of the finding during the dosing phase.

Given the small magnitude of changes, effects on mean food consumption were considered not adverse.

See attached summary tables (attachment [1]) for more information.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No abnormal ophthalmic observations were noted during the dosing phase.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

During the dosing phase, compared with controls, test article-related effects on hematology and coagulation parameters included decreased mean corpuscular
hemoglobin in females administered 300 or 600 mg/kg/day; increased monocytes in males administered 100, 300, or 600 mg/kg/day; increased large unstained cells in females administered 600 mg/kg/day; decreased platelet counts in both sexes administered 600 mg/kg/day; decreased activated partial thromboplastin time in both sexes administered 100, 300, or 600 mg/kg/day; and decreased fibrinogen in males administered 600 mg/kg/day.

Given their small magnitude, all of which were inconsistent between the sexes and reversed after a 4-week recovery period, these changes were considered not adverse. Although decreased activated partial thromboplastin time was observed in both sexes at all dose levels and persisted after the 4-week recovery period in animals previously administered 600 mg/kg/day, in the absence of any correlating microscopic findings, this finding was considered not adverse.

All other differences in test results, whether statistically significant or not, between control and animals administered the test articles or between predose and dosing phase test results for individual animals were consistent with normal variation and considered incidental. These differences were characterized by most or all of the following: small magnitude, lack of dose-response, inconsistency between sexes, absence of correlative findings, and/or similarity to differences present before initiation of dosing.

See attached summary tables (attachment [1]) for more information.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance related clinical chemistry changes included increased aspartate aminotransferase activity in both sexes administered 600 mg/kg/day; increased alanine aminotransferase activity in both sexes administered 300 or 600 mg/kg/day; increased total bilirubin in both sexes administered 300 or 600 mg/kg/day; increased cholesterol in males administered 600 mg/kg/day and females administered 300 or 600 mg/kg/day; decreased triglycerides in both sexes administered 100, 300, or 600 mg/kg/day; decreased total protein in both sexes administered 600 mg/kg/day; decreased globulin and an associated increase in albumin:globulin ratio in males administered 100, 300, or 600 mg/kg/day; increased phosphorus in males administered 100, 300 or 600 mg/kg/day and females administered 300 or 600 mg/kg/day; and increased urea in both sexes administered 300 or 600 mg/kg/day.

Liver enzyme and bilirubin changes correlated with hepatocyte hypertrophy, noted microscopically. Protein changes were consistent with stress and/or reduced food consumption. Phosphorus and urea changes correlated with microscopic kidney findings. These changes reversed following 4 weeks of recovery.

All other differences in test results, whether statistically significant or not, between control and animals administered the test articles or between predose and dosing phase test results for individual animals were consistent with normal variation and considered incidental. These differences were characterized by most or all of the following: small magnitude, lack of dose-response, inconsistency between sexes, absence of correlative findings, and/or similarity to differences present before initiation of dosing.

See attached summary tables (attachment [1]) for more information.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

During the dosing phase, markedly higher urine volume was noted for females administered 600 mg/kg/day. Although specific gravity was generally similar to controls, increased urine volume likely correlated with microscopic findings and organ weight changes in the kidney; therefore, it was considered related to test article administration. A high number of urine phosphate crystals was noted during the dosing phase in animals administered 300 or 600 mg/kg/day and during the recovery phase in animals previously administered 600 mg/kg/day. These changes correlated with microscopic kidney findings.

All other differences in test results, whether statistically significant or not, between control and animals administered the test articles or between predose and dosing phase test results for individual animals were consistent with normal variation and considered incidental. These differences were characterized by most or all of the following: small magnitude, lack of dose-response, inconsistency between sexes, absence of correlative findings, and/or similarity to differences present before initiation of dosing.

See attached summary tables (attachment [1]) for more information.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

No test substance related observations were noted during detailed clinical observational measurements in the home cage, in the hand, or in the arena. Mild to moderate vocalization and elevated tail for a few females were considered incidental as they lacked dose response or were present for controls.

During quantitative assessments, dose-dependent decreases in foot splay and forelimb grip strength were noted for males administered 100, 300, or 600 mg/kg/day. Statistical significance was achieved at one or more dose levels. During the recovery phase, foot splay and fore limb grip strength remained lower for males previously administered 600 mg/kg/day, compared with controls, suggesting the effect on these parameters was not reversed. No effect on foot splay or forelimb or hindlimb grip strength was observed for females.

During motor activity assessments, mean total activity counts and total mobile counts were generally lower for males administered 600 mg/kg/day, compared with control. During the recovery phase, these parameters remained lower for males previously administered 600 mg/kg/day, compared with controls, which suggested the effects on these parameters were not reversed following at least 3 weeks of recovery. No effects on total activity counts, total mobile counts, or number of rears were observed for females. Any statistical significance observed was considered incidental due to the lack of a consistent pattern.

See attached summary tables (attachment [1]) for more information.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test substance related organ weight changes were noted for animals administered 300 or 600 mg/kg/day. For males administered 300 mg/kg/day, kidney weights were increased, relative to body weight, and unadjusted thyroid weights were increased, relative to body and brain weights. For males administered 600 mg/kg/day, heart weights were increased, relative to body weight; kidney weights were increased, relative to body weight; liver weights were increased, relative to body and brain weights; lung weights were increased, relative to body weight; and thyroid weights were increased, relative to body weight.

For females administered 300 or 600 mg/kg/day, heart weights were increased, relative to brain weight for females administered 300 mg/kg/day and relative to body and brain weights for females administered 600 mg/kg/day; kidney weights were increased, relative to body and brain weights, and liver weights were increased, relative to body and brain weights.

Following a 4-week recovery phase, no test substance related changes in organ weight were noted.

All other organ weight and/or organ weight ratio changes were attributed to normal biological variation and were considered not test article related as they were small in magnitude, not dose-dependent, inconsistent between the sexes, due to normal inter-animal variability, and/or lacked a microscopic correlate.

See attached summary tables (attachment [2]) for more information alongside Text Table 1 in 'Any other information on results incl. tables'.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

One female administered 100 mg/kg/day, one male and two females administered 300 mg/kg/day, and four males and three females administered 600 mg/kg/day had distension of the colon at necropsy. These findings correlated with microscopic distension, which sporadically involved the rectum. Other animals administered 600 mg/kg/day had distension noted elsewhere within the gastro-intestinal tract; however, these macroscopic findings could not be definitively linked with microscopic changes.

One female administered 300 mg/kg/day and four females administered 600 mg/kg/day had dark livers at necropsy. In addition, one of these animals receiving 600 mg/kg/day also had hepatic enlargement. Two males administered 600 mg/kg/day also had enlarged livers, and one male administered 600 mg/kg/day had a mottled liver at necropsy. These findings correlated with microscopic hepatocyte hypertrophy.

One male administered 600 mg/kg/day had a dark mesenteric lymph node at necropsy. This correlated with the microscopic presence of sinus erythrocytes. One female administered 600 mg/kg/day had large ovaries at necropsy. This finding correlated with microscopically retained corpora lutea. One male administered 600 mg/kg/day had pale and enlarged thyroid glands at necropsy. These findings correlated with microscopic follicular cell hypertrophy. Most other tissues were macroscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

One male previously administered 600 mg/kg/day had pale foci within the lungs, correlating microscopically with increased alveolar macrophages. Most other tissues were macroscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

See attached summary tables (attachment [2]) for more information.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No evidence of neuropathological findings were noted.

See attached summary tables (attachment [2]) for more information.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance related microscopic findings were present within the colon, rectum, kidney, liver, lung, mandibular salivary gland, mesenteric lymph node, ovary/uterus, pancreas, and thyroid.

Incidence of distension (terminal sacrifice): One male and two females administered 100 mg/kg/day, four males and females administered 300 mg/kg/day and eight males and ten females administered 600 mg/kg/day had microscopic distension of the colon and/or rectum. This was characterized by an increased luminal diameter of the tract, with associated flattening of the lining mucosa. This variably correlated with macroscopic distension within the colon at necropsy.

Kidney (terminal sacrifice): Females administered 600 mg/kg/day had microscopic hypertrophy of the proximal tubule cells of the kidney. This was characterized by an increase in individual renal tubular epithelial cell size, primarily within the cortical region, which resulted in an overall increase in the size and weight of this organ.

Liver (terminal sacrifice): Males administered 600 mg/kg/day and females administered 300 or 600 mg/kg/day had microscopic hypertrophy within the liver. This was characterized by an increase in hepatocellular size, appeared to originate within centri-lobular areas, and extended to involve the midzonal areas at the higher severities. This finding correlated with variable enlargement, darkening, or mottling at necropsy and an increase in liver weight.

Lungs (terminal sacrifice): Animals administered 600 mg/kg/day had a minor increase in the incidence/severity of microscopic infiltrates of alveolar macrophages within the lungs. These contained abundant granular cytoplasm and were focal-to-multifocal in distribution.

Mandibular salivary gland (terminal sacrifice): Males administered 600 mg/kg/day had minor increases in acinar atrophy/degranulation within the mandibular salivary gland, which was characterized by a loss in enzymatic zymogen content.

Mesenteric lymph nide (terminal sacrifice): Animals administered 600 mg/kg/day had an increased incidence/severity of erythrocytes within the sinuses of the mesenteric lymph node. This was accompanied by variable hemolysis/pigment accumulation. This finding correlated with macroscopic darkening in one male administered 600 mg/kg/day.

Ovary (terminal sacrifice): One female administered 300 mg/kg/day and females administered 600 mg/kg/day had retained corpora lutea within the ovary. This was characterized by an increased number of luteal structures, with a concomitant increase in the incidence of diestrus in these dose groups. These changes were characteristic of prolonged diestrus. This finding correlated with macroscopic enlargement of the ovaries in one female administered 600 mg/kg/day.

Pancreas (terminal sacrifice): Two terminal sacrifice males administered 600 mg/kg/day had degranulation within the exocrine pancreas, which was characterized by a loss in enzymatic zymogen content.

Thyroid (terminal sacrifice): Finally, male animals administered 300 mg/kg/day, and males and females administered 600 mg/kg/day had hypertrophy within the follicular epithelial cells of the thyroid. This was characterized by an overall increase in cell size/cytoplasmic height, with variable alterations in accompanying colloid morphology. This finding correlated with macroscopic pallor/enlargement in one male administered 600 mg/kg/day and increases in thyroid/parathyroid weight in males.

Most other tissues were microscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

Following a 4-week recovery period, test substance related microscopic findings were present within the lungs and thyroid.

Lungs (recovery sacrifice): Within the lungs, there remained a residual minor increase in the incidence/severity of alveolar macrophages, in males previously administered 600 mg/kg/day. This finding correlated with macroscopic pale foci in one male previously administered 600 mg/kg/day.

Thyroid (recovery sacrifice): Within the thyroid, one male and one female previously administered 600 mg/kg/day had residual follicular cell hypertrophy at this time-point. All other test article-related findings noted in main phase animals administered 600 mg/kg/day had fully reversed; the tissues examined were within normal limits.

Most other tissues were microscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

See attached summary tables (attachment [2]) for more information alongside Text Table 2 in 'Any other information on results incl. tables'.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No evidence of neoplastic histopathological lesions were noted.

See attached summary tables (attachment [2]) for more information alongside Text Table 2 in 'Any other information on results incl. tables'.

Other effects:

not specified

Details on results:

Two decedents administered 600 mg/kg/day had a cause of demise considered related to the test substance.

Over the course of the dosing phase, noteworthy inlife findings were limited to salivation for animals administered ≥100 mg/kg/day; reduced food consumption and body weight gain, with correlating decreases in grip strength and foot splay, in males administered ≥100 mg/kg/day; and increased food consumption and body weight gain for females administered ≥300 mg/kg/day. Clinical pathology findings were consistent with altered food consumption, stress, and microscopic findings in the liver and kidney. During the recovery phase, most findings in animals previously administered 600 mg/kg/day were reversed, except effects on motor activity and grip strength in males, activated partial thromboplastin time, and urine phosphate crystals.

At the terminal sacrifice, variable organ weight increases were noted for the heart, kidney, liver, lung (males administered 600 mg/kg/day only), and thyroid/parathyroid (males only) of both sexes administered 300 or 600 mg/kg/day.

At necropsy, a number of substance-related macroscopic findings were noted. One female administered 100 mg/kg/day and both sexes administered 300 or 600 mg/kg/day had distension of the colon, which correlated with microscopic distension; females administered 300 mg/kg/day and both sexes administered 600 mg/kg/day had darkening/enlargement/mottling of the liver, which correlated with microscopic hepatocellular hypertrophy; one male administered 600 mg/kg/day had a dark mesenteric lymph node, which correlated with microscopic sinus erythrocytes; one female administered 600 mg/kg/day had large ovaries, which
correlated with microscopically retained corpora lutea; and one male administered 600 mg/kg/day had pallor/enlargement of the thyroid, which correlated with microscopic follicular cell hypertrophy.

Test substance related microscopic findings were present in the colon/rectum (distension; from 100 mg/kg/day), kidney (proximal tubule hypertrophy [females only], which correlated with increased kidney weight; only 600 mg/kg/day), liver (hepatocellular hypertrophy, which correlated with increased liver weight; females administered 300 mg/kg/day and males only administered 600 mg/kg/day), lung (increased alveolar macrophages, which correlated with increased lung weights in males; only 600 mg/kg/day), mandibular salivary gland (acinar atrophy/degranulation [males only]; only 600 mg/kg/day), mesenteric lymph node (sinus erythrocytes; only 600 mg/kg/day), ovary/uterus (retained corpora lutea/increased incidence of diestrus [females only]; 300 mg/kg/day), pancreas (exocrine degranulation [males only]; only 600 mg/kg/day), and thyroid (follicular cell hypertrophy, which correlated with increased thyroid/parathyroid weight for males; males administered 300 mg/kg/day and females administered 600 mg/kg/day).

Following a 4-week recovery phase, no residual test substance related organ weight changes were noted. Within the lungs, there remained a residual minor increase in the incidence/severity of alveolar macrophages in males previously administered 600 mg/kg/day (correlating with macroscopic pale foci in one male); within the thyroid, one male and one female previously administered 600 mg/kg/day had residual follicular cell hypertrophy.

All other test article-related findings noted in terminal sacrifice animals administered 600 mg/kg/day had fully reversed; the tissues examined were within normal limits.

In addition to disturbances related to the gastro-intestinal tract and associated mesenteric lymph nodes, findings in the liver and thyroid were consistent with hepatic enzyme induction/adaptation (Hall et al., 2012; Maronpot et al., 2010). The features of tubule hypertrophy in the kidney were of uncertain pathogenesis; similar findings may be associated with an adaptive response to an increase in active transcellular transport capacity (Ellison et al., 1989; Hard et al., 1999). Findings within the mandibular lymph node and pancreas were considered secondary and related to the development of cachexia in affected animals (Longnecker and Wilson, 2002).

The no observed adverse effect level (NOAEL) established from the pathology findings was considered 300 mg/kg/day, due to the finding of two decedent males administered 600 mg/kg/day, for which a test substance related cause of death could not be excluded, and the fact that many of the other findings were adaptive in nature.

References:
Ellison DH, Velázquez H, Wright FS (1989) Adaptation of the distal convoluted tubule of the rat. Structural and functional effects of dietary salt intake and chronic diuretic infusion. J Clin Invest; 83:113-126.

Hall AP, Elcombe CR, Foster JR, et al (2012) Liver hypertrophy: A review of adaptive (adverse and non-adverse) changes-conclusions from the 3rd international ESTP expert workshop. Toxicol Pathol; 40: 971-994.

Hard GC, Alden CL, Bruner RH, et al (1999) Non-proliferative lesions of the kidney and lower urinary tract in rats. Gui Toxicol Pathol; 1-32.

Longnecker DS, Wilson GL (2002) Pancreas. In: Haschek WM, Rousseaux CG, Wallig MA (eds). Handbook of toxicologic pathology. 2nd Ed; Ch. 13; San Diego (CA): Academic Press, p. 227-254.

Maronpot R, Yoshizawa K, Nyska A, et al (2010) Hepatic enzyme induction: Histopathology. Toxicol Pathol; 38(5):776-795.

Effect levels

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Text Table 1: Summary of organ weight %-Differences compared with concurrent controls – terminal sacrifice

		Males			Females
		2M	3M	4M	2F	3F	4F
	Level (mg/kg/day)	100	300	600	100	300	600
% difference
Heart	Group mean unadjusted weights	-7	-5	+2	+6	+15*	+30*
	Bodyweight ratio	0	-1	+17*	+1	+7	+19*
	Brain weight ratio	-3	-3	+6	+8	+16*	+26*
Kidney	Group mean unadjusted weights	+1	+7	+8	+6	+22*	+39*
	Bodyweight ratio	+9	+12*	+24*	0	+12*	+27*
	Brain weight ratio	+5	+10	+13	+7	+23*	+35*
Liver	Group mean unadjusted weights	-10	-1	+15*	+5	+26*	+54*
	Bodyweight ratio	-4	+3	+32*	-1	+16*	+41*
	Brain weight ratio	-7	+1	+19*	+6	+27*	+50*
Lung	Group mean unadjusted weights	-8	-1	+2	+62	+9	+7
	Bodyweight ratio	-2	+3	+16*	+58	0	-3
	Brain weight ratio	-5	+1	+6	+64	+10	+4
Thyroid/ parathyroid	Group mean unadjusted weights	+2	+38*	+18	+2	+6	+13
	Bodyweight ratio	+10	+43*	+33*	-4	-4	+2
	Brain weight ratio	+7	+41*	+22	+4	+7	+10

* statistically significant

Text table 2: Incidence of selected findings

		Males				Females
		1M	2M	3M	4M	1M	2M	3M	4M
Tissue and finding	Level (mg/kg/day)	0	100	300	600	0	100	300	600
Colon	No. examined	10	10	10	8	10	9	10	10
Distension	Unremarkable	10	9	6	0	10	7	6	6
	Minimal	0	1	4	8	0	2	4	4
Rectum	No. examined	10	10	10	8	10	9	10	10
Distention	Unremarkable	10	9	8	5	10	9	10	10
	Minimal	0	1	2	3	0	0	1	1
Kidney	No. examined	10	10	10	8	10	9	10	10
Hypertrophy, proximal tubule cell	Unremarkable	10	10	10	8	10	9	10	10
	Minimal	0	0	0	0	0	0	0	7
	Slight	0	0	0	0	0	0	0	2
Liver	No. examined	10	10	10	8	10	9	10	10
Hypertrophy, hepatocyte	Unremarkable	10	10	10	0	10	9	0	0
	Minimal	0	0	0	2	0	0	2	0
	Slight	0	0	0	6	0	0	6	1
	Moderate	0	0	0	0	0	0	2	9
Lung	No. examined	10	10	10	8	10	9	10	10
Macrophages, alveolar, increased	Unremarkable	8	8	8	3	8	9	8	4
	Minimal	2	2	2	4	2	0	2	6
	Slight	0	0	0	1	0	0	0	0
Mandibular salivary gland	No. examined	10	10	10	8	10	9	10	10
Atrophy/degranulation, acinar	Unremarkable	10	10	10	3	10	9	10	10
	Minimal	0	0	0	3	0	0	0	0
	Slight	0	0	0	2	0	0	0	0
Mesenteric Lymph node	No. examined	10	10	10	8	10	9	10	10
Erythrocytes, sinus	Unremarkable	9	7	7	0	9	8	9	4
	Minimal	1	2	3	4	1	1	1	6
	Slight	0	1	0	4	0	0	0	6
Ovary	No. examined	0	0	0	0	10	9	10	10
Corpora lutea, retained	Unremarkable					10	9	9	2
	Present					0	0	1	8
Uterus	No. examined	0	0	0	0	10	9	10	10
Proestrus	Unremarkable					5	5	7	9
	Present					5	4	3	1
Estrus	Unremarkable					9	7	10	9
	Present					1	2	0	1
Diestrus	Unremarkable					7	6	3	2
	Present					3	3	7	8
Metestrus	Unremarkable					9	9	10	10
	Present					1	0	0	0
Pancreas	No. examined	10	10	10	8	10	9	10	10
Degranulation, exocrine	Unremarkable	10	10	10	6	10	9	10	10
	Minimal	0	0	0	2	0	0	0	0
Thyroid	No. examined	10	10	10	8	10	9	10	10
Hypertrophy, follicular cell	Unremarkable	10	10	6	0	10	9	10	0
	Minimal	0	0	4	6	0	0	0	10
	Slight	0	0	0	2	0	0	0	0
Lung (recovery sacrifice)	No. examined	5	0	0	4	5	0	0	5
Macrophages, alveolar, increased	Unremarkable	3			1	3			4
	Minimal	2			3	1			1
	Slight	0			1	1			0

Applicant's summary and conclusion

Conclusions:

Administration of control article (vehicle) or 100, 300, or 600 mg/kg/day test substance to Crl:WI(Han) rats following daily oral (gavage) administration for 13 weeks resulted in mortality in animals administered 600 mg/kg/day.

A few non-adverse in life findings were observed in males administered ≥100 mg/kg/day and females administered ≥300 mg/kg/day, including salivation and changes in food consumption, body weight gain, grip strength, and foot splay (males only). Clinical pathology, macroscopic, and microscopic findings were consistent with liver and kidney toxicity and were primarily observed in animals administered ≥300 mg/kg/day. Additionally, a few macroscopic and microscopic findings in other tissues were observed primarily in animals administered 600 mg/kg/day. Most findings ranged from minimal to slight severity and were considered not adverse. The majority of findings recorded during the dosing phase were fully or partly reversible. Under the conditions of this study, 300 mg/kg/day is considered the no observed adverse effect level (NOAEL).