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Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 March 2020 to 23 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
OECD guideline for the Testing of Chemicals Number 443, Extended One-Generation Reproductive Toxicity Study (adopted 25 June 2018).
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
Species Selection and Dose Administration Rationale
The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics.

Background Information
In a previous prenatal development study in the rat (MPI Research Study 399-243; MPI Research, 2014), pregnant female rats were administered the test article at a dose level of 0, 100, 200, or 400 mg/kg/day from GD 6 to 19 and were sacrificed on GD 20. All animals survived to their scheduled termination. Salivation was observed for all test article treated groups. No adverse test article effects were observed following administration of ≤200 mg/kg/day. Following administration of 400 mg/kg/day, maternal toxicity was restricted to early in the dosing period (GD 0 through 3) and included clinical observations (i.e., decreased activity, red material around the nose/mouth, hunched posture, thin appearance, discolored brown hair on the face, and discolored brown hair on the forelimbs), low gestation weight gain, and low food consumption. No adverse test article effect was observed on pregnancy indices; uterine implantation data; fetal sex ratios; fetal body weights; or fetal external, visceral, or skeletal evaluations. In the group administered 400 mg/kg/day, low incidences of red or white foci and swollen mucosa of the non glandular portion of the stomach were observed during F0 female macroscopic evaluations and were considered test article related. The no observed adverse effect level (NOAEL) for maternal toxicity was 200 mg/kg/day and the no observed effect level (NOEL) for developmental toxicity was 400 mg/kg/day.
In a previous 90-day repeat dose study (MPI Research Study 399-241; MPI Research, 2015), male and female rats were administered the test article at a dose level of 0, 25, 100, or 325 mg/kg/day. No adverse effects were noted in-life. Test article related clinical pathology changes included reversible increases in cholesterol for males administered 100 mg/kg/day and both sexes administered 325 mg/kg/day; reversible increases in fibrinogen and globulin for males administered 325 mg/kg/day (suggestive of an inflammatory response); and reversible increases in urea nitrogen for males administered ≥100 mg/kg/day. No test article related effects were observed for either sex administered 25 mg/kg/day. Macroscopic findings included tan discoloration and enlargement of the adrenal glands in all test article treated groups. Test article-related adrenal gland weight changes were noted in males administered ≥100 mg/kg/day and in females at all dose levels. Macroscopic and organ weight changes correlated with diffuse vacuolation of the zona fasciculata, observed microscopically, and was characterized by enlarged cells with foamy cytoplasm. In addition, the zona fasciculata cells noted near the zona reticularis had large, single, clear, vacuoles that expanded outwardly, depending on severity. The adrenal gland changes were considered adverse, based on the severity and the marked increases in adrenal gland weights in females. At recovery, the diffuse vacuolation was not present; however, cells near the zona reticularis had large, single, cytoplasmic vacuoles. Test article-related organ weight changes were noted for the liver of both sexes administered ≥100 mg/kg/day, thyroid gland for males administered 325 mg/kg/day, and ovary weights for females at all dose levels. Organ weights for the liver, thyroid gland, and ovary correlated microscopically with centrilobular or pablobular hypertrophy in the liver, follicular cell hypertrophy in the thyroid glands, and interstitial cell vacuolation in the ovaries. Additionally, lower terminal body weight in males administered 325 mg/kg/day resulted in higher organ:body weights, lower absolute organ weights, and/or lower organ:brain weights for the brain, epididymides, heart, and pituitary gland. A NOEL or NOAEL was not established for that study.
In a previous OECD 421 study (MPI Research Study 1038-004; MPI Research, 2005), male and female rats were administered the test article at a dose level of 0 or 400 mg/kg/day. Following administration of 400 mg/kg/day to rats, mean pup survival after parturition and up to PND 4 was lower than controls. Microscopically, test article-associated fatty changes were observed in the adrenals of males and females administered 400 mg/kg/day. Fatty changes were similar in the adrenals of males and females and were characterized by cells in the zona fasciculata and zona reticularis that were enlarged by the presence of increased small or large clear vacuoles. Vacuolation of interstitial cells of the ovaries and centrilobular hepatocellular hypertrophy of livers was also seen in females administered 400 mg/kg/day of the test article.

Dose Level Selection
The high-dose level of 100 mg/kg/day was expected to elicit some toxicity without causing undue stress or death.
The intermediate-dose level of 25 mg/kg/day was anticipated to be a no observed adverse effect level (NOAEL).
The low-dose level of 5 mg/kg/day was anticipated to be a no observed effect level (NOEL).
The oral route of administration was chosen because it is an acceptable and commonly used route exposure for regulatory studies of this type.

Test material

Constituent 1
Chemical structure
Reference substance name:
Phenol, isopropylated, phosphate (3:1)
EC Number:
273-066-3
EC Name:
Phenol, isopropylated, phosphate (3:1)
Cas Number:
68937-41-7
Molecular formula:
CXHYO4P X and Y are variable dependant on the molecular component.
IUPAC Name:
diphenyl 4-(propan-2-yl)phenyl phosphate; phenyl bis[4-(propan-2-yl)phenyl] phosphate; triphenyl phosphate; tris[4-(propan-2-yl)phenyl] phosphate
Test material form:
liquid
Details on test material:
Test article identification: Reofos 35
Chemical Name: Isopropylated Triphenyl Phosphate
Synonyms: Phenol, isopropylated, phosphate
Batch Number: 2018089159
CAS/Chemical Reference number: 68937-41-7
EC Number: 273-066-3
Molecular weight: 452.54 g/mol
Expiration date: 21 August 2021
Appearance: Clear liquid
Purity: 100%
Correction factor: None
Storage conditions: Sealed container, 15 to 25C, protected from light
Specific details on test material used for the study:
No further details specified in the study report

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
104 male and 104 female Crl:WI(Han) rats were obtained from Charles River Laboratories, Margate, United Kingdom, in order to provide sufficient animals for F0 generation study selection.
Upon arrival, animals were 5 to 6 weeks of age. At the start of dosing, F0 animals were 6 to 7 weeks of age, and males weighed between 149.0 and 217.6 g; females weighed between 107.2 and 171.4 g.
Upon arrival, all animals were given a clinical inspection for ill health. Animals were acclimated for 13 days, and an inspection was performed by the Named Animal Care and Welfare Officer (NACWO) before the start of dosing to ensure their suitability for the study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Environmental Conditions, Diet, and Water
Housing
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).
F0 animals were housed in groups of up to four by sex and dose group (pre-pairing phase for both sexes and post-pairing phase for males). During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed or until the end of the pairing phase. Following mating, females were housed individually during gestation or post-pairing, and with their litter during the lactation phase.
During the maturation phase, F1 animals were housed in groups of up to four by sex, dose group, and cohort. For Cohort 1B, during the pairing phase, one female was housed with one male from the same dose group until mating was confirmed or until the end of the pairing phase. Following mating, males were housed in groups of up to four by sex and dose group (post-pairing phase); females were housed individually during gestation or post-pairing, and with their litter during the lactation phase.
During neurobehavioral assessments, animals remained in their home cage, except when placed in the testing apparatus.
Bedding was provided at least weekly to each cage by use of clean Aspen wood chips or European Softwood bedding during gestation and lactation phases Datesand Ltd; Manchester, United Kingdom).
Each batch of bedding was analyzed for specific constituents and contaminants. No contaminants were present in the bedding at levels which might have interfered with achieving the objective of the study. Results are retained on file at Labcorp.

Water
Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants.
No contaminants were present in the water at levels which might have interfered with achieving the objective of the study. Results are retained on file at Labcorp.

Diet
Animals had ad libitum access to VRF1 (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants.
No contaminants were present in the diet at levels which might have interfered with achieving the objective of the study. Results are retained on file at Labcorp.
Four batches of diet were used on this study. A 50 g sample of each batch of diet used on study was collected and stored at ambient temperature until study finalization.

Environment
Animals were housed in exclusive rooms. Rooms were air conditioned to provide a minimum of 15 air changes/hour. The temperature and relative humidity ranges were maintained in the specified ranges of 19 to 25 deg C and 40 to 70%, respectively.
Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark.

Environmental Enrichment
Animals were provided with wooden Aspen chew blocks, rodent retreats and paper wool nesting materials (gestation and lactation phases only) as forms of environmental enrichment. The paper wool nesting material was retained at bedding changes between GD 18 and LD 4.

Animal Identification and Assignment to the Study
Upon arrival, F0 animals were assigned to groups based on a total randomization procedure.
Animals were individually identified by electronic implant. Pups were uniquely identified from PND 1 via a back mark with an indelible pen.
During FOB assessments, applicable F1 generation, Cohort 2A animals were identified by tail marks.
F1 Cohort 2B animals were uniquely identified by tail mark with an indelible pen.
For F0 animals, following the first full weight collection during the predose phase, group mean body weights and standard deviations were calculated and inspected to ensure no unacceptable differences occurred between groups.
Cages were placed in group order across the batteries of cages, except during FOB assessments.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Formulations were prepared daily for the first 2 days and then weekly thereafter.
The test article was formulated as a suspension in corn oil following dispensary SOPs and the formulation method (Method 8415993_O_01D), as maintained in the study data.
Formulations were stored refrigerated (2 to 8 deg C) in a sealed container, protected from light.
Details on mating procedure:
F0 Generation
During the pairing phase, one male was housed for up to 14 days with one female of the same group.
Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal washing. Upon the confirmation of mating, vaginal washing was discontinued, and the male was removed from the cage. The day of mating confirmation was considered GD 0.

F1 Generation - Post weaning
During the pairing phase, one Cohort 1B male was housed for up to 14 days with one female of the same cohort/dose group.
Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal washing. Upon the confirmation of mating, vaginal washing was discontinued, and the male was removed from the cage. The day of the confirmation of mating was considered GD 0.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Procedure
The samples were analyzed in accordance with the validated Labcorp Analytical Procedure (DFA/M032/20).
The analytical method involved extraction and dilution in Acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 10 µg/mL to 100 µg/mL. Standard and sample solutions were all spiked with a known amount of Tetradecane (internal standard), and the peak area response ratio of the test item:internal standard was used for quantitation.

Concentration of Dose Formulations
The formulations for the first, middle and last dosing occasion preparations were sampled. For all groups, 1 × 5 mL was sampled at random from the formulation by Labcorp Harrogate Dose Formulation personnel.
Two aliquots from all groups were analyzed in accordance with the analytical procedure. The remainder of the sample was retained for contingency. On the first and last dosing occasion duplicate additional aliquots were taken from the samples and analysed following out of specification results being achieved in the initial analysis. Samples were disposed of once satisfactory results were achieved.
Procedural recoveries were prepared as a quality control measure and were not used to correct for the analytical results.
Duration of treatment / exposure:
F0 males were dosed once daily for 126 consecutive days (at least 10 weeks prior to pairing, during pairing, and 41 days post-pairing); females were dosed for up to 128 days (10 weeks prior to pairing, during pairing, throughout gestation, and up to LD 21).
All F1 offspring were dosed once daily from PND 14 through 21; prior to this, pups were maternally administered test article through milk. Following selection into appropriate cohorts, selected F1 animals were administered test article once daily.
Frequency of treatment:
Daily
Details on study schedule:
F0 Generation Procedures
Test Article Administration
Formulations (excluding vehicle control group) were stored at room temperature and stirred continuously for at least 30 minutes before and throughout dosing.
The test article was administered orally by gavage.
F0 males were dosed once daily for 126 consecutive days (at least 10 weeks prior to pairing, during pairing, and 41 days post-pairing); females were dosed for up to 128 days (10 weeks prior to pairing, during pairing, throughout gestation, and up to LD 21).
Dosing was omitted on one occasion for the following females as they were in or near parturition at the time of daily dosing.
• Animals R0404, R0418, R0420, R0421, and R0425 (F0 generation, Group 1)
• Animals R0502, R0508, R0516, and R0524 (F0 generation, Group 2)
• Animals R0604, R0608, R0609, R0615, and R0621 (F0 generation, Group 3)
• Animals R0701, R0706, R0715, R0717, R0719, R0720, and R0725 (F0 generation, Group 4)
All F1 offspring were dosed once daily from PND 14 through 21; prior to this, pups were maternally administered test article through milk. Following selection into appropriate cohorts, selected F1 animals were administered test article once daily.
Formulations were administered at a constant dose volume of 4 mL/kg. Dose volumes were based on the most recently recorded body weight for each animal.

F1 Generation Procedures - Post Weaning
Test Article Administration
Formulations (excluding vehicle control group) were stirred continuously for at least 30 minutes before and throughout dosing.
The test article was administered orally by gavage.
Following selection into appropriate cohorts, selected F1 animals were administered once daily as follows:
• Cohort 1A: For up to 73 days
• Cohort 1B: For up to 104 consecutive days for males (up to 75 days prior pairing, during pairing and 15 days post pairing) or for up to 108 days for females (up to 75 days prior to pairing, during pairing, throughout gestation and up to LD 3
• Cohort 2A: For 54 days
• Cohort 2B: For 1 day
Dosing was omitted for the following Cohort 1B females as they were in or near parturition at the time of daily dosing.
• Animals R1222, R1227, and R1230 (F1 generation, Group 1)
• Animals R1326 and R1328 (F1 generation, Group 2)
• Animals R1424 and R1426 (F1 generation, Group 3)
• Animals R1522, R1523, R1526, R1528, and R1532 (F1 generation, Group 4).
Formulations were administered at a constant dose volume of 4 mL/kg. Dose volumes were based on the most recently recorded body weight for each animal.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0 Generation: 25/sex per dose
F1 Generation, Cohort 1A & 1B: 20/sex per dose
F1 Generation, Cohort 2A & 2B: 10/sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The high-dose level of 100 mg/kg/day was expected to elicit some toxicity without causing undue stress or death.
The intermediate-dose level of 25 mg/kg/day was anticipated to be a no observed adverse effect level (NOAEL).
The low-dose level of 5 mg/kg/day was anticipated to be a no observed effect level (NOEL).
The oral route of administration was chosen because it is an acceptable and commonly used route exposure for regulatory studies of this type.

Examinations

Parental animals: Observations and examinations:
F0 Generation
Clinical Observations
Health Monitoring
All animals were observed in the home cage at the beginning and end (nominal) of each working day for signs of ill health or overt toxicity.

Clinical Examinations
Each animal was given a detailed physical examination once daily from the start of dosing. An individual record was maintained of the clinical condition of each animal.

Weekly Detailed Clinical Observations
All animals were assessed by detailed clinical observations within the home cage, in the hand, and in an open field arena for approximately 30 seconds. Males were assessed once weekly from the start of dosing. Females were assessed once weekly during the pre-pairing and post pairing phases; on GD 0, 6, 14, and 20; and on LD 1, 7, 14, and 21. For one of the females with no confirmation of mating but which was pregnant (Animal R0404 [F0 generation, Group 1]), the data collected during the post-pairing phase were back entered to the gestation phase.
Each animal was observed and evaluated for the following:
Activity, Aggression to cage mate, Alertness, Approach response, Acoustic startle response, Bar test, Behaviour – other & stereotype, Body tone, Discharge, Excretion, Extensor thrust, Eye closure, Eyes, Gait, Grasping loss, Grip strength, Involuntary movement, Lacrimation, Pain response, Palpebral reflex, Pelage, Pinna response, Posture, Proprioception (right hind leg), Pupil status, Pupillary response, Reactivity to handling, Respiration, Restlessness, Righting reflex, Salivation. Skin colour, Tail, Visual response, Vocalisation, Waxy ridgity.


Postdose Observations
Animals were observed daily for the first 7 days of dosing at approximately 0.5 hours postdose.

Body Weights
Body weights were recorded once during acclimation (Predose Day 1[Day-3]) for all animals.
Male body weights were recorded weekly from the first day of dosing (Pre-Pairing Day 1).
Female body weights were recorded weekly from the first day of dosing (Pre-Pairing Day 1); on GD 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; and on LD 1, 4, 7, 14, and 21.
Where an animal had completed parturition before the daily dose on LD 0, a body weight was recorded and was used to calculate the correct dose volume to be administered to avoid overdosing; then, dosing occurred.
Body weights for females with no confirmation of mating were recorded weekly during the post-pairing phase. For one of the females with no confirmation of mating but which was pregnant (Animal R0404 [Group 1] and R0510 [Group 2]), the data collected during the post pairing phase were back-entered to the gestation phase.

Food Consumption
Male food consumption was recorded twice weekly during the pre-pairing and post pairing phases.
Female food consumption was recorded twice weekly during pre-pairing; every 2 days during gestation; and from LD 1 to 4, 4 to 7, and 7 to 14.
Food consumption was not recorded during pairing (both sexes) or post-pairing (females), between GD 21 and LD 0, due to parturition, and from LD 14, due to the start of pup weaning.
Food consumption was calculated as g/animal/day.
Food conversion efficiency (body weight gain per gram of food consumed) was also calculated.


F0 Generation Clinical Laboratory Procedures
Clinical Pathology
Sample Collection and Handling
Blood samples for hematology (1 x 0.5 mL [EDTA], nominal), coagulation (1 x 0.6 mL prior to the issue of Protocol Amendment 05 [2 x 0.5 mL pots were used], then 1 x 0.5 mL thereafter [trisodium citrate], nominal), clinical chemistry (1 x 0.5 mL prior to the issue of Protocol Amendment 05, then 1 x 0.6 mL thereafter [serum separator tubes], nominal), and thyroid hormone analysis (1 x 1.2 mL [serum separator tubes], nominal) were withdrawn from the abdominal aorta at necropsy on Post Pairing Day 42 (Week 19 of the study) for at least 10 randomly selected males within each group and at necropsy on LD 22 for at least 10 randomly selected females within each group. Samples were collected after animals were fasted overnight.
Blood samples for hematology and coagulation were fully inverted several times (approximately 10), ensuring blood travelled all the way to the top and bottom of the tube each time, followed by at least 5 minutes on an automatic mixer. Blood samples for clinical chemistry were gently inverted several times (approximately 10), ensuring blood travelled all the way to the top and bottom of the tube each time to mix with the clot activator.
Blood samples for thyroid hormone analysis from unselected F1 generation pups sacrificed on PND 22 (2 x 0.6 mL [serum separator tubes], nominal) were withdrawn by cardiac puncture from two male pups/litter and two female pups/litter. Samples from one male and one female sample were analyzed in the first instance; when required, the spare sample was analyzed. Spare samples not required for additional analysis are retained in storage until report finalization, after which, they will be discarded.
Thyroid hormone sampling was performed at a similar time on each occasion (between 09:00 and 13:00); samples were protected from light until placed in frozen storage.
Each blood sample for thyroid hormone analysis was gently inverted several times (approximately 10), ensuring blood travelled all the way to the top and bottom of the tube each time to mix with the clot activator, and was stored at room temperature (15 to 25° C) for at least 30 minutes prior to processing to allow samples to clot. Each sample was then centrifuged at 2300g for 10 minutes at approximately 4° C. The resultant serum was separated, transferred to uniquely labeled amber polypropylene tubes, and frozen at < 10°C (nominal -20°C).
During sample processing, each blood sample obtained from adults for thyroid hormone analysis was split into two equal aliquots. One aliquot was analyzed in the first instance; when required, the spare sample was analyzed. Spare samples not required for additional analysis are retained in storage until report finalization, after which, they will be discarded.
Urine samples from the first six males/group from batch 1 and 2 (total of 12 males per group) and up to 13 females/group which littered first (across batch 1 and 2) were collected overnight the day prior to necropsy. Food was removed during collection.

Hematology Tests
hemoglobin, red blood cell count, packed cell volume (hematocrit), mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, reticulocyte count, red cell distribution width, hemoglobin distribution width, total and differential white cell count, platelet count*, platelet crit, mean platelet volume, platelet distribution width
*Includes platelet clump assessment. Clump counts below 100 are considered none detected; clump counts over 100 are considered platelet clumps present and are confirmed by review of Advia cytogram or blood film examination.

Blood smears were prepared from each hematology specimen and used when necessary to confirm results produced by the analyzer.

Coagulation Tests
prothrombin time, fibrinogen, activated partial thromboplastin time

Clinical Chemistry Tests
aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total cholesterol, total bilirubin, total protein, albumin, globulin, albumin:globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, enzymatic creatinine, urea, glucose, blood urea nitrogen

Urinalysis Tests
volume, color, turbidity, specific gravity, pH*, protein*, glucose*, ketones*, urobilinogen*, bilirubin*, blood*, microscopy of sediment
*Determined semi quantitatively.

Thyroid Hormone Tests
thyroxine (Total T4), thyroid stimulating hormone (TSH)

Bone Marrow Smear Evaluation
Bone marrow smears were prepared at necropsy from the femur for F0 animals. Tissues were taken into bovine serum albumin, smear-prepared, air-dried, and fixed in methanol, but not examined.

F1 Generation Procedures - Post Weaning
Clinical Observations
Health Monitoring
All animals were observed in the home cage at the beginning and end (nominal) of each working day for signs of ill health or overt toxicity.

Clinical Examinations
Each animal was given a detailed physical examination once daily from the start of dosing. An individual record was maintained of the clinical condition of each animal.

Weekly Detailed Clinical Observations
All animals of Cohorts 1A, 1B, and 2A were assessed by detailed clinical observations within the home cage, in the hand, and in an open field arena for approximately 30 seconds. Both sexes were assessed once weekly from Maturation Day 22, and Cohort 1B females were assessed on GD 0, 6, 14, and 20 and on LD 4.
Each animal was observed and evaluated for the following:
Activity, Aggression to cage mates, Alertness, Approach response, Acoustic startle response, Bar test, Behaviour – other & stereotype, Body tone, Discharge, Excretion, Extensor thrust, Eye closure, Eyes, Gait, Grasping loss, Grip strength, Involuntary movement, Lacrimation, Pain response, Palpebral reflex, Pelage, Pinna response, Posture, Proprioception (right hind leg), Pupil status, Pupillary response, Reactivity to handling, Respiration, Restlessness, Righting reflex, Salivation, Skin colour, Tail, Visual response, Vocalisation, Waxy rigidity.

Postdose Observations
Animals of Cohorts 1A, 1B, and 2A were observed daily for the first 7 days of dosing at approximately 0.5 hours postdose.

Body Weight
Body weights for Cohorts 1A, 1B (males), and 2A were recorded on PND 22, 24, 26, 28, 30, 32, 34, and 36 and then weekly thereafter.
Body weights for Cohorts 1B females were recorded on PND 22, 24, 26, 28, 30, 32, 34, and 36; weekly thereafter (maturation/pre-pairing phase); on GD 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; and on LD 1 and 4.
Body weights for Cohort 2B were recorded on PND 21 and 22 (terminal body weight).
For Cohorts 1A, 1B, and 2A, body weights were recorded on the day balano preputial separation or vaginal opening occurred.

Food Consumption
Food consumption was recorded twice weekly from Maturation Day 22, until the day prior to necropsy for Cohorts 1A and 2A. See Protocol Deviations.
Food consumption was recorded twice weekly from Maturation Day 22 until pairing (both sexes); then twice weekly during post-pairing (males); every 2 days during gestation, and from LD 1 to 4 for Cohort 1B.
Food consumption was not recorded during pairing and between GD 21 and LD 0, due to parturition.
Food consumption was calculated as g/animal/day.

Sexual Maturation
Animals were observed for vaginal opening (females) from Maturation Day 28 and cleavage of the balano preputial gland (males) from Maturation Day 37; observations lasted until these milestones were attained. Body weights were recorded on the day balano preputial separation or vaginal opening occurred.
Oestrous cyclicity (parental animals):
F0 Generation
Daily vaginal lavage (washings) was undertaken from females for 14 days prior to pairing and until confirmation of mating, and on the day of necropsy.

F1 generation - Post weaning
Daily vaginal lavage (washings) were undertaken for Cohort 1A females after vaginal opening until the first cornified smear was observed, then from Maturation Day 75 to 89 inclusive, and on the day of necropsy.
Daily vaginal lavage (washings) were undertaken for Cohort 1B females after vaginal opening until the first cornified smear was observed, then from Maturation Day 75 until confirmation of mating, and on the day of necropsy for females that littered.
Sperm parameters (parental animals):
Sperm Assessments
For each scheduled male, sperm number, motility, and velocity were recorded by Computer Assisted Sperm Analysis (CASA) from a sample of sperm from the left epididymis (vas deferens and epididymis cauda). Each sample was prepared for microscopic evaluation of sperm morphology. Slides prepared from all males for each group were read for morphological changes. The left epididymis was discarded following seminology investigations.

Cohort 1A
For each scheduled male, sperm number, motility, and velocity were recorded by Computer Assisted Sperm Analysis (CASA) from a sample of sperm from the left epididymis (vas deferens and epididymis cauda). Each sample was prepared for microscopic evaluation of sperm morphology. Slides prepared from all males for each group were read for morphological changes. The left epididymis was discarded following seminology investigations
Litter observations:
F0 Generation
Parturition
Animals were observed three times each day (at the beginning, middle, and end of each working day), starting when the first females reached GD 21 and until the last female littered. Females that were confirmed as mated but did not litter were sent to necropsy on GD 26; females that did not have a confirmation of mating and did not litter were sent to necropsy on Post-Pairing Day 25.
Females were observed for signs of the start of parturition (for example, blood in the cage). The time and date of this observation were recorded, and marked the end of gestation; when not observed, the end of gestation was the day when the completion of parturition was recorded or on the day prior to when LD 1 observations were made.
Abnormal observations of nesting, parturition, or nursing were recorded. It was considered important that F0 female was disturbed as little as possible on LD 0. Any dead pups were removed and sent to necropsy to establish if they were born alive or dead (by looking for lung inflation).

F1 Offspring Procedures - Pre-Weaning
Litter Size and Sex Determination
Litter size and pup sex were recorded on PND 0, 1, 4, 7, 14, and 21. See Protocol Deviations.
Daily records of mortality and changes in litter sizes were maintained. Pups found dead or in a moribund condition were given a macroscopic necropsy for cause of death and possible birth defects.

Clinical Observations
A detailed clinical examination was recorded for each pup on PND 1, 4, 7, 14, and 21.

Postdose Observations
All pups were observed daily from PND 14 through 21 at approximately 0.5 hours postdose.

Body Weights
Pup body weights were recorded on PND 1, 4, 7, 13, 14, 16, 18, and 20.

Anogenital Distance
The anogenital distance of all pups was recorded on PND 4.

Nipple/Areolae Count
The number of nipples/areolae for male pups was counted on PND 13. See Protocol Deviations.

Weaning
Pups were weaned on PND 21, and 240 pups/sex were randomly selected from available litters (any obvious runts [pups with a body weight more than two standard deviations below the mean pup weight of the respective litter] were not included in selection). Animals of Cohorts 1A and 1B were allocated, based on a random selection of 20 animals/sex/group from the available litters (one male and one female from each litter). Animals of Cohorts 2A and 2B were allocated, based on a random selection of 10 animals/sex/group from the available litters (one male and one female from each litter).
On PND 22, a blood sample for thyroid hormone analysis was withdrawn from all pups not selected to continue into the F1 generation. Following blood sampling, pups were sacrificed and necropsied.

F1 Generation - Post weaning
Parturition
Animals were observed three times/day (at the beginning, middle, and end of each working day), starting when the first females reached GD 21 and until the last female littered. Females confirmed as mated but that did not litter were sent to necropsy on GD 26.
Females were observed for signs of the start of parturition (for example, blood in the cage). The time and date of this observation were recorded, and marked the end of gestation; when not observed, the end of gestation was the day when the completion of parturition was recorded or on the day prior to when LD 1 observations were made.
Abnormal observations of nesting, parturition, or nursing were recorded. It was considered important that the dam was disturbed as little as possible on LD 0. Any dead pups were removed and sent to necropsy to establish if they were born alive or dead (by looking for lung inflation).

F2 Offspring Procedures
Litter Size and Sex Determination
Litter size and pup sex were recorded on PND 0, 1, and 4.
Daily records of mortality and changes in litter sizes were maintained. Pups found dead or in a moribund condition were given a macroscopic necropsy for cause of death and possible birth defects.

Clinical Observations
A detailed clinical examination was recorded for each pup on PND 1 and 4.

Body Weights
Pup body weights were recorded on PND 1 and 4.

Anogenital Distance
The anogenital distance of all pups was recorded on PND 4.

Neurotoxicity Testing
Cohort 2A animals were assessed for acoustic startle on Maturation Day 24 (±1 day).
Each animal was evaluated for startle habituation using a Kinder Auditory Startle Chamber. Prior to habituation testing, each animal was acclimatized to the chamber for 4 minutes under white noise conditions. Each animal underwent one habituation session, which consisted of 50 standard trials of 115 dB bursts of noise, with mixed inter-trial intervals.
On a separate day, prior to pre-pulse inhibition (PPI) testing, each animal was acclimatized to the chamber for 4 minutes under white noise conditions. Animals underwent testing, which consisted of 41 randomized trials. Thirty were pre-pulse trials (10 trials each of 74, 78, or 86 dB pre-pulse, followed by 115 dB startle), and 11 were standard trials (115 dB startle, with no pre-pulse), with mixed inter-trial intervals and decibel levels. The first standard trial (no pre-pulse) for each animal was used for acclimatization and was not analyzed. Data were reported as percent pre pulse inhibition for the PPI session.
Data collected for habituation included the mean maximum amplitude (five blocks of 10 trials for each group). Pre-pulse inhibition was calculated as 100 - ((maximum startle amplitude on pre-pulse trials / maximum startle amplitude on 115 dB standard trials) x 100) for each animal.

Functional Observational Battery
Cohort 2A animals underwent FOB assessments by detailed clinical observations, quantitative assessments, and elicited responses.
The FOB assessments were undertaken in a manner so the observer did not know the dose group of animals during testing. Observations were performed at approximately the same time on each occasion.

Detailed Clinical Observations
Cohort 2A animals were assessed for detailed clinical observations on Maturation Day 69 (±6 days). Males were assessed on Maturation Days 66, and 68 through 71. Females were assessed on Maturation Days 67, and 69 through 72.
Animals were observed within the home cage, in the hand, and in the arena for approximately 2 minutes. Each animal was observed and evaluated for the following.
Activity, Aggression to cage mates, Alertness, Approach response, Acoustic startle response, Bar test, Behaviour – other & stereotype, Body tone, Discharge, Excretion, Extensor thrust, Eye closure, Eyes, Gait, Grasping loss, Grip strength, Involuntary movement, Lacrimation, Pain response, Palpebral reflex, Pelage, Pinna response, Posture, Proprioception (right hind leg), Pupil status, Pupillary response, Reactivity to handling, Respiration, Restlessness, Righting reflex, Salivation, Skin colour, Tail, Visual response, Vocalisation, Waxy rigidity.
Numerical observations: Faecal boli, Rears, Urine pools, Latency to first step.

Quantitative Assessments
Cohort 2A animals were assessed for quantitative assessments on Maturation Day 69 (±6 days). Males were assessed on Maturation Days 66, and 68 through 71. Females were assessed on Maturation Days 67, and 69 through 72.
Quantitative assessment parameters are as follows.
Hindlimb foot splay, Forelimb and hindlimb grip strength.

Motor Activity
Cohort 2A animals were assessed for motor activity on Maturation Day 69 (±6 days).
Motor activity was assessed in an automated photocell activity recorder (Kinder Motor Monitor system) for 60 minutes.
Motor activity was recorded with the room lights turned off and white noise switched on. The white noise generator was maintained (level recorded at the beginning and end of each session) at an appropriate level so that a sound meter registered between 60 and 80 dB (Decibels), during the course of the scheduled sessions.
Animals were acclimated in their holding cages to the white noise for at least 10 minutes prior to any procedure taking place in the room.
Motor activity data are reported over 10-minute intervals.
The following parameters were assessed:
Basic Movement: Count of all horizontal beam breaks
Fine Movement: Measure of small movements, such as grooming and head movements; the animal remained in a fixed point (i.e., a single beam break with no other beams affected).
Total Ambulations: Measure of large movements; when a new beam was blocked and the anchor beam was broken (i.e., animal has relocated its whole body [e.g., a step forward]).
Total ambulation = X ambulation + Y ambulation
Total Distance Travelled: Record of distance travelled around the cage.
Total distance travelled = High-Density Periphery Zone Distance + Center Zone Distance
Rearing Event: Beam break on top grid (rearing)

F1 Generation Cohort 1A Clinical Laboratory Procedures
Clinical Pathology
Sample Collection and Handling
Blood samples for hematology (1 x 0.5 mL [EDTA], nominal), coagulation (1 x 0.5 mL [trisodium citrate], nominal), clinical chemistry (1 x 0.6 mL [serum separator tubes], nominal), and thyroid hormone analysis (1 x 1.2 mL [serum separator tubes], nominal) were withdrawn from the abdominal aorta at necropsy on Maturation Day 92, 93, 94, or 95 for at least 10 randomly selected males within each group and at necropsy on Maturation Day 92, 93, or 94 for at least 10 randomly selected females within each group. Samples were collected after animals were fasted overnight.
Blood samples for hematology and coagulation were fully inverted several times (approximately 10), ensuring that the blood travelled all the way to the top and bottom of the tube each time followed by at least 5 minutes on an automatic mixer. Blood samples for clinical chemistry were gently inverted several times (approximately 10), ensuring blood travelled all the way to the top and bottom of the tube each time to mix with the clot activator.
Thyroid hormone sampling was performed at a similar time on each occasion (between 09:00 and 13:00); samples were protected from light until placed in frozen storage.
Each blood sample for thyroid hormone analysis was gently inverted several times (approximately 10), ensuring blood travelled all the way to the top and bottom of the tube each time to mix with the clot activator, and was stored at room temperature (15 to 25°C) for at least 30 minutes prior to processing to allow samples to clot. Each sample was then centrifuged at 2300g for 10 minutes at approximately 4°C. The resultant serum was separated, transferred to uniquely labeled amber polypropylene tubes, and frozen at < 10°C (nominal -20°C).
During sample processing each blood sample for thyroid hormone analysis was split into two equal aliquots. One aliquot was analyzed in the first instance; when required, the spare sample was analyzed. Spare samples not required for additional analysis are retained in storage until report finalization, after which, they will be discarded.
Urine samples from at least 10 randomly selected males and females in each group were collected overnight the day prior to necropsy. Food was removed during collection.

Hematology Tests
hemoglobin, red blood cell count, packed cell volume (hematocrit), mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, reticulocyte count, red cell distribution width, hemoglobin distribution width, total and differential white cell count, platelet count*, platelet crit, mean platelet volume, platelet distribution width
*Includes platelet clump assessment. Clump counts below 100 are considered none detected; clump counts over 100 are considered platelet clumps present and are confirmed by review of Advia cytogram or blood film examination.
Blood smears were prepared from each hematology specimen and used when necessary to confirm results produced by the analyzer.

Coagulation Tests
prothrombin time, activated partial thromboplastin time, fibrinogen

Clinical Chemistry Tests
aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total cholesterol, total bilirubin, total protein, albumin, globulin, albumin:globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, enzymatic creatinine, urea, glucose, blood urea nitrogen

Urinalysis Tests
volume, color, turbidity, specific gravity, pH*, protein*, glucose*, ketones*, urobilinogen*, bilirubin*, blood*, microscopy of sediment
*Determined semi quantitatively.

Thyroid Hormone Tests
thyroxine (Total T4), thyroid stimulating hormone (TSH)

Bone Marrow Smear Evaluation
Bone marrow smears were prepared at necropsy from the femur for F1 Cohort 1A animals. Tissues were taken into bovine serum albumin, smear-prepared, air-dried, and fixed in methanol, but not examined.
Postmortem examinations (parental animals):
F0 Generation Terminal Procedures
With the exception of fasting, these procedures were also followed for unscheduled sacrifices.

Adult Necropsy
F0 males were sacrificed by isoflurane anesthesia on Post-Pairing Day 42 (Week 19) after an overnight period without food. Sacrifices were carried out in a controlled randomization order (i.e. one cage of animals from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed). Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
F0 females were sacrificed by isoflurane anesthesia after the completion of weaning on LD 22 (those that littered) or GD 26 (those with confirmation of mating that did not litter) or on Post-Pairing Day 25 (those with no confirmation of mating that did not litter). Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Females sacrificed on LD 22 were starved overnight prior to necropsy.
Females with the same day of mating/littering were sacrificed in a controlled, randomization order (i.e. one animal from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed). Females that did not litter were sacrificed in animal identification order.
The number of implantation sites for all paired females were recorded. The uterus of any apparently non-pregnant female was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.
Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded. Macroscopic examinations were performed under the general supervision of a pathologist. For tissue collection.
Organ weights, were recorded at each scheduled sacrifice, excluding decedents and paired females that mated but did not litter. Organs were dissected free from fat and other contiguous tissue and weighed before fixation. Left and right organs were weighed together. Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated.
Tissue List
Adrenal1 [WE], animal identification, bone marrow smear (femur)2, brain [WE], cecum [E], colon [E], duodenum [E], epidydymis1,3 [WE], eye4 [E], femur with bone marrow and femorotibial joint [E], gross lesions [E], head, heart [WE], ileum [E], jejunum [E], kidney1 [WE], liver [WE], lungs with main stem bronchi and bronchioles [E], mammary gland [E], muscle, biceps femoris [E], nerve, optic [E], nerve, sciatic [E], ovary1 [WE], oviduct [W5E], pituitary [WE], prostate [WE], rectum [E], seminal vesicle with coagulating glands and fluid [WE], spinal cord, cervical [E], spinal cord, lumbar [E], spinal cord, thoracic [E], spleen [WE], stomach [E], testis1,3 [WE], thymus [WE], thyroid with parathyroid6 [WE], trachea [E], urinary bladder [E], uterus with cervix [WE], vagina [E].
E = Processed and examined microscopically; W = Weighed
Note: bone designated for microscopic examination was decalcified using Kristenson’s fluid.
1 Tissue weighed as a pair
2 Smear prepared, air dried, the fixed in methanol
3 Tissue taken into Modified Davidsons fixative and processed to at least the block stage
4 Tissue taken into Davidsons fixative
5 Weighed with uterus
6 Tissues weighed together

Reproductive and Endocrine Tissue List
Animal identification, epididymis1 [E], gross lesions [E], mammary gland [E], ovary [E], oviduct [E], pituitary [E], prostate [E], seminal vesicle [E], testis1 [E], uterus with cervix [E], vagina [E].
E = Processed and examined microscopically
1 Tissue taken into Modified Davidsons fixative and processed to at least the black state


Histology and Microscopic Examinations
All tissues denoted by E in the Table from Groups 1 and 4 and all decedents and the adrenal glands from Groups 2 and 3 were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
All tissues denoted by E in the table from males in Groups 2 and 3 that did not sire and females in Groups 2 and 3 that were not mated and/or not pregnant were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
Additional sections of testes and epididymides were also stained with periodic acid Schiff (PAS) for spermatogenic staging for all males.
Due to suspected test article-related effects in Group 4, thyroids, liver, spleen (both sexes), ovaries (females), and kidneys (males), for all Group 2 and 3 animals, were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
Qualitative assessment of spermatogenesis and testicular interstitial cell structure was performed for all males, where appropriate.

F1 Generation Terminal Procedures
With the exception of fasting, these procedures were also followed for unscheduled sacrifices.

Cohort 1A
Animals were sacrificed by isoflurane anesthesia between Maturation Days 92 and 95 (Week 11) after an overnight period without food. Sacrifices were carried out in a controlled randomization order (i.e. one cage of animals from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed). Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Upon sacrifice, macroscopic examinations were conducted and all lesions were recorded. Macroscopic examinations were performed under the general supervision of a pathologist. For tissue collection, see Text Table.
Organ weights, as indicated in Text Table, were recorded at each scheduled sacrifice. Organs were dissected free from fat and other contiguous tissue and weighed before fixation. Left and right organs were weighed together. Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated.
Animal R1103 (Group 4, F1 Cohort 1A male) was found dead and replaced. Tissues as indicated in Text Table 3.8 were retained in 10% neutral buffered formalin, unless otherwise indicated. Tissues were not processed or examined microscopically by the Study Pathologist.
Tissue List
Adrenal1 [WE], animal identification, bone marrow smear (femur)2, brain [WE], cecum [E], colon [E], duodenum [E], epidydymis1,3 [WE], eye4 [E], femur with bone marrow and femorotibial joint [E], gross lesions [E], head, heart [WE], ileum [E], jejunum [E], kidney1 [WE], liver [WE], lungs with main stem bronchi and bronchioles [E], lymph node, mandibular8 [WE], lymph node, mesentric8 [WE], mammary gland [E], muscle, biceps femoris [E], nerve, optic [E], nerve, sciatic [E], ovary1 [WE], oviduct [W5E], pituitary [WE], prostate [WE], rectum [E], seminal vesicle with coagulating glands and fluid [WE], spinal cord, cervical [E], spinal cord, lumbar [E], spinal cord, thoracic [E], spleen7 [WE], stomach [E], testis1,3 [WE], thymus [WE], thyroid with parathyroid6 [WE], trachea [E], urinary bladder [E], uterus with cervix [WE], vagina [E].
E = Processed and examined microscopically; W = Weighed
Note: bone designated for microscopic examination was decalcified using Kristenson’s fluid.
1 Tissue weighed as a pair
2 Smear prepared, air dried, the fixed in methanol
3 Tissue taken into Modified Davidsons fixative and processed to at least the block stage
4 Tissue taken into Davidsons fixative
5 Weighed with uterus
6 Tissues weighed together
7 Tissue weighed for all animals. For at least 10 animals/sex/group, half of the spleen was provided to Labcorp Immunology and Immunotoxicity then used for splenic lymphocyte subpopulation analysis.
8 Tissue weighed and examined for 10/sex/group (males with the lowest identification number; females with the highest identification number.

Histology and Microscopic Examinations
All tissues denoted by E in Text Table 3.8 from Groups 1 and 4, were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
For at least 10 animals/sex from Groups 2 and 3, the mandibular and mesenteric lymph nodes, adrenals, thymus, spleen, and bone marrow (femur) were processed for immunotoxicity assessments. All macroscopic lesions were processed.
Additional sections of testes and epididymides were also stained with periodic acid Schiff (PAS) for spermatogenic staging for all males in Groups 1 and 4 and all decedents.
Qualitative assessment of spermatogenesis and testicular interstitial cell structure was performed for all males, where appropriate.
Qualitative assessment of the stages of follicular development to regression from small growing follicles to corpora lutea was performed in the ovaries for all females.
Due to suspected test article-related effects in Group 4, thyroids, liver (both sexes), ovaries (females), and spleens from remaining males and females, where not processed as part of immunotoxicity assessments, were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
All processed tissues were examined microscopically by the Study Pathologist.

Immunotoxicity Assessments
Prenatally and postnatally induced immunotoxic effects were evaluated by assessment of the mandibular and mesenteric lymph nodes, adrenals, thymus, spleen, and bone marrow (femur) from at least 10 animals/sex/group from all Cohort 1A groups.

Cohort 1B
Males were sacrificed by isoflurane anesthesia on Post Pairing Day 16 (Week 15), but food was not removed. Sacrifices were carried out in a controlled randomization order (i.e. one cage of animals from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed). Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
Females were sacrificed by isoflurane anesthesia on LD 4 (those that littered) or GD 26 (those with confirmation of mating that did not litter). Food was not removed prior to sacrifice. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
Females with the same day of mating/littering were sacrificed in a controlled randomization order (i.e. one animal from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed). Females that did not litter were sacrificed in animal identification order.
The number of implantation sites and corpora lutea for all paired females were recorded. The uterus of any apparently non-pregnant female was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.
Decedent animals were sacrificed by isoflurane anesthesia. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Tissues from decedent animals were retained as indicated in Text Table. Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated.
Upon sacrifice, macroscopic examinations were conducted and all lesions were recorded. Macroscopic examinations were performed under the general supervision of a pathologist.
Organ weights were recorded at each scheduled sacrifice, excluding decedents. Organs were dissected free from fat and other contiguous tissue and weighed before fixation. Left and right organs were weighed together. Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated.

Tissue List for Cohort 1B
Animal identification, epidudymis1,2 [WE], gross lesions [E], mammary gland [E], ovary [WE], oviduct [W3E], pituitary [WE], prostate [WE], seminal vesicle [WE], testis1,2[WE], uterus with cerviz [WE], vagnia [E].
E=Processed and examined microscopically
W=Weighed
1 Tissue weighed as a pair
2 Tissue taken into Modified Davidsons fixative and processed to at least the block stage
3 Weighed with uterus

3.13.2.1 Histology Processing and Microscopic Examinations
All tissues denoted by E in Text from males in Groups 1 through 4 that did not sire and females in Groups 1 through 4 that were not pregnant were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
Processed tissues were not examined microscopically by the Study Pathologist.
The ovary from all scheduled females in Groups 1 through 4 were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin. The tissues were examined microscopically by the Study Pathologist.
Postmortem examinations (offspring):
Pup Necropsy
Unselected pups on PND 22, and pups in a moribund condition were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Sacrifices were carried out in group order.
Full macroscopic examinations with particular attention to the reproductive genitals, were conducted for all decedents and pups sacrificed on PND 22.
Organ weights, were recorded from each pup sacrificed on PND 22, and were dissected free from fat and other contiguous tissue and weighed before fixation.
The following tissues from each pup sacrificed on PND 22 were preserved in 10% neutral buffered formalin. Tissues are retained until report finalization, then discarded.
Adrenal glands [W], brain [W], gross lesions, kidneys [W], mammary tissue, spleen [W], stomach, thymus [W].
W = Weighed

3.13.3 F2 Generation Pup Necropsy
Pups on PND 4 were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Sacrifices were carried out in a controlled randomization order, (i.e. one cage of animals from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed).
Macroscopic examinations of the abdominal cavity, adrenal, brain, gastrointestinal tract, gross lesions, head, kidney, leg, tail and thoracic cavity were conducted for all decedents and pups sacrificed on PND 4. Macroscopic examinations were performed under the general supervision of a pathologist.
The kidney weight was recorded for each pup sacrificed on PND 4, and was dissected free from fat and other contiguous tissue and weighed before fixation.
The adrenal glands, kidneys and animal identification from each pup sacrificed on PND 4 were preserved in 10% neutral buffered formalin. Tissues are retained until report finalization, then discarded.

3.13.4 Cohort 2A
Animals were sacrificed on Maturation Day 76, by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal; food was not removed. Sacrifices were carried out in a controlled randomization order (i.e. one cage of animals from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed).
Upon sacrifice, macroscopic examinations were conducted (which included all orifices, cranial cavity, external surfaces of the brain and spinal cord, cervical tissues and organs, thoracic, abdominal and pelvic cavities and viscera, external surface of the body and nasal cavity and paranasal sinuses), and all lesions were recorded. Macroscopic examinations were performed under the general supervision of a pathologist.
The brain from all animals was weighed and retained in 10% neutral buffered formalin, prior to fixed tissue dissection to remove tissues. Animals were subjected to whole body perfusion fixation with 50% Karnovsky’s fluid.

Tissue List for Cohort 2A
Olfactory bulb [H&E*; MSSa*], Forebrain (including hippocampus [H&E*; MSSa*], Basal ganglia (including caudate nucleus) [H&E*; MSSa*], Hypothalamus/thalamus [H&E*; MSSa*], Midbrain [H&E*; MSSa*], Brain stem (including pons and medulla oblongata) [H&E*; MSSa*], Cerebellum [H&E*; MSSa*], Cerebral cortex [H&E*; MSSa*], Entire spain cord (including cervical [to include enlargement at C4 level], thoracic and lumbar [to include enlargement at L4/5 level] regions) [H&E* – Left TS and LS from all three sections], Cervical dorsal root ganglia [H&E*; TB*† ], Lumbar dorsal root ganglia [H&E*; TB*†], Trigeminal ganglia [H&E*], Eye including retina [H&E*], Optic nerve [H&E*], Proximal and distal sciatic nerve [H&E* – Left TS and LS; TB – Right TS*† ], Proximal and distal tibial nerve [H&E* – Left TS and LS; TB – Right TS*† ], Sural nerve [H&E* – Left TS and LS; TB – Right TS*† ], Anterior tibialis muscle (left side) [H&E*], Gastrocnemius muscle (left side) [H&E*], Macroscopic lesions [H&E*]
H&E = Hematoxylin and eosin; TB = Toluene Blue; MSS = Myelin and Silver Stain; LS = Longitudinal section; TS = Transverse section.
a Luxol fast blue and Cresyl violet stains
The tissue listed were collected from all animals
The animal identification was also preserved

Histology and Microscopic Examinations
Those tissues indicated by * in Text were trimmed, processed, and embedded in paraffin wax (all animals); however, neuropathological evaluation by hematoxylin and eosin (H&E) was performed on Groups 1 and 4 only.
Those tissues indicated by † in Text were trimmed, post-fixed in osmium tetroxide, processed, and embedded in epoxy resin (all animals); however, neuropathological evaluation by toluidine blue was performed on Groups 1 and 4 only.

Cohort 2B
Animals were sacrificed on Maturation Day 22, by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Sacrifices were carried out in a controlled randomization order (i.e. one cage of animals from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed). Food was not removed.
Upon sacrifice, macroscopic examinations were conducted (which included all orifices, cranial cavity, external surfaces of the brain and spinal cord, cervical tissues and organs, thoracic, abdominal and pelvic cavities and viscera, and external surface of the body), and all lesions were recorded and retained in 10% neutral buffered formalin. Macroscopic examinations were performed under the general supervision of a pathologist.
The brain from all animals was weighed and then retained in 10% neutral buffered formalin, prior to fixed tissue dissection to remove tissues .


Tissue List for Cohort 2A
Olfactory bulb [H&E*; MSSa*], Forebrain (including hippocampus [H&E*; MSSa*], Basal ganglia (including caudate nucleus) [H&E*; MSSa*], Hypothalamus/thalamus [H&E*; MSSa*], Midbrain (including tectum, tegmentum and cerebral peduncles) [H&E*; MSSa*], Brain stem (including pons and medulla oblongata) [H&E*; MSSa*], Cerebellum [H&E*; MSSa*], Cerebral cortex [H&E*; MSSa*], Macroscopic lesions [H&E*]
H&E = Hematoxylin and eosin; MSS = Myelin and Silver Stain
a Luxol fast blue and Cresyl violet stains
The tissue listed were collected from all animals
The animal identification was also preserved

Histology and Microscopic Examinations
Those tissues indicated by * in Text were trimmed, processed, and embedded in paraffin wax (all animals); however, neuropathological evaluation by H&E was performed on Groups 1 and 4 only.
Statistics:
See below under "any other information"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):

Nonadverse, test article related clinical observations were confined to instances of salivation noted following administration of 100 mg/kg/day, with one instance also noted for one male administered 25 mg/kg/day. This was considered attributable to test article palatability and was not a toxic effect of the test article.
Instances of increased salivation were noted for 11 of 25 females administered 100 mg/kg/day. Isolated instances of increased salivation were noted for two males administered 100 mg/kg/day (Animal R0306 on Pre-Pairing Day 71 and Pairing Days 1 and 2; Animal R0321 on Pre-Pairing Days 57 and 60 to 65) and one male administered 25 mg/kg/day on Pre-Pairing Day 57 (Animal R0225). Such observations are occasionally observed following gavage administration of a slightly irritant or unpalatable test article formulation; as such, this finding was considered not to have represented toxicity.
Remaining clinical observations were generally associated with pelage changes or skin lesions which were evident throughout groups, with no dose relationship and are commonly observed in studies of this type. These were not related to the test article.
No observations were recorded postdose or during the weekly detailed clinical examinations.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Animal Fate
No test article-related unscheduled deaths occurred.
A total of seven unscheduled deaths occurred in the F0 generation. The cause of demise for these decedents was not attributed to the test article, and the findings noted in these animals were generally similar to those observed for animals that survived to the scheduled sacrifice.
One control female (Animal R0409) was sacrificed on Pre-Pairing Day 17 following clinical observations of labored and audible respiration. Macroscopic examinations revealed abnormal contents in the thoracic cavity and an esophageal rupture, which were consistent with gavage dosing trauma.
One female administered 25 mg/kg/day (Animal R0602) was sacrificed on Pre Pairing Day 21 following clinical observations of hunched posture, labored respiration, and decreased activity. Macroscopic findings included abnormal contents in the oral cavity, which were considered procedure related and not a consequence of the test article.
One male administered 25 mg/kg/day (Animal R0202) was sacrificed on Post-Pairing Day 24 following whole body convulsions that lasted less than 1 minute. The whole body convulsions were also observed on Post-Pairing Day 11. The animal was, therefore, sacrificed for humane reasons in accordance with test facility welfare standards. In isolation at this dose level, this was not test article related. One female administered 5 mg/kg/day (Animal R0522) was sacrificed on LD 19 following a whole body convulsion that lasted more than 1 minute. The animal was, therefore, sacrificed for humane reasons in accordance with test facility welfare standards. In the absence of a dose response, the isolated instances in these dose groups, was considered unrelated to the test article.
One female administered 5 mg/kg/day (Animal R0516) and one female administered 25 mg/kg/day (Animal R0609) were sacrificed on LD 0 following clinical observations of dystocia (difficulty in parturition). Macroscopic findings for Animal R0516 included abnormal contents in the stomach, with appearance of placental/pup tissue. The two incidences in two groups were isolated, and dystocia is occasionally observed in studies of this type and the finding was not observed in the high dose group; as such, in the absence of a dose response, these findings were considered incidental in nature, with no relationship to the test article.
One female administered 100 mg/kg/day (Animal R0715) was sacrificed on LD 1 following total postnatal litter loss. This is occasionally observed in studies of this type, and in isolation, was unrelated to the test article.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test article-related effects on mean body weight or body weight gain were noted.
The occasional statistically significant intergroup differences in mean body weight gains did not show any trends and were inconsistent across phases and sexes; as such, they were considered to have arisen incidentally, with no relationship to the test article.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test article-related effect on food consumption was observed.
The occasional statistically significant intergroup differences did not show any trends and were inconsistent across phases and sexes; as such, they were considered to have arisen incidentally, with no relationship to the test article.
Food efficiency:
no effects observed
Description (incidence and severity):
No overt differences in food conversion efficiency were evident for test article-treated animals, compared with controls.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related hematology changes were noted at any dose level.
White blood cells, specifically in the neutrophil, lymphocyte fractions, were marginally higher for all test article treated male groups, compared with controls; however, in the absence of a convincing dose response or the absence of similar changes noted for females, these intergroup differences were considered to have arisen incidentally and were not associated with the test article.
Females administered 100 mg/kg/day exhibited increased red cell distribution width and decreased packed cell volume (P< 0.05), compared with controls. Remaining red cell parameters were unaffected, and a similar change was not evident for males. In the absence of a clear hematopoietic effect, these intergroup differences were considered to have arisen incidentally and were not test article related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related clinical chemistry changes were noted at any dose level.
Total protein was significantly increased for males administered 100 mg/kg/day, compared with controls (P< 0.05). In the absence of any supporting data or similar changes noted for females, this was considered to have arisen incidentally, with no association with the test article.
Females administered 100 mg/kg/day were noted with decreased glucose levels on LD 22, compared with controls (P < 0.05). A similar change was not evident for F0 males. In the absence of any supporting data to suggest an effect of the test article, this was considered to have arisen incidentally.

Thyroid Hormone Analysis
No test article-related changes in T4 or TSH were noted at any dose level, compared with controls.
The statistically significant increase in TSH was observed for males administered 25 mg/kg/day, compared with controls, but was not supported by a concurrent and expected reduction in T4 levels, and similar changes were not evident for females. Furthermore, no thyroid weight changes were noted at this dose level, and, as such, these were considered to have arisen incidentally.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test article-related changes were noted in the urine parameters investigated.
Statistical analysis of the quantitative data did not show any statistically significant intergroup differences.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Upon microscopic examination, test article-related findings were recorded for the adrenal glands, ovary, thyroid glands, liver, spleen, and kidneys.
An increased incidence and severity of diffuse zona glomerulosa vacuolation was recorded for the adrenal glands of F0 animals from all test article-treated groups, compared with controls. A similar increased incidence and severity of diffuse zona fasciculata vacuolation was recorded for both sexes administered 25 or 100 mg/kg/day. Zona glomerulosa vacuolation was characterized by cells within this zone having an increased number and/or size of large, pale, cytoplasmic vacuoles, which increased the depth of this zone at a moderate grade. Zona fasciculata vacuolation was characterized at lower grades by cells within this zone with increased numbers of pale, cytoplasmic vacuoles and at higher grades by expansion of the zone by cells with large, pale, cytoplasmic vacuoles and/or large, clear vacuoles distorting the border with the zona reticularis. These findings generally correlated with the organ weight changes and macroscopic finding of large adrenal glands.
Slight to moderate ovarian interstitial cell vacuolation was recorded for all F0 females administered 25 or 100 mg/kg/day and was characterized by interstitial cells with moderate to large, pale, cytoplasmic vacuoles. This finding generally correlated with the changes in organ weights and organ weight ratios for females administered 100 mg/kg/day.
Follicular cell hypertrophy was recorded for the thyroid glands of males administered 5 mg/kg/day and both sexes administered 25 or 100 mg/kg/day (more notably in F0 males). Follicular cell hypertrophy was characterized by thyroid follicular cells with increased amounts of pale cytoplasm, without any associated microscopic changes in colloid production. This finding generally correlated with the changes in organ weights and organ weight ratios and the macroscopic finding of large thyroids.
In addition, cystic follicular cell hyperplasia was recorded for the thyroid gland from one male (Animal R0118) and one female (Animal R0723), administered 5 and 100 mg/kg/day, respectively, and a benign follicular cell adenoma was recorded for one F0 male administered 100 mg/kg/day (Animal R0309).
Centrilobular hepatocellular hypertrophy/eosinophilia was recorded for some F0 animals administered 100 mg/kg/day. This finding was characterized by centrilobular hepatocytes with increased amounts of eosinophilic cytoplasm, occasionally causing an increase in cellular size. This finding generally correlated with the changes in organ weights and organ weight ratios and the macroscopic finding of large liver.
An increase in the incidence and severity of pigmented macrophages was recorded for the spleen of males administered 25 or 100 mg/kg/day, compared with controls, and was characterized by macrophages with increased amounts of yellow-brown, granular pigment within the red pulp. Pigmented macrophages were noted in spleens from F0 males. This finding may be observed as a background change in rats. It was only observed in one sex in the F0 generation; as such, it was considered of unlikely toxicological significance, and, therefore, considered nonadverse.
An increased severity of renal hyaline droplet accumulation was recorded for F0 males administered 100 mg/kg/day, compared with controls. Hyaline droplet accumulations were characterized by renal cortical tubular cells with small to moderate amounts of granular, eosinophilic, cytoplasmic material, without any associated changes in cellular structure, tubular architecture, or inflammation. This finding generally correlated with the macroscopic finding of pale kidney.
No other test article-related microscopic findings were recorded for F0 animals.
Microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain, age, and at this stage of reproduction.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycles were unaffected by the test article at any dose level prior to pairing or prior to scheduled sacrifice on Lactation Day 22.
Statistical analysis of the data did not show any statistically significant intergroup differences.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related effects were noted on sperm analysis or sperm morphology parameters at any dose level.
Straightness of sperm was significantly lower for males administered 100 mg/kg/day, compared with control males (P < 0.001), with mean values outside the HCD range (65 to 73%). Straight line velocity was also reduced by 11%, compared with controls, although mean values did not attain statistical significance and were within the HCD range (85.4 to 118.2 um/sec). These reductions were due to two zero (0) values for Animals R0302 and R0310) and the mated female partners achieved a pregnancy, therefore, and as such, did not represent an effect of the test article.
Caudal epididymis weights used for sperm analysis were statistically significantly lower than those of controls (P < 0.01), although mean values were within the HCD range (0.224 to 0.270 g). Individual data across groups showed some values that were higher and lower than the HCD range; five control values were lower than the HCD and six control values were higher than the HCD range, which may have resulted in the statistically significant intergroup differences. In the absence of any supporting testes or epididymis weight changes at the terminal sacrifice, this change in caudal weights was considered to have arisen incidentally and unrelated to the test article.
Total abnormal sperm counts were increased in a dose-related manner for all male groups, compared with controls, although a large standard deviation was evident and resulted in a lack of statistical significance in the group administered 100 mg/kg/day. Statistically significant increases in percentage abnormal sperm were evident following administration of 5 or 25 mg/kg/day, compared with controls (P < 0.01 or P < 0.001, respectively). The percentage change in the group administered 100 mg/kg/day was also increased in a dose related manner, although the large standard deviation resulted in a lack of statistical significance at this dose level. The number of normal sperm was unaffected at any dose level; as such, these intergroup differences were considered to have arisen incidentally.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
A test article-related reduction in fertility was evident following administration of 100 mg/kg/day, compared with controls. Fertility index was reduced, compared with controls, and was slightly lower than the historical control data (HCD) range (85 to 100%), although values did not attain statistical significance, compared with controls.
Following mating, 23, 23, 23, and 21 females administered control article (vehicle) or 5, 25, or 100 mg/kg/day, respectively, were pregnant.
No test article-related effects on mating performance were noted. The majority of animals mated within 4 days of pairing.
The lower fertility index observed following administration of 100 mg/kg/day, resulted in a lower fecundity index, compared with controls, although the value was within the HCD range (85 to 100%); as such, this was considered not to have represented an effect of the test article. Of the four non-pregnant females from the group administered 100 mg/kg/day, Animals R0718, R0721, and R0722 mated on the day of pairing. Animal R0718 was in estrus from the previous night, therefore it was considered likely that a decaying estrus was the cause of the lack of pregnancy following mating and unrelated to the test article. Animal R0716 mated on the second day of pairing. One, two, and one non-pregnant females, respectively, were in the groups administered control article (vehicle) or 5 or 25 mg/kg/day.

Parturition and Litter Data
Following parturition, 23, 22, 22, and 21 females administered control article (vehicle) or 5, 25, or 100 mg/kg/day, respectively, produced a live litter.
Adverse test article-related increased numbers of stillborn pups were noted following administration of 100 mg/kg/day compared with controls. However, there was no effect of the test article on implantation losses in utero or on pup survival after PND 1 until weaning; values were similar to controls. A total of 14 stillborn pups were observed in litters from the group administered 100 mg/kg/day, compared with controls. Although values did not attain statistical significance, the number of stillborn pups was outside the HCD range (0 to 6 pups). These stillborn pups were from five litters of the group administered 100 mg/kg/day; this number was also outside the HCD range (0 to 4 litters).
Two stillborn pups from one control article (vehicle) litter, seven stillborn pups from two 5 mg/kg/day litters and five stillborn pups from two 25 mg/kg/day litters were observed. Although a dose response was evident following administration of 25 mg/kg/day, these numbers were within the historical control data ranges, and, as such, were considered to have arisen incidentally.
No effect on the duration of gestation was noted. Two females (Animal R0516 administered 5 mg/kg/day and Animal R0609 administered 25 mg/kg/day), were sacrificed early due to clinical observations of dystocia, and the number of pups born was small, however at necropsy, no fetuses were found in utero. In the absence of a dose response and no increase in the duration of gestation, these findings were considered related to difficulties during parturition for these females and unrelated to test article administration.
The increased incidence of stillborn pups, resulted in a lower live birth index for litters of the group administered 100 mg/kg/day, compared with controls, although mean values were within the HCD range (92.9 to 100%).

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F1 Generation Offspring Observations (Pre-weaning)
Pup Clinical Observations
No test article-related clinical observations were noted for pups from all groups.
The incidence of missing pups was slightly higher in litters of the group administered 100 mg/kg/day, compared with controls (four versus one), although this was within the HCD range (0 to 7 pups) and, as such, was considered incidental.
No postdose observations were noted following direct dosing from PND 14.

F1 Generation
No postdose observations were noted.
No test article-related clinical observations or weekly detailed observations were noted.
Excessive salivation was observed during the maturation phase for two females administered 100 mg/kg/day (Cohort 1A Animal R1502 on Maturation Day 77, Animal R1503 on Maturation Day 59). These were isolated incidences and, taking into the consideration the duration of the dosing phase, were considered to have arisen incidentally.
Detailed weekly clinical observations showed isolated incidences of high stepping gait, generally for females, with incidences also noted for controls. This was evident on occasion during the maturation, pairing, and gestation phases. Isolated incidences of elevated tail were noted for a few animals in each group, including controls. In the absence of a convincing dose relationship, these findings were considered transient in nature as they were not evident on each weekly testing occasion; as such, no relationship to the test article was noted.

F2 Pup
No test article-related clinical observations were noted for pups at any dose level.
The minor observations noted were considered low incidence findings, and, as such, were considered to have arisen incidentally.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
F1 Generation Offspring Observations (Pre-weaning)
No test article-related effect on postnatal pup survival was observed.

F1 Generation
No test article-related unscheduled deaths occurred.
One Cohort 1A male administered 100 mg/kg/day (Animal R1103) was found dead on PND 22. A cause of demise was not determined for this animal as it was not examined microscopically; however, the macroscopic finding was consistent with findings in rats of this strain and age and, in isolation, this death was considered to have arisen incidentally, with no relationship to the test article.
One Cohort 1B male administered 5 mg/kg/day (Animal R0928) was sacrificed on PND 71 following a convulsion that lasted more than 1 minute. The cause of demise was not confirmed as it was not examined microscopically. Another Cohort 1B male administered 5 mg/kg/day (Animal R0931) was sacrificed on Pairing Day 14. This was considered attributable to a gavage dosing error, as confirmed by esophageal rupture of the lung and fluid present in the thoracic cavity upon macroscopic examination.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
F1 Generation Offspring Observations (Pre-weaning)
No test article-related effects on pup body weights were noted at any dose level, compared with controls.
Statistical analysis of the data did not show any statistically significant intergroup differences.

F1 Generation
No test article-related effects on body weights or body weight gains were observed.
Mean body weights on PND 22 were essentially similar across groups and remained similar throughout the dosing period.
Mean body weights were essentially similar to control for females during gestation; however, mean body weight gain was higher for all test article-treated groups over the lactation phase (LD 1 to 4), and a dose relationship was evident. However, this was most likely attributable to variation within the control group, as evidenced by a larger standard deviation value, compared with remaining groups.

F2 Pup
No test article-related effect on pup body weights was evident on PND 1 and 4.
Statistical analysis of the data did not reveal any significant intergroup differences.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
F1 Generation
No test article-related effects on food consumption were observed at any dose level.
Statistical analysis of the data did not reveal any statistically significant intergroup differences.
Food efficiency:
no effects observed
Description (incidence and severity):
No overt differences in food conversion efficiency were evident for test article-treated animals, compared with controls.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 Generation
No test article-related hematology changes were noted.
Statistically significant increases in white cell counts were evident for females at all dose levels and were supported by statistically significant increases in lymphocyte counts for females administered 25 or 100 mg/kg/day, compared with controls, and a similar trend was evident for males. A similar pattern was evident following the immunophenotyping from the spleen. However, a dose response was not evident, and in the absence of any microscopic correlates, these increases were considered not to have represented an immune response and were, therefore, nonadverse in nature.
Females administered 100 mg/kg/day were noted with an increase in fibrinogen, compared with controls (P < 0.05). This was an isolated finding and, in the absence of similar changes for males, was considered to have arisen incidentally.
Females administered 25 or 100 mg/kg/day were noted with a dose-related increase in percentage red cell distribution width (RDWG %), compared with controls (P < 0.05). In the absence of any supporting changes in red cell mass, and because similar changes not observed for the males, these intergroup differences were considered to have arisen incidentally and were unrelated to the test article.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
F1 Generation Offspring Observations (Pre-weaning)
No test article-related changes in T4 or TSH were evident following maternal or direct administration of the test article at any dose level, compared with controls.
Statistical analysis of the data did not show any statistically significant intergroup differences. Marginal increases in TSH were not accompanied by a reduction in T4 levels, and were, therefore, considered to have arisen incidentally.

F1 Generation
Test article-related increases in globulin levels were noted for animals administered 100 mg/kg/day and males administered 25 mg/kg/day and increases in cholesterol levels were noted for females administered 100 mg/kg/day, compared with controls.
Total protein levels were elevated for females administered 100 mg/kg/day (P < 0.05), characterized by an increase in globulin level (P < 0.01). A:G ratio was consequently reduced (P < 0.01), compared with controls.
Globulin levels were increased for males administered 25 or 100 mg/kg/day (P < 0.01 or P < 0.05, respectively), and A:G ratio was reduced, although statistical significance was only attained following administration of 25 mg/kg/day, with no associated increase in total protein. These were considered to be nonadverse, test article-related changes.
Cholesterol levels were increased for females administered 100 mg/kg/day, compared with controls (P < 0.01). A similar change was evident in a previous 90-day toxicity study (MPI Research Study 399-241; MPI Research, 2015), and, as such, was considered test article related.
Reduced glucose levels were evident for males administered 100 mg/kg/day, compared with controls (P< 0.01). This was not evident for F1 females; as such, it was considered to have arisen incidentally.

F1 Generation: Thyroid Hormone Analysis
No test article-related TSH and T4 changes were noted at any dose level.
The statistically significant increase in TSH for males administered 25 mg/kg/day did not show an expected reduction in T4 levels. Furthermore, a similar change was not evident for females or at the high dose level and was not supported by any thyroid weight changes; as such, this was considered to have arisen incidentally, with no relationship to the test article.
Urinalysis findings:
no effects observed
Description (incidence and severity):
F1 Generation
No test article-related changes were evident in urine parameters measured.
Statistical analysis of the quantitative data did not show any statistically significant intergroup differences.
Sexual maturation:
no effects observed
Description (incidence and severity):
No test article-related effects on age of sexual maturity or body weights at attainment, were evident at any dose level.
Statistical analysis of the data did not reveal any statistically significant intergroup differences.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
F1 Generation Offspring Observations (Pre-weaning)
No test article related effects on anogenital distance were noted for males or females from all test article-exposed litters, compared with controls.
Statistical analysis of the data did not show any statistically significant intergroup differences.

F2 Pup
No test article-related effects were evident on anogenital distance.
Statistical analysis of the data did not reveal any statistically significant intergroup differences.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
F1 Generation Offspring Observations (Pre-weaning)
Nipple retention was not observed for any male at any dose level.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1 Generation Offspring Observations (Pre-weaning)
No test article-related organ weight changes were recorded for PND 22 offspring from F0 animals, compared with controls.
Organ weight and organ weight ratio changes for these animals were attributed to biological variation.

F2 Pup
No changes in kidney weight or kidney:body weight ratio were noted for F2 offspring from test article-treated F1 animals.
Changes in organ weights and organ weight ratios for these animals were attributed to biological variation.

F2 Pup
Test article-related changes in organ weights and organ weight ratios were recorded for the adrenal glands and liver of Cohort 1A animals. Test article-related ovary weight changes were recorded for Cohort 1A and 1B females.
Increased adrenal weights and/or adrenal weight ratios and liver weights and/or liver weight ratios were recorded for both sexes administered 100 mg/kg/day, compared with concurrent controls. Increased ovarian weights and ovarian weight ratios were recorded for F1 Cohort 1A females administered 100 mg/kg/day, compared with concurrent controls. The observed changes generally correlated with findings noted macroscopically and/or microscopically.
Test article-related increases in ovarian weights and ovarian weight ratios were recorded for F1 Cohort 1B females administered 25 or 100 mg/kg/day, compared with concurrent controls, which generally correlated with microscopic findings .
All other organ weight and organ weight ratio changes noted for Cohort 1A and 1B animals were attributed to normal biological variation and were considered not test article related as they were small in magnitude, not dose-dependent, inconsistent between sexes, attributed to normal inter-animal variability, and/or lacked a microscopic correlate.
No test article-related brain weight changes were recorded for Cohort 2A and 2B animals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 Generation Offspring Observations (Pre-weaning)
No test article-related macroscopic abnormalities were noted for any tissues on PND 22.
An increase in the number of stillborn pups was noted for F1 offspring (more notable in female pups) from F0 females administered 100 mg/kg/day, compared with controls. However, no consistent, associated macroscopic findings were noted to suggest an underlying cause.
Most tissues in pups that died or were sacrificed between PND 0 and 22 were macroscopically unremarkable, and/or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age and at this stage of development.

F2 Pup
No test article-related macroscopic findings were recorded for F2 offspring from test article-treated F1 animals.
Most tissues in pups that died or were sacrificed between PND 0 and 4 were macroscopically unremarkable and/or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age and at this stage of development.

Test article-related macroscopic findings were noted in the adrenal glands, liver, and ovaries of Cohort 1A animals.
Large adrenal glands and large ovaries were noted in F1 Cohort 1A females administered 100 mg/kg/day, which generally correlated with increased organ weights/weight ratios and microscopic findings.
Large liver was noted for F1 Cohort 1A males administered 100 mg/kg/day, which also generally correlated with increased organ weights/weight ratios and microscopic findings.
No other test article-related macroscopic findings were recorded.
Other tissues from Cohort 1A animals were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.
No test article-related macroscopic findings were recorded for Cohort 1B animals. Tissues were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain, age, and at this stage of reproduction.
No test article-related macroscopic findings were recorded for Cohort 2A or 2B animals. Most tissues were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test article-related microscopic findings were recorded for the adrenal glands, thyroid glands, and liver of Cohort 1A animals. Test article-related microscopic changes were also recorded for the ovaries of F1 Cohort 1A and 1B females.
An increased incidence and severity of diffuse zona glomerulosa vacuolation of the adrenal gland was recorded for F1 Cohort 1A animals administered 25 or 100 mg/kg/day, compared with controls. A similar increased incidence and severity of diffuse zona fasciculata vacuolation was recorded for F1 Cohort 1A animals from all test article-treated groups. Zona glomerulosa vacuolation was characterized by cells within this zone having an increased number and/or size of large, pale, cytoplasmic vacuoles, which increased the depth of this zone at a moderate grade. Zona fasciculata vacuolation was characterized at lower grades by cells within this zone with increased numbers of pale, cytoplasmic vacuoles and at higher grades by expansion of the zone by cells with large, pale, cytoplasmic vacuoles and/or large, clear vacuoles distorting the border with the zona reticularis. These findings generally correlated with the organ weight changes and macroscopic findings.
An increased incidence and severity of follicular cell hypertrophy was recorded for the thyroid glands of males administered 25 mg/kg/day and both sexes administered 100 mg/kg/day, more notably in F1 Cohort 1A males. Follicular cell hypertrophy was characterized by thyroid follicular cells, with increased amounts of pale cytoplasm, without any associated microscopic changes in colloid production.
Centrilobular hepatocellular hypertrophy/eosinophilia was recorded for both sexes administered 100 mg/kg/day. Animals administered 5 or 25 mg/kg/day were generally comparable with controls. This finding was characterized by centrilobular hepatocytes with increased amounts of eosinophilic cytoplasm, occasionally causing an increase in cellular size.
Slight to marked ovarian interstitial cell vacuolation was recorded for all F1 Cohort 1A females administered 25 or 100 mg/kg/day. Interstitial cell vacuolation was characterized by ovarian interstitial cells with moderate to large, pale, cytoplasmic vacuoles and generally correlated with the organ weight changes and macroscopic finding of large ovaries.
No other test article-related microscopic findings were recorded for F1 Cohort 1A animals. Microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain, age, and at this stage of development.
Minimal to marked ovarian interstitial cell vacuolation was noted for all terminal sacrifice F1 Cohort 1B females administered 25 or 100 mg/kg/day, which generally correlated with the observed organ weight and organ weight ratio changes recorded for this cohort.
No other tissues were examined from terminal sacrifice F1 Cohort 1B females.
In addition, moderate interstitial cell vacuolation was noted for the ovaries of one F1 Cohort 1B female administered 25 mg/kg/day, and slight to moderate interstitial cell vacuolation was noted in the ovaries from three F1 Cohort 1B females administered 100 mg/kg/day, which were examined due to failure to conceive. This finding generally correlated with the organ weight and weight ratio changes for these groups.
No other test article-related microscopic findings were recorded for the limited tissue list examined in F1 Cohort 1B females that did not conceive, as microscopic findings in the examined reproductive tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain, age, and at this stage of reproduction.
No test article-related microscopic findings were recorded for Cohort 2A and 2B animals. Microscopic findings were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
F1 Generation
Estrous Cycles
No test article-related effects on the number or duration of the estrous cycles were evident.
The statistically significant increase in mean cycle length for females administered 25 mg/kg/day (P < 0.01) did not show an expected reduction in the number of cycles, and in the absence of a dose response, was considered to have arisen incidentally.

Fertility and Reproductive Performance
No test article-related effects on mating were evident at any dose level.
All animals mated, with the majority of females within each dose group mating within the first 6 days of pairing.
Fertility and fecundity indices were slightly lower following administration of 100 mg/kg/day, compared with controls. Seventeen of 20 females administered 100 mg/kg/day were pregnant, compared with 19 of 20 from each of the remaining groups. This resulted in lower fecundity and fertility indices at this dose level, which were just within the HCD range for both indices (85 to 100%).
Three non-pregnant females were noted in the group administered 100 mg/kg/day (Animals R1530, R1533, and R1535), compared with one each from the remaining groups. Animals R1530 and R1535 mated on the day of pairing and were in estrus from the previous night, therefore it was considered likely that a decaying estrus was the cause of the lack of pregnancy following mating for these animals, and unrelated to the test article. One female administered 25 mg/kg/day (Animal R1440) mated on Pairing Day 9. The remaining non-pregnant animals did not mate on the first day of pairing and were not in estrous on the day of mating; as such, the slightly higher non pregnancy rate in the group administered 100 mg/kg/day was considered to have arisen incidentally and was not related to the test article as non-pregnancies are occasionally observed in studies of this type.
One control female (Animal R1239) was pregnant but had a total in utero litter loss, which is occasionally observed in studies of this type.

Parturition and Litter Data
Parturition, gestation length, and gestation index were unaffected.
Live birth index and prenatal and postnatal pup survival were unaffected. No stillborn pups were evident following administration of 5 or 100 mg/kg/day. Four stillborn pups were evident from two control litters and two stillborn pups observed from two litters from the group administered 25 mg/kg/day. Pup mortality (missing or dead) was limited to four, one, two, or four pups from groups administered control article (vehicle) or 5, 25, or 100 mg/kg/day, respectively; therefore, the PND 4 Viability index was similar between control and the group administered 100 mg/kg/day. One control female (Animal R1239) was sent to necropsy on GD 24. This animal had an in utero total litter loss. No total postnatal litter losses were evident at any dose level.
Litter size and sex ratio were unaffected by the test article.
No test article-related effect on the number of implantation sites was evident. Post implantation loss was higher following administration of 100 mg/kg/day, compared with controls (mean number 1.71 and 14.16% for the group administered 100 mg/kg/day, compared with 0.94 or 8.57% for controls), although these values were within the historical control data range (0.23 to 2.67 implantations losses; 1.7 to 19.83 post implantation loss) and, as such, were considered to have arisen incidentally.
A total of 18, 19, 19, or 17 females administered control article (vehicle) or 5, 25, or 100 mg/kg/day, respectively, survived to their scheduled sacrifice.

F1 Generation
Seminology Evaluations
No test article-related changes in sperm analysis or sperm morphology parameters were observed at any dose level.
A statistically significant reduction in sperm motility was evident for males administered 100 mg/kg/day, compared with control (P < 0.05), although mean values were within the historical control data range (66 to 93%), which was attributable to seven lower than expected values at this dose level, compared with three low and two high values from the control group. In the absence of any supporting changes to suggest an effect on sperm, this intergroup difference was considered to have arisen incidentally and was not associated with the test article.

Ovarian Follicle Count Evaluations
Test article-related reductions in ovarian follicle counts, primarily primordial follicles, were evident following administration of 25 or 100 mg/kg/day, compared with controls.
Small reductions in primordial ovarian follicles were evident for F1 females administered 25 or 100 mg/kg/day, compared with controls, although a dose relationship was not evident. This resulted in statistically significant reductions in total follicle counts (P < 0.05) following administration of 25 or 100 mg/kg/day, compared with controls.
Small reductions in primordial ovarian follicles were evident following administration of 5 mg/kg/day, compared with controls, although statistical significance was not attained for the overall ovarian counts, and as such, this was considered to be incidental and unrelated to the test article.

Brain Histomorphometry Evaluations
No test article-related changes in brain histomorphometry were noted on PND 22 (Cohort 2B) or PND 76 (Cohort 2A) for animals administered 100 mg/kg/day, compared with controls.
The statistically significant changes in some histomorphometry measurements between controls and animals administered 100 mg/kg/day generally affected different brain regions and were inconsistent between sexes and between F1 Cohort 2A and Cohort 2B animals. Outliers within the data were noted and were probably due to slightly oblique or damaged sections. Therefore, in the absence of behavioral or clinical neurological observations and no corresponding macroscopic or microscopic findings, these changes were considered to have arisen incidentally, with no relationship to the test article.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
F2 Pup
Functional Observational Battery
No test article-related effects were observed following the FOB assessments.
Statistical analysis of the quantitative data did not reveal any statistically significant intergroup differences.

Motor Activity
No test article-related effects on locomotor activity parameters were evident.
The statistically significant intergroup differences detected for males, females, and combined sex throughout groups, compared with controls, did not show any dose relationship, and, in the absence of any supporting data to suggest a neurotoxic effect of the test article, were considered to have arisen incidentally.

Acoustic Startle
No test article-related deficits in pre-pulse inhibition or startle habituation were noted, compared to controls.
The statistically significant increase in startle habituation in 1 of 5 blocks (Block 3) for males administered 5 mg/kg/day, compared with controls, was minimal (P< 0.05) and were not part of a dose response; as such, the increase was considered to have arisen incidentally

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
effects observed, treatment-related
Description (incidence and severity):
F2 Pup
An immunotoxic response was not evident at any dose level.
Test article-related increases in T lymphocytes, Helper T lymphocytes, Cytotoxic T lymphocytes, and B lymphocyte populations were evident at all dose levels. These increases, however, did not show a dose response and, at the magnitude observed, were considered not immunotoxic and, therefore, nonadverse in nature.
In F1 Cohort 1A animals administered 5, 25, or 100 mg/kg/day, total T lymphocytes (CD3+) and Helper T lymphocytes (CD3+CD4+) had slight to marked increases in absolute numbers on Maturation Days 92, 3, 4, or 5, compared to controls. The latter effects were not consistently reflected within the relative frequencies or were dose dependent in nature. A similar level of a test article-related increase was also observed in the Cytotoxic T lymphocyte population (CD3+CD8+), compared to controls, and the highest increase in absolute numbers was noted in F1 Cohort 1A animals administered 100 mg/kg/day, although a dose-dependent effect was observed for this population in males only.
The spleen natural killer (NK) lymphocytes (CD3-CD161a+) of Reofos 35-treated F1 Cohort 1A animals were generally comparable to those of controls. Moderate increases in absolute NK numbers (and slight increases in relative frequencies) were observed in F1 Cohort 1A females administered 5 mg/kg/day and were associated with high inter-animal variability and considered not biologically significant.
Splenic B lymphocyte (CD45RA+) absolute numbers (and, for the most part, relative frequencies) were slightly to moderately increased in F1 Cohort 1A males administered >5 mg/kg/day, compared to controls. Similarly, B lymphocyte absolute numbers and relative frequencies typically increased to a slight extent in F1 Cohort 1A females administered >5 mg/kg/day, compared to controls, with the exception of B lymphocyte numbers in F1 Cohort 1A females administered 25 mg/kg/day, which were comparable to those of controls. Aforementioned differences in B lymphocyte populations of Reofos 35-treated groups and controls were independent of dose.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental neurotoxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
neuropathology
histopathology: non-neoplastic
developmental immunotoxicity
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

Any other information on results incl. tables

F0 Generation


Summary incidence of the stage of estrus on Lactation Day 22




































 



Diestrus



Estrus



Proestrus



Control



19



4



0



5 mg/kg/day



16



3



2



25 mg/kg/day



20



0



1



100 mg/kg/day



18



1



1



 


Summary of Organ Weight % Difference Compared with Concurrent Controls – Terminal Sacrifice







































































































































 



 



Males



Females



 



 



2M



3M



4M



2F



3F



4F



Tissue



Level (mg/kg/day)



5



25



100



5



25



100



 



 



% difference



Adrenal



Group mean absolute weights



0.147



8.516



16.630***



-1.741



13.564*



32.485***



Body weight ratio



-1.475



8.055



17.507***



1.740



19.127**



33.431***



Thyroid/parathyroid



Group mean absolute weights



8.830



7.161



22.382***



-1.177



-8.101



-3.836



Body weight ratio



6.418



6.293



23.222***



1.331



-4.480



-3.608



Liver



Group mean absolute weights



3.863



7.797**



19.194***



-2.322



-2.129



1.316



Body weight ratio



1.461



7.353**



19.622***



0.374



2.232



2.042



Kidney



Group mean absolute weights



2.162



2.914



6.837



-2.076



-2.905



2.522



Body weight ratio



-0.202



2.236



6.919*



1.045



1.581



3.717



Ovary



Group mean absolute weights



-



-



-



-7.985



1.689



34.323***



Body weight ratio



-



-



-



-5.332



5.884



34.881***



F = Female; M = Male


* = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001


Note: Organ weights were not taken from decedents or excluded animals, based on reproductive status



 


Incidence of Selected Findings: Adrenal Gland – Terminal Sacrifice
























































































































































 



 



Males



Females



 



 



1M



2M



3M



4M



1F



2F



3F



4F



Tissue and finding



Level (mg/kg/day)



0



5



25



100



0



5



25



100



Adrenal gland



No. examined



25



25



24



25



23



21



21



20



Vacuolation, zona glomerulose, increased, diffuse



Grade -



20



11



2



2



19



9



0



0



1



5



13



10



5



4



9



5



0



2



0



1



12



15



0



3



12



12



3



0



0



0



3



0



0



4



8



Vacuolation, zona fasciculata, increased diffuse



Grade-



16



11



1



0



23



21



4



0



1



9



13



9



4



0



0



11



6



2



0



1



12



7



0



0



6



10



3



0



0



2



12



0



0



0



4



4



0



0



0



2



0



0



0



0



- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; 4 = Marked; F = Female; M = Male



 


Incidence of Selected Findings: Ovary – Terminal Sacrifice











































































 



 



Females



 



 



 



1F



2F



3F



4F



 



Tissue and finding



Level (mg/kg/day)



0



5



25



100



 



Ovary



No. examined



23



21



21



20



 



Vacuolation, interstitial cell



Grade -



23



21



1



0



 



1



0



0



5



0



 



2



0



0



12



6



 



3



0



0



3



14



 



- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; 4 = Marked; F = Female



 


Incidence of Selected Findings: Thyroid Gland – Terminal Sacrifice





















































































 



 



Males



Females



 



 



1M



2M



3M



4M



1F



2F



3F



4F



Tissue and finding



Level (mg/kg/day)



0



5



25



100



0



5



25



100



Thyroid Gland



No. examined



25



25



24



25



23



21



21



20



Hypertrophy, follicular cell



Grade -



25



19



6



5



23



21



16



15



1



0



5



16



6



0



0



5



4



2



0



1



2



14



0



0



0



1



- = Finding not present; 1 = Minimal; 2 = Slight; F = Female; M = Male



 


Incidence of Selected Findings: Liver – Terminal Sacrifice





















































































 



 



Males



Females



 



 



1M



2M



3M



4M



1F



2F



3F



4F



Tissue and finding



Level (mg/kg/day)



0



5



25



100



0



5



25



100



Liver



No. examined



25



25



24



25



23



21



21



20



Hypertrophy/eosinophilia, hepatocyte, centrilobular



Grade -



25



25



24



19



22



21



20



12



1



0



0



0



3



0



0



1



4



2



0



0



0



3



1



0



0



4



- = Finding not present; 1 = Minimal; 2 = Slight; F = Female; M = Male



 


Incidence of Selected Findings: Spleen – Terminal Sacrifice
































































































 



 



Males



Females



 



 



1M



2M



3M



4M



1F



2F



3F



4F



Tissue and finding



Level (mg/kg/day)



0



5



25



100



0



5



25



100



Spleen



No. examined



25



25



24



25



23



21



21



20



Pigmented macrophage, increased



Grade -



5



6



1



0



1



5



2



1



1



14



13



7



7



13



8



9



8



2



6



5



15



13



9



7



8



9



3



0



1



1



5



0



1



2



2



- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; F = Female; M = Male



 


Incidence of Selected Findings: Kidney – Terminal Sacrifice





















































































 



 



Males



Females



 



 



1M



2M



3M



4M



1F



2F



3F



4F



Tissue and finding



Level (mg/kg/day)



0



5



25



100



0



5



25



100



Kidney



No. examined



25



25



24



25



23



0



0



20



Accumulation, hyaline droplet



Grade -



0



1



0



0



23



 



 



1



1



20



24



23



4



0



 



 



0



2



5



0



1



21



0



 



 



0



- = Finding not present; 1 = Minimal; 2 = Slight; F = Female; M = Male



 


Summary of Organ Weight % Difference Compared with Concurrent Controls – Terminal Sacrifice (Cohort 1A)




























































































































 



 



Males



Females



 



 



2M



3M



4M



2F



3F



4F



Tissue



Level (mg/kg/day)



5



25



100



5



25



100



 



 



% difference



Adrenal



Group mean unadjusted weights



2



1



8



-8



5



24***



Body weight ratio



2



3



12*



-6



7



28***



Brain weight ratio



3



3



8



-7



7



25***



Liver



Group mean unadjusted weights



2



6



15***



-3



0



10



Body weight ratio



2



8



20***



0



3



13***



Brain weight ratio



3



7



15***



-1



3



11*



Ovary



Group mean unadjusted weights



-



-



-



14



7



28***



Body weight ratio



-



-



-



18



10



32***



Brian weight ratio



-



-



-



15



10



29***



F = Female; M = Male


* = P ≤ 0.05; *** = P ≤ 0.001


Note: Organ weights were not taken from decedents.



 


Summary of Organ Weight % Difference Compared with Concurrent Controls – Terminal Sacrifice (Cohort 1B)














































 



 



Females



 



 



2F



3F



4F



Tissue



Level (mg/kg/day)



5



25



100



 



 



% difference



Ovary



Group mean unadjusted weights



9



15*



19**



Body weight ratio



11



19**



20**



F = Female


* = P ≤ 0.05; ** = P ≤ 0.01


Note: Organ weights were not taken from decedents or excluded animals.



 


Incidence of Selected Findings: Adrenal Gland – Terminal Sacrifice (Cohort 1A)













































































































































 



 



Males



Females



 



 



1M



2M



3M



4M



1F



2F



3F



4F



Tissue and finding



Level (mg/kg/day)



0



5



25



100



0



5



25



100



Adrenal gland



No. examined



20



12



13



20



20



11



16



20



Vacuolation, zona glomerulosa, increased, diffuse



Grade -



12



1



0



1



14



5



1



0



1



8



9



8



7



6



6



7



2



2



0



1



5



12



0



0



7



18



3



0



1



0



0



0



0



1



0



Vacuolation, zona fasciculata, increased diffuse



Grade-



20



6



3



0



18



6



1



0



1



0



5



5



7



2



5



5



1



2



0



1



5



12



0



0



10



12



3



0



0



0



1



0



0



0



7



- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; F = Female; M = Male



 


Incidence of Selected Findings: Thyroid Gland – Terminal Sacrifice (Cohort 1A)





















































































 



 



Males



Females



 



 



1M



2M



3M



4M



1F



2F



3F



4F



Tissue and finding



Level (mg/kg/day)



0



5



25



100



0



5



25



100



Thyroid Gland



No. examined



20



20



20



20



20



20



20



20



Hypertrophy, follicular cell



Grade -



18



18



10



6



20



19



19



13



1



2



2



8



7



0



1



1



6



2



0



0



2



7



0



0



0



1



- = Finding not present; 1 = Minimal; 2 = Slight; F = Female; M = Male



 


Incidence of Selected Findings: Liver – Terminal Sacrifice (Cohort 1A)





















































































 



 



Males



Females



 



 



1M



2M



3M



4M



1F



2F



3F



4F



Tissue and finding



Level (mg/kg/day)



0



5



25



100



0



5



25



100



Liver



No. examined



20



20



20



20



20



20



20



20



Hypertrophy/eosinophilia, hepatocyte, centrilobular



Grade -



17



16



14



2



20



20



17



5



1



3



4



6



15



0



0



3



8



2



0



0



0



3



0



0



0



7



- = Finding not present; 1 = Minimal; 2 = Slight; F = Female; M = Male



 


Incidence of Selected Findings: Ovary – Terminal Sacrifice (Cohort 1A)



















































































 



 



Females



 



 



 



1F



2F



3F



4F



 



Tissue and finding



Level (mg/kg/day)



0



5



25



100



 



Ovary



No. examined



20



20



20



20



 



Vacuolation, interstitial cell



Grade -



20



19



0



0



 



1



0



1



3



0



 



2



0



0



14



1



 



3



0



0



3



12



 



4



0



0



0



7



 



- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; 4 = Marked; F = Female



 


Incidence of Selected Findings: Ovary – Terminal Sacrifice (Cohort 1B)



















































































 



 



Females



 



 



 



1F



2F



3F



4F



 



Tissue and finding



Level (mg/kg/day)



0



5



25



100



 



Ovary



No. examined



18



19



19



17



 



Vacuolation, interstitial cell



Grade -



18



19



0



0



 



1



0



0



7



0



 



2



0



0



12



3



 



3



0



0



0



8



 



4



0



0



0



6



 



- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; 4 = Marked; F = Female



 


 

Applicant's summary and conclusion

Conclusions:
Following, once daily oral gavage administration of 5, 25, or 100 mg/kg/day Reofos 35 to Han Wistar rats for up to two consecutive generations, test article related effects were noted at all dose levels investigated.
Fertility was reduced following administration of 100 mg/kg/day to the F0 generation, which was accompanied by an increased incidence of stillborn pups and litters with stillborn pups at this dose level. Fertility was also slightly reduced for the F1 generation following administration of 100 mg/kg/day. Furthermore, microscopic examination of the ovaries revealed interstitial cell vacuolation for F0 and F1 generations following administration of 25 or 100 mg/kg/day, and fewer ovarian follicles (specifically, primordial follicles) were evident following administration of 25 or 100 mg/kg/day. In the absence of an effect on fertility following administration of 5 or 25 mg/kg/day, the no observed adverse effect level (NOAEL) for reproductive toxicity is 25 mg/kg/day. Postnatal pup development was unaffected for F1 and F2 generations at any dose level; as such, the no observed effect level (NOEL) for offspring growth and development is 100 mg/kg/day.
No neurotoxic effects were noted in F1 animals; as such, the NOEL is 100 mg/kg/day for developmental neurotoxicity.
Systemic effects were confined to microscopic changes in the adrenal glands, which were considered to have represented toxicity at all dose levels. Based on this finding, neither a no observed effect level (NOEL) nor a no observed adverse effect level (NOAEL) was established for systemic toxicity. Reversibility of these findings was not investigated
Executive summary:

The objective of the study was to determine the effects of the test article, Reofos 35, on the gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, lactation, weaning and the growth and development of the offspring when administered orally, by gavage, to two successive generations. Developmental neurotoxicity was also assessed in the F1 offspring.


 


Methods


For the parental (F0) generation, male and female Crl:WI(Han) rats were assigned to four groups and administered 0 (control article [vehicle]), 5, 25, or 100 mg/kg/day Reofos 35 via oral gavage, for up to 126 consecutive days for males (at least 10 weeks prior to pairing, during pairing and 41 days post-pairing) or up to 128 days for females (10 weeks prior to pairing, during pairing, throughout gestation, and up to Lactation Day [LD] 21), until the day before necropsy. Direct dosing to the F1 offspring was initiated from Postnatal Day (PND) 14. The control article (vehicle) was corn oil, and a constant dose volume of 4 ml/kg was used.


Assessment of toxicity for the F0 generation was based on mortality, clinical observations, body weights, food consumption, estrous cycles, pregnancy indices, parturition, offspring development, and clinical and anatomic pathology. Litters were observed from birth, and the number of pups, sexes, body weights, and any clinical observations were monitored. Anogenital distance was measured on PND 4, and areolae (nipple) counts were recorded for male offspring on PND 13. F0 males were sacrificed during Week 19 (Study Day 127), and F0 females and F1 pups not selected for the next study phase were sacrificed on LD 22 (PND 22). Blood samples were taken for thyroid hormone assessments; selected organs were weighed, and macroscopic examinations were performed on the unselected offspring.


For the F1 generation, male and female Crl:WI(Han) rats were assigned to four groups in four cohorts and dosed as indicated in the following table: Animals were dosed via oral gavage at a volume of 4 mL/kg, once daily from PND 14 until the day before necropsy. The control article (vehicle) was corn oil.


 


Cohort 1A – Systemic and Reproductive Toxicity







































Group



Dose Level


(mg/kg/day)



Number of Animals/Group


F1 generation Cohort 1A



Males



Females



1 (Control)



0



20



20



2 (Low)



5



20



20



3 (Intermediate)



25



20



20



4 (High)



100



20



20



 


Cohort 1B – Reproductive Toxicity







































Group



Dose Level


(mg/kg/day)



Number of Animals/Group


F1 generation Cohort 1B



Males



Females



1 (Control)



0



20



20



2 (Low)



5



20



20



3 (Intermediate)



25



20



20



4 (High)



100



20



20



 


Cohort 2A – Developmental Neurotoxicity







































Group



Dose Level


(mg/kg/day)



Number of Animals/Group


F1 generation Cohort 2A



Males



Females



1 (Control)



0



10



10



2 (Low)



5



10



10



3 (Intermediate)



25



10



10



4 (High)



100



10



10



 


Cohort 2B – Developmental Brain Neuropathology Assessments







































Group



Dose Level


(mg/kg/day)



Number of Animals/Group


F1 generation Cohort 2B



Males



Females



1 (Control)



0



10



10



2 (Low)



5



10



10



3 (Intermediate)



25



10



10



4 (High)



100



10



10



 


Mortality, clinical observations, and body weights were recorded for F1 animals. Food consumption and sexual maturation were assessed for Cohorts 1A, 1B, and 2A. In addition, estrous cycles, ovarian follicle counts, seminology analysis, ovarian follicle counts, and clinical and anatomic pathology assessments were performed for Cohort 1A. Estrous cycles, pregnancy indices, parturition, offspring development, and anatomic pathology were assessed for Cohort 1B. Acoustic startle, motor activity, learning behavior, and brain histomorphometry were assessed for Cohort 2A. Brain histomorphometry was performed for Cohort 2B.


 


Results


Parental (F0) Generation


No test article-related unscheduled deaths occurred.


Test article-related clinical observations were confined to instances of salivation noted following administration of 100 mg/kg/day, with one instance also noted for one male administered 25 mg/kg/day.


No test article-related effects on body weights, body weight gain, or food consumption were noted.


Estrous cycles were unaffected, and no effect on mating performance was observed at any dose level.


A test article-related reduction in fertility was evident following administration of 100 mg/kg/day, compared with controls, and an increased incidence of stillborn pups was also evident at this dose level.


Hematology, clinical chemistry, and urine parameters were unaffected by the test article, and no test article-related changes in thyroxine (T4) or thyroid stimulating hormone (TSH) were evident for F0 males or females.


Sperm assessments did not reveal any test article-related effects for F0 males.


Increased adrenal weights and weight ratios were recorded for F0 animals administered 100 mg/kg/day and F0 females administered 25 mg/kg/day, compared with concurrent controls. Increased liver weights and weight ratios were recorded for F0 males administered 25 or 100 mg/kg/day. Increased thyroid weight and weight ratios and kidney:body weight ratios were recorded for F0 males administered 100 mg/kg/day, and increased ovarian weight and weight ratios were recorded for F0 females administered 100 mg/kg/day, compared with concurrent controls.


Macroscopically, an increased incidence of enlarged adrenals were recorded for both sexes from all test article-treated groups, more notably in females, and an increased incidence of pale adrenal was recorded for both sexes administered 25 or 100 mg/kg/day. An increased incidence of large thyroid gland and pale kidney was recorded for F0 males administered 100 mg/kg/day, compared to controls, and an increased incidence of large liver was recorded for males administered 25 or 100 mg/kg/day, compared to controls.


Microscopically, an increased incidence and severity of diffuse zona glomerulosa vacuolation was noted for the adrenal glands of F0 animals from all test article‑treated groups, compared with controls, and an increased incidence and severity of diffuse zona fasciculata vacuolation was recorded for F0 animals administered 25 or 100 mg/kg/day. Slight to moderate ovarian interstitial cell vacuolation was recorded for all F0 females administered 25 or 100 mg/kg/day.


Also microscopically, an increased incidence and severity of thyroid follicular cell hypertrophy was recorded for the thyroid glands of males administered 5 mg/kg/day and both sexes administered 25 or 100 mg/kg/day. Centrilobular hepatocellular hypertrophy/eosinophilia in the liver were recorded for F0 animals administered 100 mg/kg/day. An increased incidence and severity of pigmented macrophages was recorded for the spleen of F0 males administered 25 or 100 mg/kg/day, and an increased severity of renal hyaline droplet accumulation was recorded for F0 males administered 100 mg/kg/day.


 


F1 Offspring (Pre-Weaning)


Postnatal pup survival was not affected, and no clinical observations were associated with the test article. Furthermore, pup body weights and anogenital distance were unaffected. Nipple retention was not evident for male pups from any group.


No test article-related changes in T4 or TSH were evident, and no test article-related macroscopic changes or organ weight changes were evident for the PND 22 pups.


 


F1 Generation (All Cohorts)


No test article-related unscheduled deaths occurred.


No postdose observations were noted. No test article-related clinical observations or weekly detailed observations were noted.


Body weights, body weight gains, and food consumption were not affected by the test article.


No test article-related effects on age of sexual maturity or body weights at attainment were evident at any dose level. The number or duration of estrous cycles was also not affected.


 


F1 Generation Systemic and Reproductive Toxicity


No test article-related changes to urine parameters were evident for Cohort 1A animals.


Nonadverse test article-related increases in cholesterol, total protein, and globulin, and reductions in albumin:globulin (A:G) ratio were evident for Cohort 1A animals following administration of 100 mg/kg/day, compared with controls.


No test article-related TSH and T4 changes were noted for Cohort 1A animals.


Mating was unaffected by the test article for all Cohort 1B animals.


Fertility and fecundity indices were marginally lower for Cohort 1B animals administered 100 mg/kg/day, compared with controls.


Parturition, gestation length, and gestation index were unaffected, and no test article‑related effects on litter size or sex ratio were evident for Cohort 1B animals.


No test article-related clinical observations or effects on body weight or anogenital distance were noted for F2 pups, and no test article-related macroscopic findings or changes in kidney weight or kidney:body weight ratio were evident.


No test article-related changes to sperm analysis or sperm morphology parameters were observed for Cohort 1A males at any dose level.


Test article-related statistically significant reductions in ovarian follicle counts, primarily in primordial follicles, were evident for Cohort 1A females following administration of 25 or 100 mg/kg/day, compared with controls.


Increased adrenal weights and/or adrenal weight ratios and increased liver weights and/or liver weight ratios were recorded for F1 Cohort 1A animals administered 100 mg/kg/day, compared with concurrent controls.


Increased ovarian weights and ovarian weight ratios were recorded for F1 Cohort 1A and 1B females administered 100 mg/kg/day, compared with concurrent controls. Increases in ovarian weights and ovarian:body weight ratios were also recorded for F1 Cohort 1B females administered 25 mg/kg/day, compared with concurrent controls.


Macroscopically, enlarged adrenal glands and large ovaries were noted in F1 Cohort 1A females administered 100 mg/kg/day, and large liver was noted in F1 Cohort 1A males administered 100 mg/kg/day.


Microscopically in the adrenal glands, an increased incidence and severity of diffuse zona glomerulosa vacuolation was noted for F1 Cohort 1A animals administered 25 or 100 mg/kg/day, compared with controls, and an increased incidence and severity of diffuse zona fasciculata vacuolation was observed for F1 Cohort 1A animals from all test article-treated groups.


Slight to marked ovarian interstitial cell vacuolation was recorded for all F1 Cohort 1A females, and minimal to marked interstitial cell vacuolation was noted for the ovaries of F1 Cohort 1B females administered 25 or 100 mg/kg/day.


Thyroid follicular cell hypertrophy was recorded for F1 Cohort 1A males administered 25 mg/kg/day and both sexes administered 100 mg/kg/day, and centrilobular hepatocellular hypertrophy/eosinophilia was noted in the liver of F1 Cohort 1A animals administered 100 mg/kg/day.


No adverse test article-related differences in lymphocyte populations were evident in spleens, and no evidence of an immune response were evident following microscopic examinations from selected tissues of Cohort 1A animals.


 


F1 Generation Neurotoxicity and Neuropathology


No test article-related effects on functional observation battery (FOB) or motor activity parameters were evident for Cohort 2A animals, and no test article-related deficits in pre-pulse inhibition or startle habituation were noted for test article treated animals, compared with controls.


No test article-related brain weight changes or macroscopic or microscopic findings were recorded for F1 Cohort 2A or 2B animals, and no test article-related changes in brain histomorphometry were noted on PND 22 (Cohort 2B) or PND 76 (Cohort 2A) for F1 animals administered 100 mg/kg/day, compared with controls.


 


Conclusion


Following, once daily oral gavage administration of 5, 25, or 100 mg/kg/day Reofos 35 to Han Wistar rats for up to two consecutive generations, test article‑related effects were noted at all dose levels investigated.


Fertility was reduced following administration of 100 mg/kg/day to the F0 generation, which was accompanied by an increased incidence of stillborn pups and litters with stillborn pups at this dose level. Fertility was also slightly reduced for the F1 generation following administration of 100 mg/kg/day. Furthermore, microscopic examination of the ovaries revealed interstitial cell vacuolation for F0 and F1 generations following administration of 25 or 100 mg/kg/day, and fewer ovarian follicles (specifically, primordial follicles) were evident following administration of 25 or 100 mg/kg/day. In the absence of an effect on fertility following administration of 5 or 25 mg/kg/day, the no observed adverse effect level (NOAEL) for reproductive toxicity is 25 mg/kg/day. Postnatal pup development was unaffected for F1 and F2 generations at any dose level; as such, the no observed effect level (NOEL) for offspring growth and development is 100 mg/kg/day.


No neurotoxic effects were noted in F1 animals; as such, the NOEL is 100 mg/kg/day for developmental neurotoxicity.


Systemic effects were confined to microscopic changes in the adrenal glands, which were considered to have represented toxicity at all dose levels. Based on this finding, neither a no observed effect level (NOEL) nor a no observed adverse effect level (NOAEL) was established for systemic toxicity. Reversibility of these findings was not investigated.