Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
28 Jul 2011
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: 70 days
- Basis for dose level selection: Dosage levels were selected based on the range-finding study. In that study, dosage levels of 40, 160, and 400 mg/kg/day were tested. Animals in the high-dosage group showed clinical signs (salivation and red material around the eyes, nose, and mouth), which were still observed to a lesser extent in the mid-dosage group, and the males were more affected than the females. Macroscopic examinations showed thickened and eroded stomach in the 400 mg/kg/day group males and thickened stomach was still observed in the 160 mg/kg/day group animals (predominantly in males). In the current study, the animals were dosed for a longer time period. Therefore, based on the results of the range-finding study, dosage levels of 20, 50, and 150 mg/kg/day were selected for the current study. The high-dosage level of 150 mg/kg/day was expected to show parental toxicity, whereas the low-dosage level of 20 mg/kg/day was not expected to show toxic effects.
- Inclusion/exclusion of extension of Cohort 1B: not performed as P0 and F1 Cohort 1A did not reveal any results
- Termination time for F2: not performed as P0 and F1 Cohort 1A did not reveal any results
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: not performed as P0 and F1 Cohort 1A did not reveal any results
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: not performed as P0 and F1 Cohort 1A did not reveal any results
- Route of administration: oral

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
- Lot No. F534801GB
- Exp. date: 11 Dec 2016
- Colorless, clear liquid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 8 weeks
- Weight at study initiation: Male body weights ranged from 264 g to 378 g and female body weights ranged from 154 g to 228 g on the initial day of test substance administration
- Housing: F0 parental animals were housed 2–3 per cage by sex in clean, solid-bottom cages with heat-treated aspen bedding material (Aspen Bed™ 1, Northeastern Products Corporation, Warrensburg, NY). Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly
- Diet: the basal diet was provided ad libitum throughout the acclimation period and during the study. The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5K96 Advanced Protocol® Verified Casein Diet 10 IF, was a certified feed with appropriate analyses performed by the manufacturer.
- Water: Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system was provided ad libitum throughout the acclimation period and during the study.
- Acclimation period: 24 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% carboxymethylcellulose (medium viscosity), 0.014% Kolliphor® EL, and 0.0035% hydrochloric acid in deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared daily as single formulations for each dosage level and maintained on wet ice, protected from light. The test substance formulations were stirred continuously on wet ice throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Concentration in vehicle: 0, 4, 10 and 30 mg/mL (corresponding to dosage levels of 0, 20 50 and 150 mg/kg/day)
- Amount of vehicle: 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: The animals were paired on a 1:1 basis within each treatment group after a minimum of 70 days of treatment.
- Length of cohabitation: 14 day mating period
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Following positive evidence of mating, the F0 males and females were individually housed in solid-bottom cages with bedding material until the scheduled necropsy. The dams were housed in these cages with their litters until weaning on Lactation Day 21. Following weaning of the F1 litters, the weaned F1 pups were housed together by litter for 1 week. Beginning on PND 28, the F1 offspring were housed 2–3 per cage by sex in clean, solid-bottom cages with bedding material until necropsy. Females for which there was no evidence of mating were placed in clean, solid-bottom cages with bedding material upon completion of a 14-day mating period with no further opportunity for mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the 4, 10, and 30 mg/mL dosing formulations and from the middle stratum of the control group dosing formulations prepared on Study Day 0, 7, 15, and 30 for the first month and once a month thereafter (total of 9 sets). Analysis was performed using a validated gas chromatography method using flame ionization detection. The analysed dosing formulations were, with minor exceptions, within the range for suspensions (85% to 115%) and were homogeneous. Analysed concentrations above the acceptance criteria had no impact on the study as the no-observed-adverse-effect level (NOAEL) was determined to be 150 mg/kg/day, the highest dose evaluated on this study.
Duration of treatment / exposure:
The vehicle and test substance formulations were administered to the F0 males and females for a minimum of 70 consecutive days prior to mating. Dose administration for the F0 males continued throughout mating and through the day prior to euthanasia, for a total of 129-131 doses. The F0 females continued to be dosed throughout mating, gestation, and lactation, through the day prior to euthanasia, for a total of 132-136 doses. Animals selected for the F1 generation were directly administered the vehicle or test substance following weaning (beginning on PND 21) and continuing through the day prior to euthanasia (PND 91 [Cohort 1A] and between PND 103-110 [Cohort 1B]).
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
30
Control animals:
yes, concurrent vehicle
Details on study design:
Dosage levels were selected based on the range-finding study (see any other information on materials and methods)

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS
Detailed physical examinations were recorded weekly, beginning 1 week prior to the initiation of test substance administration, for all parental animals throughout the study period. In addition, detailed physical examinations were conducted on Gestation Days 0, 7, 14, and 20 for all females with evidence of mating and on Lactation Days 1, 7, 14, and 21. In addition, animals were observed for signs of toxicity approximately 1 hour following dose administration. Special attention was paid to the degree of salivation and lacrimation, presence or absence of urination and defecation (including polyuria and diarrhoea), pupil size, degree of palpebral closure, presence of convulsions, tremors, or abnormal movements, presence of posture and gait abnormalities, the presence of any unusual or abnormal behaviours, and any repetitive actions (stereotypies). Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labour, delayed labour) or other difficulties. In addition, the social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal.

BODY WEIGHT
Individual F0 male body weights were recorded weekly, beginning 1 week prior to the initiation of test substance administration, throughout the study and prior to the scheduled necropsy. Individual F0 female body weights were recorded weekly, beginning 1 week prior to the initiation of test substance administration, until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating treatment period (males and females) and for the entire F0 treatment period (males only). Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 10, 14, 17, and 21. After weaning (Lactation Day 21), weekly body weights were recorded for these females until the scheduled necropsy. For F0 females with no evidence of mating, weekly body weights were recorded until necropsy.

FOOD CONSUMPTION
F0 male and female food consumption was measured weekly, beginning 1 week prior to the initiation of test substance administration and continuing until cohabitation. Food consumption was measured on a per cage basis for the corresponding body weight intervals. Food consumption was normalized to the number of animals/cage and was reported as g/animal/day. Food intake was not recorded during the breeding period. Following the breeding period, individual food consumption for males and for females with no evidence of mating was measured on a weekly basis until the scheduled necropsy. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, 14, 17, and 21. Food intake was reported for the corresponding intervals of gestation and lactation, and also for Gestation Days 0–20 and Lactation Days 1–21. Food efficiency (body weight gained as a percentage of food consumed) was also calculated

CLINICAL PATHOLOGY
Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected prior to the scheduled necropsy (Study Days 129-136 for the F0 males and females). All animals were fasted overnight prior to blood collection while in metabolism cages for urine collection blood and urine samples were collected from 10 randomly selected F0 animals/sex/group sent to necropsy. Blood for serum chemistry and haematology was collected from the jugular vein using the hand-held technique following anaesthesia with isoflurane inhalation. Blood for coagulation parameters was collected from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation.

- Haematology and coagulation parameters examined: Total leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (Platelet), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count Percent (RETIC), Absolute (RETIC Absolute), Mean platelet volume (MPV), Differential leukocyte count Percent and absolute, Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC), Red cell distribution width (RDW), Haemoglobin distribution width (HDW), Platelet estimate, Red cell morphology (RBC Morphology).
- Serum chemistry parameters examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total BILI), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyl transferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Appearance, Bile Acids
- Urinalysis parameters examined: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Colour (COL), Clarity (CLA), Protein (PRO), Glucose (GLU)

THYROID HORMONE ANALYSIS
Blood (approximately 1.5 mL) for thyroid hormone levels was collected via the retro-orbital sinus following isoflurane anaesthesia from F0 males and females (10/sex/group) following the completion of weaning of all F0 females
- Parameters evaluated: Thyroxine (T4) and Thyroid Stimulating Hormone (TSH)
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the oestrous cycle of each F0 female for 14 days prior to cohabitation and continuing until evidence of mating was observed or until the end of the mating period. The average cycle length was calculated for complete oestrous cycles (i.e., the total number of returns to metoestrous [M] or dioestrous [D] from oestrus [E] or prooestrous [P], beginning 14 days prior to initiation of the mating period and continuing until the detection of evidence of mating). Oestrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual oestrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the oestrous cycle. At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.
Sperm parameters (parental animals):
Immediately upon euthanasia, the reproductive tract of each F0 male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed separately. An incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a 10 to 20-minute incubation period, a sample of sperm was loaded onto a slide with a 100-μm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all slides and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyser. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported:
Percent Motile (or Progrressively Molite) Sperm = Number of Motile (or Progressively Motile) Sperm / Total Number of Sperm Counted x 100

The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and cauda epididymis from all F0 males from the control and high-dose groups were weighed, stored frozen, homogenized, and analysed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each sample was added to a solution containing a DNA-specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-μm chamber depth. Illumination from a xenon lamp within the analyser allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample. The sperm production rate was calculated as follows:
Sperm Production Rate = No. of Sperm Per Gram of Tissue / 6.1 days
6.1 days = The rate of turnover of the germinal epithelium
Litter observations:
STANDARDISATION OF LITTERS
To reduce variability among the litters, 10 pups/litter, 5 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 10 pups. All selections were performed by computerized randomization. Following selection, culled pups were euthanized by decapitation (those pups used for blood/thyroid collection) or an intraperitoneal injection of sodium pentobarbital, and examined macroscopically; tissues with gross lesions were preserved in 10% neutral-buffered formalin.

PARAMETERS EXAMINED PRE-WEANING
- A daily record of litter size was maintained. Intact offspring that were found dead during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels.7 Pups found dead on PND 0 or 1 had the lungs removed and placed in a saline-filled jar. If the lungs sank to the bottom of the jar, the pup had no prior documentation of being viable, and there was no evidence of milk in the stomach, the pup was considered to be stillborn. If the lungs floated, the pup was considered to be found dead on the originally documented day (PND 0 or 1).
- Litters were examined daily for survival and any adverse changes in appearance or behaviour. Each pup received a detailed physical examination on PND 1, 4, 7, 14, and 21.
- Pups were individually weighed on PND 1, 4 (before culling), 7, 14, and 21.
- Pups were individually sexed on PND 0, 4 (before culling), 7, 14, and 21.
- The anogenital distance of all F1 pups was measured on PND 1
- On PND 12, all F1 male offspring were evaluated for the presence of thoracic nipples/areolae.

GROSS EXAMINATION OF DEAD PUPS
A detailed gross necropsy was performed on any pup found dead after PND 4 and prior to weaning. Tissues were preserved for possible future histopathological examination only as deemed necessary by the gross findings.

POST-WEANING DEVELOPMENTAL LANDMARKS
- Balanopreputial Separation: Each male was observed for balanopreputial separation beginning on PND 35. The age at which balanopreputial separation was first observed was recorded for each pup. Examination of the pups continued daily until balanopreputial separation was present. Body weights were recorded at the age of attainment of this landmark. In addition, the appearance of a partial and complete balanopreputial separation or a persistent thread of tissue between the glans and prepuce was recorded.
- Vaginal Patency: Each female was observed for vaginal perforation beginning on PND 25. The age at which the vaginal lumen was first observed to open was recorded for each pup. Examination of the females was continued daily until vaginal patency was present. Body weights were recorded at the age of attainment of this landmark. In addition, the appearance of a small “pin hole”, a vaginal thread, and complete vaginal opening were recorded.
- Oestrous Cyclicity (Cohort 1A): Beginning on the day vaginal opening was observed, vaginal lavages were performed daily for all F1 females assigned to Cohort 1A and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female until the first sign of oestrus (cornified cells) was observed. The age of first vaginal oestrus after vaginal opening was recorded. Vaginal lavages were also performed daily for all F1 females assigned to Cohort 1A and the slides were evaluated microscopically to determine the stage of the oestrous cycle of each female for 2 weeks during PND 75–91 (the day of necropsy). The average cycle length was calculated and reported for complete oestrous cycles (i.e., the total number of returns to metaoestrus [M] or dioestrus [D] from oestrus [E] or prooestrus [P]). Oestrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.

POST WEANING OBSERVATIONS
- Clinical observations: Following weaning, all animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Detailed physical examinations were performed weekly until necropsy. In addition, animals were observed for signs of toxicity approximately 1 hour following dose administration.
- Body weights: F1 males and females were weighed weekly following weaning, and on the day of euthanasia.
- Food Consumption: F1 male and female food consumption was measured weekly, beginning on PND 28, until the day prior to euthanasia. Food consumption was normalized to the number of animals/cage and was reported as g/animal/day. Food efficiency (body weight gained as a percentage of food consumed) was also calculated and reported for these intervals.

CLINICAL PATHOLOGY (Cohort 1A)
Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected prior to the scheduled necropsy (PND 91 for the Cohort 1A F1 males and females). All animals were fasted overnight prior to blood collection while in metabolism cages for urine collection blood and urine samples were collected from10 randomly selected F0 and F1 Cohort 1A animals/sex/group sent to necropsy. Blood for serum chemistry and haematology was collected from the jugular vein using the hand-held technique following anaesthesia with isoflurane inhalation. Blood for coagulation parameters was collected from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation.

- Haematology and coagulation parameters examined: Total leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (Platelet), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count Percent (RETIC), Absolute (RETIC Absolute), Mean platelet volume (MPV), Differential leukocyte count Percent and absolute, Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC), Red cell distribution width (RDW), Haemoglobin distribution width (HDW), Platelet estimate, Red cell morphology (RBC Morphology).
- Serum chemistry parameters examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total BILI), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyl transferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Appearance, Bile Acids
- Urinalysis: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Colour (COL), Clarity (CLA), Protein (PRO), Glucose (GLU)
Postmortem examinations (parental animals):
SACRIFICE
The surviving F0 males and females were euthanized on Study Days 129-136

GROSS NECROPSY
- A complete necropsy was conducted on all F0 animals that were found dead, euthanized in extremis, or at termination. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. Clinical findings that were verified at necropsy were designated CEO. For F0 females that delivered, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. Numbers of corpora lutea were also recorded for females necropsied during gestation through Lactation Day 4. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.
- The following F0 parental tissues and organs were collected and were placed in 10% neutral-buffered formalin: Adrenal glands (2), Aorta (1), Bone with marrow (sternebrae), Brain (7 levels), Coagulating glands (2), Eyes with optic nerve (2) [a], Gastrointestinal tract, Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Lacrimal/Harderian glands (2), Liver (sections of 2 lobes fixed and also 2 sections frozen) [b], Lungs (including bronchi, fixed by inflation with fixative) (5), Lymph node (mandibular and mesenteric) (1), Ovaries [c] and oviducts (2), Pancreas (1), Peripheral nerve (tibial), Pituitary (1), Prostate (1), Mandibular salivary gland (1), Seminal vesicles (2), Skeletal muscle (rectus femoris) (1), Skin with mammary gland and subcutis (1)[d], Spinal cord (cervical, thoracic, lumbar) (3), Spleen (1), Testes with epididymides (1) and, vas deferens (1) [e,f],Thymus, Thyroids with trachea [g] [with parathyroids if present (1)], Urinary bladder, uterus with cervix and vagina (4), All gross lesions (all groups).
The number in parentheses is the minimum number of sections to be examined if the tissue was chosen for histopathologic examination.
[a] Eyes with optic nerves were fixed in Davidson’s solution.
[b] Sections of 2 liver lobes were placed in 10% neutral-buffered formalin and 2 sections of liver lobes were flash frozen in liquid nitrogen and stored frozen at -70°C for possible future analysis.
[c] Ovaries were fixed in 10% neutral-buffered formalin for approximately 48 hours following which all ovaries were transferred to 70% ethanol. The ovaries were processed to blocks.
[d] For females, a corresponding section of skin was taken from the same anatomic area for males.
[e] Testis (right only) and epididymides (right and left caput and body) were fixed in modified Davidson’s solution. Care was taken to ensure separation between the left and right organs.
[f] If the left testis or epididymis was noted with a gross lesion, the left testis and epididymis were fixed in modified Davidson’s solution and the right testis and epididymis were homogenized
[g] Thyroids and parathyroids (if present) were removed from the trachea following fixation to ensure tissue structure was maintained during prosection.

ORGAN WEIGHTS
Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported. The following organs were weighed from all F0 animals at the scheduled necropsies: Adrenal glands, Brain, Epididymides (total and cauda) [a], Heart, Kidneys, Levator ani and bulbocavernosus (LABC) muscle group, Liver, Ovaries, Pituitary gland, Prostate gland, Seminal vesicles with coagulating glands (with accessory fluids), Spleen, Testes [a], Thyroids with parathyroids [b], Thymus gland, Uterus with oviducts and cervix.
[a] These paired organs were weighed separately.
[b] Tissues were weighed after fixation in 10% neutral-buffered formalin.

HISTOPATHOLOGY
Microscopic examination was performed on all tissues from all F0 parental from the control and high-dose groups and for all adult animals found dead and euthanized in extremis. In addition, reproductive organs of all F0 animals suspected of reduced fertility (e.g., those that failed to mate, conceive, sire, or deliver healthy offspring, or for which oestrous cyclicity or sperm number, motility, or morphology were affected) were subjected to histopathological examination. Additionally, all gross lesions were examined microscopically, irrespective of group.
Postmortem examinations (offspring):
SACRIFICE
- Euthanasia was scheduled PND 91 [Cohort 1A] and between PND 103-110 [Cohort 1B]
- All nonselected F1 weanlings were euthanized on PND 21.
- These animals were subjected to post-mortem examinations (macroscopic and microscopic examination) as follows:

GROSS NECROPSY
- A complete necropsy was conducted on all F1 animals in Cohorts 1A and 1B that were found dead, euthanized in extremis, or at termination. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. Clinical findings that were verified at necropsy were designated CEO.
- The following F1 Cohorts 1A and 1B tissues and organs were collected and were placed in 10% neutral-buffered formalin: Adrenal glands (2), Aorta (1), Bone with marrow (sternebrae), Brain (7 levels), Coagulating glands (2), Eyes with optic nerve (2) [a], Gastrointestinal tract, Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Lacrimal/Harderian glands (2), Liver (sections of 2 lobes fixed and also 2 sections frozen) [b], Lungs (including bronchi, fixed by inflation with fixative) (5), Lymph node (mandibular and mesenteric) (1), Ovaries [c] and oviducts (2), Pancreas (1), Peripheral nerve (tibial), Pituitary (1), Prostate (1), Mandibular salivary gland (1), Seminal vesicles (2), Skeletal muscle (rectus femoris) (1), Skin with mammary gland and subcutis (1)[d], Spinal cord (cervical, thoracic, lumbar) (3), Spleen (1), Testes with epididymides (1) and, vas deferens (1) [e,f],Thymus, Thyroids with trachea [g] [with parathyroids if present (1)], Urinary bladder, uterus with cervix and vagina (4), All gross lesions (all groups).
The number in parentheses is the minimum number of sections to be examined if the tissue was chosen for histopathologic examination.
[a] Eyes with optic nerves were fixed in Davidson’s solution.
[b] Sections of 2 liver lobes were placed in 10% neutral-buffered formalin and 2 sections of liver lobes were flash frozen in liquid nitrogen and stored frozen at -70°C for possible future analysis.
[c] Ovaries were fixed in 10% neutral-buffered formalin for approximately 48 hours following which all ovaries were transferred to 70% ethanol. The ovaries were processed to blocks.
[d] For females, a corresponding section of skin was taken from the same anatomic area for males.
[e] Testis (right only) and epididymides (right and left caput and body) were fixed in modified Davidson’s solution. Both testes and epididymides from animals euthanized in extremis and from all males in Cohort 1B were fixed in modified Davidson’s solution. Care was taken to ensure separation between the left and right organs.
[f] If the left testis or epididymis was noted with a gross lesion, the left testis and epididymis were fixed in modified Davidson’s solution and the right testis and epididymis were homogenized
[g] Thyroids and parathyroids (if present) were removed from the trachea following fixation to ensure tissue structure was maintained during prosection.
- Gross necropsies with emphasis on developmental morphology and organs of the reproductive system were performed on all nonselected F1 pups euthanized on PND 21.
-The following tissues/organs from 10 F1 nonselected pups/sex/group were retained in 10% neutral-buffered formalin for possible histopathologic examination: Brain, Mammary gland, Spleen, Thymus, Thyroids, All gross lesions

ORGAN WEIGHTS
- Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported. The following organs were weighed from all F1 animals in Cohort 1A at the scheduled necropsies: Adrenal glands, Brain, Epididymides (total and cauda) [a,c], Heart, Kidneys, Levator ani and bulbocavernosus (LABC) muscle group[c], Liver, Ovaries [c], Pituitary gland [c], Prostate gland [c], Seminal vesicles with coagulating glands (with accessory fluids) [c], Spleen, Testes [a,c], Thyroids with parathyroids [b], Thymus gland, Uterus with oviducts and cervix [c]
[a] These paired organs were weighed separately.
[b] Tissues were weighed after fixation in 10% neutral-buffered formalin.
[c] Also weighed for F1 Cohort 1B animals.
- The brain, spleen and thymus were weighed from 10 nonselected F1 pups/sex/group on PND 21. In addition, the thyroids were weighed (after fixation) from the same 10 pups/sex/group that were used for blood collection

HISTOPATHOLOGY
- Microscopic examination was performed on all tissues from all F1 Cohort 1A animals from the control and high-dose groups and for all adult animals found dead and euthanized in extremis. Additionally, all gross lesions were examined microscopically, irrespective of group.

SPERM PARAMETERS (Cohort 1A)
Immediately upon euthanasia, the reproductive tract of each F1 Cohort 1A male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed separately. An incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a 10 to 20-minute incubation period, a sample of sperm was loaded onto a slide with a 100-μm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all slides and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyser. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported:
Percent Motile (or Progrressively Molite) Sperm = Number of Motile (or Progressively Motile) Sperm / Total Number of Sperm Counted x 100

The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and cauda epididymis from all F1 Cohort 1A males from the control and high-dose groups were weighed, stored frozen, homogenized, and analysed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each sample was added to a solution containing a DNA-specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-μm chamber depth. Illumination from a xenon lamp within the analyser allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample. The sperm production rate was calculated as follows:
Sperm Production Rate = No. of Sperm Per Gram of Tissue / 6.1 days
6.1 days = The rate of turnover of the germinal epithelium
Statistics:
see any other information on materials and methods
Reproductive indices:
- Male Mating Index (%) = No. of Males with Evidence of Mating / Total No. of Males Used for Mating x 100
- Female Mating Index (%) = No. of Females with Evidence of Mating or Females Confirmed Pregnant / Total No. of Females Used for Mating x 100
- Male Fertility Index (%) = No. of Males Siring a Litter/ Total No. of Males Used for Mating x 100
- Male Copulation Index (%) = No. of Males Siring a Litter/ No. of Males with Evidence of Mating (or Females Confirmed Pregnant) x 100
- Female Fertility Index (%) = No. of Females with Confirmed Pregnancy/ Total No. of Females used for Mating x 100
- Female Conception Index (%) = No. of Females with Confirmed Pregnancy/ No. of Females with Evidence of Mating (or Females Confirmed Pregnant) x 100
Offspring viability indices:
- Mean Live Litter Size = Total Viable Pups on PND 0/ No. Litters with Viable Pups on PND 0
- Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection)(% Per Litter) = Sum of (Viable Pups/Litter on PND 0 or PND 4 [Pre-Selection]/No. of Pups Born/Litter)/ No. of Litters/Group x 100
- Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups/Litter at End of Interval N/Viable Pups/Litter at Start of Interval N)/ No. of Litters/Group x 100

Where N = PND 0–1, 1–4 (Pre-Selection), 4 (Post-Selection)–7, 7–14, 14–21, Birth to PND 4 (Pre-Selection), or 4 (Post-Selection)–21

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related clinical observations were noted during the F0 generation at the daily examinations, weekly detailed physical examinations, and/or approximately 1 hour following dose administration. Clonic convulsions were noted for 1 male in the 20 mg/kg/day group, 1 female in the 50 mg/kg/day group, and 1 male and 1 female in the 150 mg/kg/day group sporadically throughout the treatment period; this finding was noted at the daily examinations, weekly detailed physical examinations, at the time of dose administration, and/or approximately 1 hour following dose administration. This finding was transient and was not observed in the F1 generation, and therefore was not considered test substance-related. Other observations noted in the test substance-treated groups, including red and/or yellow material, scabbing, and/or hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test substance-related effects on survival were noted for F0 males and females at any dosage level. One female (in the 50 mg/kg/day group) was euthanized in extremis on Gestation Day 26 due to poor general health. The cause of morbidity was the acute inflammation of the uterus observed microscopically and the partially decomposed fetuses observed grossly. Because all animals in the high-dose group (150 mg/kg/day) survived to the scheduled necropsy, the moribundity in the 50 mg/kg/day group was not considered test substance-related. All other F0 parental animals in the control, 20, 50, and 150 mg/kg/day groups survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related effects on F0 mean body weights, body weight gains, and cumulative body weight gains were noted in the 20, 50, and 150 mg/kg/day groups. Differences from the control group were not statistically significant, with the following exceptions. For F0 males, significantly (p < 0.01) lower mean body weight gains were noted in the 20 mg/kg/day group during Study Days 112–119 and in the 150 mg/kg/day group during Study Days 91–98 compared to the control group; these differences were transient, did not occur in a dose-related manner, and/or were not of sufficient magnitude to affect the entire generation interval (Study Days 0–126) or mean body weights in these groups, and therefore were not considered test substance-related. A significantly (p < 0.05) higher mean body weight was noted for the 150 mg/kg/day group F0 females compared to the control group on Study Day 14; this difference was transient and no corresponding effect on mean body weight gain was noted in this group, and therefore was not considered test substance-related.
- Mean F0 maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance administration during gestation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant, with the following exception. A significantly (p < 0.05) lower mean body weight gain was noted in the 50 mg/kg/day group F0 females compared to the control group during Gestation Days 17–20; this difference was transient, did not occur in a dose-related manner, was not of sufficient magnitude to affect the overall gestation treatment interval (Gestation Days 0–20) or mean body weights in this group, and therefore was not considered test substance-related.
- Mean F0 maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance administration during lactation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- Mean F0 food consumption in the 20, 50, and 150 mg/kg/day groups was unaffected by test substance administration. The values in the test substance-treated groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males). Differences from the control group were not statistically significant, with the following exceptions. In F0 females, significantly (p < 0.05 or p < 0.01) higher mean food consumption was noted in the 20 mg/kg/day group during Study Days 7–14 and in the 150 mg/kg/day group during Study Days 0–7 and 14–21; these differences were transient, did not occur in a dose-related manner, and/or were not of sufficient magnitude to affect mean body weights at these dosage levels, and therefore were not considered test substance-related.
- Mean F0 maternal food consumption was unaffected by test substance administration during gestation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant.
- Mean F0 maternal food consumption was unaffected by test substance administration during lactation. Differences between the control, 20, 50, and 150 mg/kg/day groups were slight and not statistically significant.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
- Mean F0 food efficiency in the 20, 50, and 150 mg/kg/day groups were unaffected by test substance administration.
- Differences from the control group were not statistically significant, with the following exceptions. Significantly (p < 0.01) lower mean food efficiency was noted for F0 males in the 20 mg/kg/day group during Study Days 112–119 and in the 150 mg/kg/day group during Study Days 91-98 compared to the control group; these differences were transient, did not occur in a dose-related manner, and/or a corresponding effect on mean food consumption was not noted at these dosage levels, and therefore they were not considered test substance-related.
- Mean F0 maternal food efficiency was unaffected by test substance administration during gestation.
- Mean F0 maternal food efficiency was unaffected by test substanceadministration during lactation.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related alterations in serum chemistry parameters. Significantly (p < 0.05) higher mean serum albumin was observed in the 150 mg/kg/day group F0 females when compared to the concurrent control group. The mean and individual animal values were within the Charles River Ashland historical control ranges for female rats. The finding was considered a result of individual animal variation rather than a true test substance-related change.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related alterations in organ weights. However, the mean brain weight relative to final body weight was significantly (p < 0.01) lower in the 150 mg/kg/day group F0 females when compared to the concurrent control group; however, the mean absolute brain weight and mean final body weight in this group were not significantly different from concurrent controls, and thus the lower mean brain weight relative to body weight was considered spurious.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related gross observations were observed in the 150 mg/kg/day group F0 males. The test substance-related gross observation of thickened stomach occurred in 3 of 30 males in the 150 mg/kg/day group.
- An apparent dose-related increased incidence of depressed areas in the kidneys was observed in F0 females administered the test substance at all dosage levels. While not observed in the concurrent control group females, it was observed at a higher incidence (5 of 30) in control group males than in the 150 mg/kg/day group F0 females (4 of 30). The finding is also observed in the test lab historical control database for animals of similar age. The finding lacked a consistent histologic correlate and was considered incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related microscopic findings were noted in the nonglandular stomach and liver of the 150 mg/kg/day group F0 males and females and the kidneys in the 150 mg/kg/day group F0 females.
- Nonglandular stomach findings were limited to minimal to moderate epithelial hyperplasia and hyperkeratosis. The hyperplasia was characterized by increased thickness (increased number of cell layers) of the squamous epithelium with an increased thickness of the keratinized oute layers of the epithelium (hyperkeratosis). In one 150 mg/kg/day group F0 female, minimal edema and congestion were observed in the submucosa adjacent to the hyperplasia and hyperkeratosis. These changes were not associated with any clinical pathology changes. Although considered an adaptive change, the mild to moderate severity of the hyperplasia and/or hyperkeratosis in the F0 generation were considered adverse in the 150 mg/kg/day group males and females.
- Liver changes observed microscopically included increased incidence of minimal to mild biliary hyperplasia in the 150 mg/kg/day group F0 males and females and minimal to mild randomly scattered hepatocellular necrosis in the 150 mg/kg/day group F0 males. Biliary hyperplasia was characterized by increased numbers of small bile duct profiles in the portal triads. Biliary hyperplasia can occur as a normal age related finding in rats and was observed in the concurrent control group in this study. The severity was minimal to mild and a clinical pathology correlate for the finding was not observed. Similarly, randomly distributed hepatocellular necrosis can be observed spontaneously in rats and was observed in the control group in this study. The severity of the necrosis was minimal to mild and the change lacked clinical pathology correlates. There was no association of the biliary hyperplasia to the necrosis. Changes observed in the liver were considered nonadverse.
- Increased severity of mineralization at the corticomedullary junction of the kidneys was observed in the 150 mg/kg/day group F0 females. This increase in severity (minimal to mild) did not result in alterations to the clinical pathology parameters related to renal function and were considered nonadverse.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on serum levels of T4 (thyroxine) or TSH (thyroid stimulating hormone) in F0 males or females. Differences from the control group were slight, did not occur in a dose-related manner, and/or were not statistically significant.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean lengths of oestrous cycles in the test groups were also similar to the control group value. None of these differences were statistically significant. All females were noted to be cycling, and the percentage of females cycling regularly was similar across all groups.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage level. Differences from the control group were slight and were not statistically significant, with the following exceptions. Left cauda epididymal sperm concentration was significantly (p < 0.05) lower in the 150 mg/kg/day group compared to the control group. However, there were no effects on reproductive performance (mating and fertility) and no other effects on spermatogenic endpoints at this dosage level; therefore, the lower epididymal sperm concentration noted at 150 mg/kg/day was not considered test substance-related. Significantly (p < 0.05) lower mean motility and progressive motility were noted in the 20 mg/kg/day group compared to the control group; these differences did not occur in a dose-related manner, and therefore were not considered test substance-related.
Reproductive performance:
no effects observed
Description (incidence and severity):
No test substance-related effects on F0 reproductive performance were observed at any dosage level. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value.

Details on results (P0)

No test substance-related effects were noted on mean gestation lengths or the process of parturition at any dosage level.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive effects observed
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local effects (nonglandular stomach)

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by test substance administration.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects on survival were noted for F1 animals at any dosage level. In the 50 mg/kg/day group, 1 female was found dead on PND 82 following clinical observations of gasping prior to and following dose administration on the day of death. Based on the findings noted for this female at necropsy (esophageal perforation and clear fluid contents in the thoracic cavity [approximately 8.0 mL]), the cause of death was determined to be intubation error, and therefore was not considered test substance-related. In the control group, 1 male was euthanized in extremis on PND 28 due to clinical observations of head tilt and circling on the day of euthanasia; at necropsy, this male was noted with dilated lateral ventricles in the brain. This correlated to the marked chronic active inflammation in the brain. All other F1 animals in the control, 20, 50, and 150 mg/kg/day groups survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Mean F1 male and female pup body weights and body weight changes in the 20, 50, and 150 mg/kg/day groups were unaffected by test substance administration throughout the postnatal period.
- No test substance-related effects on mean body weights, body weight gains, and cumulative body weight gains were noted in the 20, 50, and 150 mg/kg/day group F1 males and females. Differences from the control group were slight and not statistically significant, with the following exception. A significantly (p < 0.01) lower mean body weight gain was noted in the 20 mg/kg/day group F1 males compared to the control group during PND 42-49; this difference was transient, did not occur in a dose-related manner, and was not of sufficient magnitude to affect mean body weights at this dosage level, and therefore was not considered test substance-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Mean F1 food consumption in the 20, 50, and 150 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exceptions. Significantly (p < 0.05 or p < 0.01) lower mean food consumption was noted in the 20 mg/kg/day group F1 males during PND 42–49 and 63–70, in the 50 mg/kg/day group F1 males during PND 70–77, and in the 50 mg/kg/day group F1 females during PND 49–63 and 70–77 compared to the control group; these differences were transient, did not occur in a dose-related manner, and were not of sufficient magnitude to affect mean body weights at these dosage levels, and therefore were not considered test substance-related.
Food efficiency:
no effects observed
Description (incidence and severity):
Mean F1 food efficiency in the 20, 50, and 150 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
- There were no test substance-related effects on serum chemistry parameters.
Urinalysis findings:
no effects observed
Sexual maturation:
no effects observed
Description (incidence and severity):
- Mean estrous cycle lengths in the test substance-treated groups were similar to the control group.
- No test substance-related effects were observed on F1 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage level.
- There were no test substance-related effects on ovarian primordial follicle counts in the 150 mg/kg/day group F1 females in Cohort 1A compared to the control group.
- Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance administration.
- Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance administration.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed for PND 21 pups at any dosage level.
-There were no test substance-related changes in organ weights for F1 males and females in Cohort 1A. The absolute and relative pituitary weights were significantly (p < 0.05 or p < 0.01) higher in the 50 and 150 mg/kg/day group F1 females when compared to the concurrent control. These values fell within the historical control reference ranges, were not associated with any microscopic findings, and/or were considered a result of individual animal variation rather than a true test substance-related effect.

-No test substance-related effects on organ weights (absolute and relative to final body weight) were observed at any dosage level for F1 animals in Cohort 1B (necropsied on PND 103–110). Differences from the control group were slight and not statistically significant, with the following exceptions. A higher mean absolute left testis weight was noted in 50 mg/kg/day group F1 males and higher mean relative (to final body weight) left and right testis weights were noted in the 20 and 50 mg/kg/day group F1 males compared to the control group; these differences did not occur in a dose-related manner, and therefore were not considered test substance-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- The numbers of pups (litters) found dead from PND 0 through the selection of the F1 generation were 50(13), 46(14), 24(5), and 31(11) in the control, 20, 50, and 150 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration of the test substance were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach and lungs that did or did not float, internal findings included renal papilla(e) not fully developed (Woo and Hoar Grade 1) for 2 pups in the 50 mg/kg/day group and red fluid contents in the bladder for 1 pup in the 20 mg/kg/day group. Because the findings in the 20 and 50 mg/kg/day groups were noted infrequently and in a non-dose-related manner, they were not considered test substance-related.
- No internal findings that could be attributed to parental administration of the test substance were noted at the necropsy of culled pups euthanized on PND 4. Internal findings included the developmental variations of distended ureters noted for one pup in the 50 mg/kg/day group and hemorrhagic ring around the iris noted for 1 pup each in the control and 20 mg/kg/day groups. These findings occurred in single pups and in a non-dose-related manner, and therefore were not considered test substance-related.
- No internal findings that could be attributed to parental administration of the test substance were noted at the necropsy of pups euthanized on PND 21. Dilated renal pelvis was noted for 1, 2 , and 1 pups in the 20, 50, and 150 mg/kg/day groups, respectively; 1 of these same pups in the 50 mg/kg/day group was also noted with distended ureters. In the 150 mg/kg/day group, 1 pup was noted with opacity of the eyes. Foamy contents in the trachea was noted for groups, respectively; this finding was also noted for 1 pup in the control group. A short tail was noted for 1 pup each in the control and 50 mg/kg/day groups. In the 50 mg/kg/day group, 1 up was noted with yellow areas on the liver and 1 Pup was noted with a fractured tail. Dark red discoloration of the eyes was noted for 1 pup each in the control and 20 mg/kg/day groups; 11 pup was also noted with a small left eye. The aforementioned findings were noted insingle pups or litters, occurred infrequently or similarly in the control group, and/or did not occur in a dose-related manner, and therefore were not considered test substance-related.
- At the PND 21 necropsy of F1 weanlings selected for hormone analysis, no internal findings that could be attributed to parental administration of the test substance were noted at any dosage level. Internal findings were limited to dark red discoloration on the thyroid glands for a Male Pup in the 20 mg/kg/day group and an accessory spleen for a Female Pup in the 150 mg/kg/day group. Because these findings were limited to a single fetus and/or did not occur in a dose-related manner, they were not considered test substance-related.
- Thickened stomachs were observed in the 20, 50, and 150 mg/kg/day group F1 males and the 150 mg/kg/day group F1 females in Cohort 1A and were considered test substance-related. At the scheduled F1 male and female necropsies for Cohort 1B, thickened stomachs were noted in the 50 and 150 mg/kg/day group F1 males and in the 150 mg/kg/day group F1 females and were considered test substance-related; given the lack of systemic toxicity
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related microscopic changes were observed in the nonglandular stomach for F1 males and females in Cohort 1A at all dosage levels. Hyperkeratosis was observed in all test substance-administered groups while epithelial hyperplasia was observed in the 50 and 150 mg/kg/day groups. The findings were not associated with clinical pathology changes but were of similar, although slightly less, severity when compared to the F0 generation and were considered adverse in the 150 mg/kg/day group males and females.
There were no test substance-related effects on ovarian primordial follicle counts in the 150 mg/kg/day group F1 females in Cohort 1A compared to the control group.
Other effects:
no effects observed
Description (incidence and severity):
- No test substance-related effects on anogenital distance were noted for F1 pups in the 20, 50, and 150 mg/kg/day groups. Longer anogenital distances (absolute and relative to the cube root of pup body weight) were noted for F1 male pups in the test substance-treated groups compared to the control group; differences were generally significant (p < 0.05 or p < 0.01). However, the aforementioned differe ces did not occur in a dose-related manner and the mean absolute values in the 20, 50, and 150 mg/kg/day groups (4.07 mm, 4.19 mm, and 4.17 mm, respectively) were within the range of values in the Charles River Ashland historical.
- Areolae/nipple anlagen in the F1 male pups was not affected by test substance administration. There was no retention of nipples noted in any male pup on study on PND 12.
- There were no test substance-related changes in total T4 or TSH in pups that were culled on PND 4.
- There were no test substance-related changes in serum T4 or TSH in F1 weanlings on PND 21.
- There were no test substance-related changes in serum T4 or TSH in F1 animals assigned to Cohort 1A.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: local effects (nonglandular stomach)

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion