Constitutional isomers of penta-O-allyl-β-D-fructofuranosylα-D-glucopyranoside; Constitutional isomers of hexa-O-allyl-β-D-fructofuranosylα-D-glucopyranoside; Constitutional isomers of hepta-O-allyl-β-D-fructofuransoylα-D-glucopyranoside

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-06-02 to 2015-03-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Constitutional isomers of penta-O-allyl-β-D-fructofuranosylα-D-glucopyranoside; Constitutional isomers of hexa-O-allyl-β-D-fructofuranosylα-D-glucopyranoside; Constitutional isomers of hepta-O-allyl-β-D-fructofuransoylα-D-glucopyranoside

EC Number:

419-640-0

EC Name:

Constitutional isomers of penta-O-allyl-β-D-fructofuranosylα-D-glucopyranoside; Constitutional isomers of hexa-O-allyl-β-D-fructofuranosylα-D-glucopyranoside; Constitutional isomers of hepta-O-allyl-β-D-fructofuransoylα-D-glucopyranoside

Cas Number:

68784-14-5

Molecular formula:

C3H4O to C36H54O11

IUPAC Name:

2-{[3,4-bis(prop-2-en-1-yloxy)-2,5-bis[(prop-2-en-1-yloxy)methyl]oxolan-2-yl]oxy}-3,4,5-tris(prop-2-en-1-yloxy)-6-[(prop-2-en-1-yloxy)methyl]oxane

Details on test material:

- Physical state: Light orange viscous liquid
- Lot/batch No.: 299664
- Expiration date of the lot/batch: 02-Jun-2016
- Purity: 100%
- Storage condition of test material: room temperature, protected from light and was considered stable under these conditions.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Crl:CD(SD) rats (66 males and 66 females) were received in good health from Charles River Laboratories, Inc., Stone Ridge, NY and Saint Constant, Quebec, Canada, respectively, on 05-Jun-2014. The animals were approximately 65 days old upon receipt.
Each animal was examined by a qualified biologist on the day of receipt. The day following receipt, all animals were weighed and clinical observations were recorded. At the initiation of dose administration (study day 0), the males and females were approximately 11 weeks old. Male body weights ranged from 314 g to
409 g and female body weights ranged from 238 g to 302 g on study day 0. The animals were approximately 13 weeks old when paired on study day 13; female body weights ranged from 259 g to 332 g on gestation day 0.
The animals were housed for an acclimation period of 11 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior, and the testes were palpated at least once for all males.
Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week. The 5 rats/sex in the control and 400 mg/kg/day groups that were assigned to the recovery phase were not paired and remained in clean, stainless steel wire-mesh cages until euthanasia. The breeding phase rats (10 rats/sex/group) were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The nesting material is periodically analyzed by the manufacturer for contaminants.
The dams and their litters were housed in
these cages until euthanasia on lactation day 4. Females that failed to deliver were housed in plastic maternity cages until post-mating day 25.
The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer. Feeders were changed and sanitized once per week.
Municipal water supplying the facility was sampled for contaminants.
Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study, with the following exception. All males and recovery phase females were fasted prior to clinical pathology blood collection when food, but not water, was withheld.
All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Actual mean daily temperature ranged from 70.6°F to
70.9°F (21.4°C to 21.6°C) and mean daily relative humidity ranged from 41.7% to 49.4% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

The experimental design consisted of 4 test item-treated groups and 1 control group composed of 10 rats/sex/group. An additional 5 rats/sex in the control, 400, and 750 mg/kg/day groups were selected to be evaluated following a 14-day non-dosing recovery period.
(Due to toxicity noted in the 750 mg/kg/day group following the first 3 days of dose administration, 5 rats/sex were arbitrarily added to the 400 mg/kg/day group and treated as recovery animals. Animals were dosed on the same dosing regimen as the animals that were initially assigned to study, beginning on 19-Jun-2014.)
The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily. The males selected for pairing were dosed during study days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females selected for pairing were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-43 doses.
Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39-40 doses. The extra 5 rats/sex in the control and 400 mg/kg/day groups were not used for mating, but were treated on a comparable regimen beginning on study day 0; following 28 doses for males and 39 doses for females, these animals were assigned to the recovery period and remained on study for a 14-day non-dosing period. The dosage volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
In a range-finding study male and female Sprague Dawley rats were dosed for 28 consecutive days at dosage levels of 15, 150, and 650 mg/kg/day. Slight body weight effects and increased liver weights (absolute and relative to final body weight)
were noted in both sexes at 650 mg/kg/day. No mortality was noted at any dosage level.
Based on these results, a high-dosage level of 750 mg/kg/day was chosen for the current study. Low- and mid-dosage levels were selected to evaluate the dose response of the test item.
The selected route of administration for this study was oral (gavage) because this is the potential route of human exposure. Historically, this route has been used extensively for studies of this nature.

Details on mating procedure:

The 10 rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained. Each female was housed in the home cage of the male during cohabitation. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages.
For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.
Mating, fertility, and copulation/conception indices were calculated as follows:
Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant)/ Total No. of Males (Females) Used for Mating x 100
Male Fertility Index (%) = No of males siring a litter / Total No. males used for mating x 100
Male Copulation Index (%) = No. of Males Siring a Litter/ No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy) x 100
Female Fertility Index (%) = No. of females with a confirmed pregnancy/Toptal 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 Confirmed Pregnancy) x 100

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for concentration and/or homogeneity determinations were collected from the top, middle, and bottom strata of each test item dosing formulation and the middle stratum of the control formulation prepared during the first and last weeks of dose administration. One set of samples from each collection was subjected to the appropriate analyses. The remaining set of samples was stored at room temperature, protected from light.

Formulations prepared at target concentrations of 20, 40, 80, and 150 mg/mL and used for dose administration were analyzed to assess test item
homogeneity and concentration acceptability. The results met the acceptance criteria for homogeneity, i.e., the (RSD) for the mean concentration was ≤10% at a
concentration within the acceptable limits (85% to 115% of the target concentration) and concentration acceptability for suspension formulations, i.e., the analyzed concentration was 85% to 115% of the target concentration. No test item was detected in the analyzed vehicle administered to the control group.
Method
PREPARATION OF CALIBRATION STOCK SOLUTION
A calibration stock solution is prepared at a concentration of 25.0 mg/mL as follows. Weigh approximately 50 mg of test item (no correction for purity) in a tared glass weigh boat and transfer to a 2-mL volumetric flask with rinses of ACN. Mix and/or sonicate the preparation as necessary to achieve complete dissolution. Add additional ACN to yield the desired concentration, and thoroughly mix the solution.
A secondary calibration stock solution is prepared at a concentration of 0.250 mg/mL by diluting the calibration stock solution 100-fold with DI water.
PREPARATION OF QC STOCK SOLUTION
A QC stock solution is prepared at a concentration of 25.0 mg/mL as follows. Weigh approximately 0.125 g of test item (no correction for purity) in a tared glass weigh boat and transfer to a 5-mL volumetric flask with rinses of ACN. Mix and/or sonicate the preparation as necessary to achieve complete dissolution. Add additional ACN to yield the desired concentration, and thoroughly mix the solution.
PREPARATION OF CALIBRATION STANDARDS
Prepare calibration standards at concentrations ranging from 20.0 to 200 μg/mL by combining aliquots of the calibration stock solution and DI water. Prepare at least triplicate calibration standards at each concentration for any validation sessions; prepare at least single calibration standards at each concentration for routine analyses.
PREPARATION AND PROCESSING OF QC SAMPLES
Prepare QC samples to simulate the processing of formulations at concentrations of 10.0 to 200 mg/mL (nominal QC concentrations) by combining aliquots of the appropriate QC stock solution, vehicle, and ACN in polypropylene tubes. Mix the samples with vortex action, inversion, shaking, and/or sonication. Further dilute the QC samples as needed with DI water to achieve a final diluted concentration within the calibration range. Prepare the QC samples in triplicate at each concentration; prepare a single vehicle blank sample. Alternate QC concentrations may be prepared within the validated range and using the same general dilution schemes.
FORMULATION SAMPLE COLLECTION AND PROCESSING
Collect samples from each formulation using a syringe and dosing cannula and place in polypropylene tubes. Process at least 2 samples from each set for analysis. Process the formulation samples by adding ACN and mix with vortex action. Further dilute portions of the processed samples as needed with DI water to achieve a final diluted concentration within the calibration range. Store processed formulation samples under appropriate conditions.
HPLC
Instrument: Agilent 1100 liquid chromatograph equipped with a variable wavelength detector and Refractive Index Detector, autosampler, and Dionex Chromeleon® software version 6.8, or equivalent system
Column: Phenomenex Shodex SUGAR, KS-801, 300 x 8.00 mm, 6-μm particle-size
Mobile Phase A: 100% DI water
Flow Rate: 1.00 mL/minute
Column Temperature: Ambient
Polarity: Positive
Autosampler Temperature: Ambient
Detector Temperature: 35°C
Injection Volume: 40 μL
Retention Time: Approximately 3.7 minutes for OS299664
Run Time: 20.0 minutes

Results
Under the described chromatographic conditions, the retention time of the test item was approximately 3.1 minutes.
Assay specificity/selectivity was confirmed when HPLC/RI analysis of processed control group formulation samples revealed no significant peaks (with S/N >10) at or near the retention time for the test item (approximately 3.1 minutes).
In addition to the experimental samples, each analytical session consisted of (but was not limited to) calibration standards at 6 concentrations and triplicate QC samples prepared at each of 3 concentrations. In this study, the formulations were prepared at target concentrations of 0, 20, 40, 80, and 150 mg/mL, and the QC samples were prepared at nominal concentrations of 10.0, 100, and 200 mg/mL. For an analytical session to be considered valid at least two-thirds of the calculated QC concentrations with at least 1 sample at each concentration had to be 85% to 115% of the nominal QC concentration.
All reported results were from analytical sessions that met the acceptance criteria.
Homogeneity and Concentration Assessment: The analyzed formulations met the acceptance criteria for homogeneity, i.e., the RSD for the mean concentration was ≤10% at a concentration within the acceptable limits (85% to 115% of target concentration) and concentration acceptability for suspension formulations, i.e., the analyzed concentration was 85% to 115% of the target concentration. No test item was detected in the analyzed vehicle administered to the control group (Group 1).

Duration of treatment / exposure:

28 days for males, 39 - 43 days for females

Frequency of treatment:

Once daily

Details on study schedule:

The males selected for pairing were dosed during study days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females selected for pairing were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-43 doses.
Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39-40 doses. The extra 5 rats/sex in the control and 400 mg/kg/day groups were not used for mating, but were treated on a comparable regimen beginning on study day 0; following 28 doses for males and 39 doses for females, these animals were assigned to the recovery period and remained on study for a 14-day non-dosing period.
SCHEDULED EUTHANASIA
On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded without further examination.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Control

Doses / concentrations 2

Remarks:

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

Doses / concentrations 3

Remarks:

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

Doses / concentrations 4

Remarks:

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

Doses / concentrations 5

Remarks:

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

No. of animals per sex per dose:

Control, 400 and 750 mg/kg bw/day dose level: 15 males/15 females
100, 200 and 400 mg/kg bw/day: 10 males/10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on result of a range finding study.

Positive control:

Not applicable.

Examinations

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS AND SURVIVAL
All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual detailed physical examinations were recorded weekly (prior to test item administration during the treatment period). Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.
BODY WEIGHTS
Individual male body weights were recorded weekly, beginning 1 week prior to test item administration, on the first day of dosing, and weekly thereafter until euthanasia.
Individual female body weights were recorded weekly, beginning 1 week prior to test item administration, on the first day of dosing, and weekly thereafter until evidence of copulation was observed or euthanasia (for females assigned to the recovery phase).
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 period (males and females) and for the entire generation (males and recovery phase females 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 0 (when possible), 1, and 4. Mean gestation body weights and corresponding mean body weight changes are presented for these intervals and for the overall gestation interval (days 0-20); mean lactation body weight changes are presented for lactation days 1-4. When body weights could not be determined for an animal during a given interval (due to an unscheduled death, as females enter gestation, weighing error, etc.), group mean values were calculated for that interval using the available data.
FOOD CONSUMPTION
Individual food consumption was recorded on the corresponding weekly body weight days until pairing (for animals paired for breeding) or euthanasia (for animals assigned to the recovery period). Food intake was not recorded during the breeding period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. Food consumption was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage.
When food consumption could not be determined for an animal during a given interval (due to an unscheduled death, weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data.
PARTURITION
All females selected for pairing were allowed to deliver naturally and rear their young to PND 4. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.
FOB ASSESSMENTS
FOB assessments were recorded for 5 animals/sex/group following approximately 28 days of dose administration (males selected for pairing) and on lactation day 4 (females). The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB. All animals were observed for the following parameters:
HOME CAGE OBSERVATIONS: Posture, Convulsions/Tremors, Feces consistency, Biting, Palpebral (eyelid) closure.
HANDLING OBSERVATIONS: Ease of removal from cage, Lacrimation/Chromodacryorrhea, Piloerection, Palpebral closure, Eye prominence, Red/Crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/Eye/Skin color, Muscle tone.
OPEN FIELD OBSERVATIONS: Mobility, Rearing, Convulsions/Tremors, Grooming, Bizarre/Stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/Defecation, Gait score, Backing, Note: Open field observations were evaluated over a 2-minute observation period.
SENSORY OBSERVATIONS: Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eyeblink response, Hindlimb extension, Olfactory orientation.
NEUROMUSCULAR OBSERVATIONS: Hindlimb extensor strength, Hindlimb foot splay, Grip strength-hind and forelimb, Rotarod performance
PHYSIOLOGICAL OBSERVATIONS: Catalepsy, Body temperature, Body weight
MOTOR ACTIVITY
Motor activity was assessed for 5 animals/sex/group following approximately 28 days of dose administration (males selected for pairing) or on lactation day 4 (females). Motor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilizes a series of infrared photobeams surrounding an amber plastic, rectangular cage to quantify each animal’s motor activity.
Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB. Each animal was tested separately. Data were collected in 5-minute epochs (print intervals) and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).
CLINICAL PATHOLOGY
Blood samples for clinical pathology evaluations (hematology and coagulation and serum chemistry) were collected from 5 animals/sex/group at the scheduled necropsies (study day 28 for males and lactation day 4 for females that were selected for pairing) and from 5 animals/sex in the control and 400 mg/kg/day groups following a 14-day non-dosing recovery period. The same animals selected for FOB and locomotor activity assessments were selected for clinical pathology assessment. All males (including those not scheduled for clinical pathology assessments) and the recovery phase females were fasted overnight prior to blood collection, with water available ad libitum. Blood for serum chemistry and hematology was collected from the retro-orbital sinus following isoflurane anesthesia. Blood for coagulation parameters was collected from the inferior vena cava at the time of necropsy. Blood was collected into tubes containing EDTA (hematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry).
HEMATOLOGY AND COAGULATION: Total leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin, (MCH), Mean corpuscular hemoglobin, concentration (MCHC), Platelet count (PLATELET), 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), Hemoglobin distribution width, (HDW), Platelet estimate, Red cell morphology (RBC Morphology),
SERUM CHEMISTRY: 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 glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium Chloride, Phosphorus, Potassium, Sodium Triglycerides (Triglyceride), Bile acids, Appearance (Includes degree of hemolysis, lipemia, and icterus)
( ) = Designates tabular abbreviation

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not examined

Litter observations:

LITTER VIABILITY AND DEATHS
Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring that were found dead were necropsied using a fresh dissection technique, which included examination of the heart and major vessels.
CLINICAL OBSERVATIONS
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nesting and nursing behavior were recorded.
BODY WEIGHTS
Pups were individually weighed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by dose group. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data.
SEX DETERMINATION
Pups were individually sexed on PND 0 and 4.

Postmortem examinations (parental animals):

ANATOMIC PATHOLOGY
MACROSCOPIC EXAMINATION: A complete necropsy was conducted on all F0 parental animals found dead, euthanized in extremis, euthanized due to early termination, or at the scheduled termination. All surviving F0 adults were euthanized by carbon dioxide inhalation. Males selected for pairing were euthanized following completion of the mating period. Females that delivered were euthanized on lactation day 4; the number of former implantation sites and corpora lutea were recorded. The number of implantation sites and corpora lutea were recorded for the female that was euthanized in extremis during lactation; recognizable retained fetuses were examined for gross abnormalities. Females that failed to deliver were euthanized on post-mating day 25; uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss. Males and females not selected for pairing were euthanized following a 14-day non-dosing recovery period. Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.
Clinical findings that were verified at necropsy were designated CEO. At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin (except as noted); no tissues were collected from animals in the 750 mg/kg/day group that were euthanized due to early termination.
Adrenal glands (2), Lungs (including bronchi, fixed by inflation with fixative), Aorta, Bone with marrow (sternebrae), Ovaries and oviducts (2), Brain Pancreas, Coagulating glands (2), Peripheral nerve (sciatic), Eyes with optic nerve (2)a, Pituitary gland, Gastrointestinal tract; Esophagus Stomach Duodenum Jejunum Ileum Cecum Colon Rectum, Prostate gland, Salivary gland [mandibular (2)], Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary gland b, Spinal cord (cervical), Spleen, Testes with epididymides c (2) and vas deferens, Heart, Thymus gland, Kidneys (2), Thyroids [with parathyroids if present (2)], Liver, (sections of 2 lobes) , Lymph node; Axillary (2) Mandibular (2) Mesenteric, Trachea, Urinary bladder, Uterus d with vagina, All gross lesions.
a = Placed in Davidson’s solution
b = For females; a corresponding section of skin was taken from the same anatomic area for males.
c = Placed in modified Davidson’s solution. Care was taken to ensure separation between the left and right organs.
d = Any uterus that was placed in 10% ammonium sulfide solution for detection of implantation sites was discarded and not preserved in 10% neutral-buffered formalin.
ORGAN WEIGHTS
The following organs were weighed from all F0 animals at the scheduled necropsies:
Adrenal glands, Ovaries with oviducts, Brain, Spleen, Epididymides a, Testes a, Heart, Thymus gland, Kidneys, Thyroids with parathyroids b, Liver
a = These paired organs were weighed separately. Care was taken to ensure separation between the left and right organs.
b = Fixed in 10% neutral-buffered formalin prior to weighing.
Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight ratios and organ to brain weight ratios were reported.
HISTOLOGY AND MICROSCOPIC EXAMINATIONS: After fixation, protocol-specified tissues were trimmed. Trimmed tissues were processed into paraffin blocks, sectioned, mounted on glass microscope slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.
Microscopic examination was performed on all tissues listed above from all animals in the control and 400 mg/kg/day groups at the scheduled necropsies and from all animals found dead or euthanized in extremis. In addition, the liver was identified as a potential target tissue and was examined from all males and females in all groups at the primary and recovery necropsies. Gross lesions from animals in all groups were also examined.

Postmortem examinations (offspring):

Not performed

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group by sex.
Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, pre-coital intervals, gestation length, numbers of former implantation sites, corpora lutea, and unaccounted-for sites, number of pups born, live litter size on PND 0, absolute and relative organ weights, clinical pathology values, and FOB data values were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test item-treated groups to the control group. FOB parameters that yield scalar or descriptive data in the test item-treated groups were compared to the
control group using Fisher’s Exact test (Steel and Torrie, 1980). Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test item-treated groups to the control group. Histopathologic findings in the test item-treated groups were compared to the control group using a two-tailed Fisher’s Exact test (Steel and Torrie, 1980).
FOB
All analyses were conducted using SAS version 9.2

Reproductive indices:

For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.
Mating, fertility, and copulation/conception indices were calculated as follows:
Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant)/ Total No. of Males (Females) Used for Mating x 100
Male Fertility Index (%) = No of males siring a litter / Total No. males used for mating x 100
Male Copulation Index (%) = No. of Males Siring a Litter/ No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy) x 100
Female Fertility Index (%) = No. of females with a confirmed pregnancy/Toptal 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 Confirmed Pregnancy) x 100

Offspring viability indices:

Litter parameters were defined as follows:
Mean Live Litter Size = Total No. of Viable Pups on PND 0 / No. of Litters with Viable Pups PND 0
Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) = Sum of (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter)* / No. of Litters Per Group x 100
Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)* / No. of Litters Per Group x 100
Where N = PND 0-1 and 1-4
* = Pups that were euthanized due to death of the dam were excluded from pup viability calculations.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mortality at 750 mg/kg bw/day

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

significantly (p<0.05) higher mean body weight gain in the 400 mg/kg/day group during study days 35-41 of the recovery period. Not considered adverse

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

significantly (p<0.05) higher mean body weight gain in the 400 mg/kg/day group during study days 35-41 of the recovery period. Not considered adverse

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see detailed results section

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL OBSERVATIONS AND SURVIVAL
Four males and 3 females in the 750 mg/kg/day group were found dead or euthanized in extremis on study day 3 or 4. Due to the excessive toxicity observed at 750 mg/kg/day, as evidenced by clinical findings that included slight to severe tremors, prostration, cool and pale body, labored respiration, unkempt appearance, and red, clear, and/or yellow material on various body surfaces at the detailed physical examinations and/or approximately 1 hour post-dosing observations, the surviving males and females in this group was euthanized on study day 4.
Female no. 20622 in the 400 mg/kg/day group was found dead on study day 12. The only remarkable clinical findings noted for female no. 20622 prior to death were red material around the nose and mouth at 1 hour following dose administration during study days 1-10. Microscopic findings for this female determined the cause of death to be severe renal tubular necrosis. Because similar microscopic findings were not observed in the 750 mg/kg/day group, the death in the 400 mg/kg/day group was not attributed to the test item. In the 200 mg/kg/day group, female no. 20626 was euthanized in extremis on lactation day 1. This female was noted with red and/or yellow material around the nose and mouth during study day 7 to gestation day 12 and had an unkempt appearance and yellow material around the urogenital area on study day 10 at 1 hour following dose administration. Based upon macroscopic and microscopic findings the cause of moribundity of this female was determined to be moderate hepatocellular and severe renal tubular necrosis and was not attributed to test item administration because similar microscopic findings were not observed at 750 mg/kg/day. All other animals survived to the scheduled necropsies.
Test item-related clinical findings of red material around the nose and red, yellow, and/or clear material around the mouth were noted in a dose-related manner for males and females in the 100, 200, and 400 mg/kg/day groups at approximately 1 hour following dose administration, generally throughout the dosing period. These findings generally did not persist to the following day and in the absence of any other signs of toxicity for F0 animals the aforementioned material findings were not considered to be adverse.
Other clinical findings noted in the test item-treated groups at the detailed physical examinations or approximately 1 hour following dose administration, including hair loss on various body surfaces and yellow material on the anogential and urogenital areas, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related and therefore were not attributed to the test item. Clinical findings noted during the recovery period in the 400 mg/kg/day group were limited to hair loss on various body surfaces.
BODY WEIGHTS
Males: Mean body weights and body weight gains in the 100, 200, and 400 mg/kg/day group males were unaffected by test item administration throughout the study. None of the differences from the control group were statistically significant, with the exception of a significantly (p<0.05) higher mean body weight gain in the 400 mg/kg/day group during study days 35-41 of the recovery period.
Females: Mean body weights and body weight gains in the 100, 200, and 400 mg/kg/day group females were unaffected by test item administration during the pre-mating or entire dosing and recovery periods. Differences from the control group were slight and not statistically significant, with the following exceptions. A significantly (p<0.05) higher mean body weight gain was noted for the 400 mg/kg/day group females assigned to the recovery period during study days 35-39 and a significant (p<0.01) mean body weight loss was noted for the 400 mg/kg/day group females during study days 39-46 of the recovery period, which resulted in a significantly (p<0.05) lower mean body weight gain when the entire recovery period (study days 39-52) was evaluated. In the absence of body weight effects during the dosing period and effects on absolute body weights, these differences were not attributed to the test item.
GESTATION: Mean body weights and body weight gains in the 100, 200, and 400 mg/kg/day groups were unaffected by test item administration during gestation. Differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner
LACTATION: Mean body weights and body weight gains in the 100, 200, and 400 mg/kg/day groups were unaffected by test item administration during lactation days 1-4. None of the differences from the control group were statistically significant.
FOOD CONSUMPTION
Males: Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 200, and 400 mg/kg/day group males was similar to that in the control group throughout the study.
Differences from the control group were slight and not statistically significant, with the following exceptions. Significantly (p<0.05) lower mean food consumption (g/kg/day value only) was noted for the 200 mg/kg/day group males during study days 0-7. In the absence of a dose response and lack of corresponding effects on body weights and body weight gains, this difference was not attributed to test item administration. During the recovery period, a significantly (p<0.05) higher g/kg/day value was noted for the 400 mg/kg/day group males compared to the control group during study days 28-35. In the absence of test item-related effects on food consumption during the dosing period, this difference was not attributed to test item administration.
Females:
Weekly: Significantly (p<0.01) lower mean food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 400 mg/kg/day group females compared to the control group during the first week of the dosing period (study days 0-7) which resulted in significantly (p<0.05 or p<0.01) lower mean food consumption for these animals when the pre-mating period (study days 0-13) was evaluated. This effect on mean food consumption during the first week of dosing was considered test item-related. However, because the lower food consumption was not sustained and in the absence of corresponding effects on body weight change, the lower food consumption at 400 mg/kg/day was not considered adverse.
Mean food consumption in the 100 and 200 mg/kg/day group females was unaffected by test item administration during the pre-mating period. None of the differences from the control group were statistically significant.
Mean food consumption in the 400 mg/kg/day group during the recovery period was similar to the control group. Differences from the control group were slight and not statistically significant.
Gestation: Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 200, and 400 mg/kg/day groups was unaffected by test item administration during gestation. Differences from the control group were not statistically significant and/or did not occur in a dose-related manner.
Lactation: Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 200, and 400 mg/kg/day groups was unaffected by test item administration during lactation days 1-4. None of the differences from the control group were statistically significant.
FOB
Home cage observations: Home cage parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group on study day 25 (males) or on lactation day 4 (females).
Handling operations: Handling parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group on study day 25 (males) or on lactation day 4 (females).
Open field observations: Open field parameters were unaffected by test item administration. A significantly (p<0.05) higher number of grooming counts were noted for the 400 mg/kg/day group males. However, there were no corresponding clinical findings noted at the detailed physical examination or post-dosing observations and there were no effects on similar endpoints in the FOB or on motor activity; therefore, this finding was not considered test item-related. There were no other statistically significant differences for the test item-treated groups when compared to the control group on study day 25 (males) or on
lactation day 4 (females).
Sensory observations: Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group on study day 25 (males) or on lactation day 4 (females).
Neuromuscular observations: Neuromuscular parameters were unaffected by test item administration. Significantly (p<0.05 or p<0.01) lower rotarod performance was noted for the 100 and 200 mg/kg/day group males. In the absence of a dose response, the differences in rotarod performance observed in the 100 and 200 mg/kg/day groups were not attributed to test item administration. There were no other statistically significant differences for the test item-treated groups when compared to the control group on study day 25 (males) or on lactation day 4 (females).
Physiological observations: Physiological parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group on study day 25 (males) or on lactation day 4 (females).
Motor activity: Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test item administration at all concentrations when evaluated on study day 25 (males) and lactation day 4 (females). Values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the historical control data. Differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the historical control data ranges, and/or did not occur in a dose-related manner.
No remarkable shifts in the pattern of habituation occurred in any of thetest item treated groups when the F0 animals were evaluated on study day 25 (males) or on lactation day 4 (females).
CLINICAL PATHOLOGY
HEMATOLOGY AND COAGULATION: Test item-related higher reticulocyte counts were present.
Significantly (p<0.05) higher percent reticulocyte and absolute reticulocyte counts were noted at 400 mg/kg/day in both dosing (study day 28) and recovery (study week 6) sacrifice males.
Recovery 400 mg/kg/day group males had a significantly (p<0.01) lower hemoglobin (Hb) value. Minor shifts in the leukogram were also noted in 400 mg/kg/day group recovery males. The percent of neutrophils was higher and the percent of lymphocytes was lower; differences from the control group were significant (p<0.05). Similar statistically significant changes in the leukogram were not noted for females at either scheduled sacrifice; however, significantly (p<0.05 or p<0.01) lower mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and percent reticulocytes were present in 400 mg/kg/day group recovery females. The 400 mg/kg/day group females had a lower activated partial thromboplastin time (APTT) and higher HDW on lactation day 4; differences from the control group were significant (p<0.05 or p<0.01). Recovery males had lower prothrombin time (PT), higher MPV, and higher RDW; differences from the control group were significant (p<0.05 or p<0.01). Significantly (p<0.05) higher platelet counts were noted for the 400 mg/kg/day group recovery females. Notably, almost all statistically significant hematology and coagulation parameters were within the historical control ranges for rats 13-15 weeks.
There were no other test item-related effects on hematology or coagulation parameters although some statistically significant differences were observed when the control and test item-treated groups were compared.
SERUM CHEMISTRY: The test item was associated with dose-related higher protein levels in the blood of males on study day 28.
Significantly (p<0.05 or p<0.01) higher protein findings included higher albumin (400 mg/kg/day), total protein (200 and 400 mg/kg/day), and globulin (200 and 400 mg/kg/day) for males. These findings were not mirrored in females. And all were within or close to the historical control ranges for rats of this age group.
A significantly (p<0.05) higher ALT and glucose occurred in 400 mg/kg/day group recovery males. Significantly (p<0.05) higher phosphorus occurred in 200 and 400 mg/kg/day group females on lactation day 4.
Statistically significantly lower chloride (p<0.01) in the 400 mg/kg/day males, lower creatinine (p<0.05) in the 400 mg/kg/day females, and lower total bilirubin (p<0.01) in the 400 mg/kg/day recovery females were all within normal historical control ranges.

REPRODUCTIVE PERFORMANCE
See attached document for tables
No test item-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test item-treated groups. One male each in the control and 200 mg/kg/day groups did not sire a litter. One female each in the same respective groups had evidence of mating but did not deliver.
The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. None of these differences were statistically significant.
GESTATION LENGTH AND PARTURITION
Mean gestation lengths in the 100, 200, and 400 mg/kg/day groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.

ANATOMIC PATHOLOGY
MACROSCOPIC EXAMINATIONS: Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test item.
Most gross necropsy observations which occurred in early death 750 mg/kg/day group rats were characteristic for rats of this age and/or reproductive status or were of an incidence and/or dose group distribution insufficient to be considered a test item effect.
A uterine mass in 400 mg/kg/day group female no. 20627 was composed of retained placental tissue. The swollen liver of a 400 mg/kg/day group female had no histologic correlate.
The mean numbers of unaccounted-for sites and implantation sites in the 100, 200, and 400 mg/kg/day groups were similar to the control group values.
ORGAN WEIGHTS: Test item-related higher liver weights were noted in the 100, 200, and 400 mg/kg/day
dose groups of both sexes in a dose-related manner.
Significantly (p<0.05 or p<0.01) higher absolute liver (200 and 400 mg/kg/day), liver to body weight, and liver to brain weight ratios were present in males at 100, 200, and 400 mg/kg/day. Absolute mean liver weights were higher (compared to concurrent controls) by 18.2%, 21%, and 51.9% in the respective male 100, 200, and 400 mg/kg/day dose groups. In females liver absolute and relative to body and brain weight ratios were higher in a dose-dependent way with significant (p<0.01) increases at 200 and 400 mg/kg/day. Absolute mean liver weights were higher (compared to concurrent controls) by 14.9%, 28.5%, and 50.7% in the respective female 100, 200, and 400 mg/kg/day dose groups. These statistically significantly higher values persisted in recovery 400 mg/kg/day animals of both sexes (males absolute and liver to body weight) (females absolute and relative to both body and brain weight). Absolute mean liver
weights were higher (compared to concurrent controls) by 22.8% and 17.5% in the respective male and female 400 mg/kg/day dose groups.
Significantly (p<0.05 or p<0.01) higher absolute thyroid/parathyroid weights, thyroid/parathyroid to body weight, and thyroid/parathyroid to brain weight ratios were present in males at 400 mg/kg/day (also relative to brain weight at 100 mg/kg/day) on study day 28 and in females at 400 mg/kg/day during the recovery period. The relationship to test item administration is inconclusive.
There were no other test item-related effects on organ weights. A decreased left epididymis to brain weight ratio was significant (p<0.05) in 400 mg/kg/day group males during the recovery period, but this was considered spurious.
MICROSCOPIC EXAMINATIONS: Test item-related microscopic findings were limited to the liver in those animals surviving to the primary necropsy.
Early death animals had additional observations in the stomach, kidney, and secondarily the thymus.
Early Death Animals:
Significant observations are presented in the following tabular form. Every early death animal had liver observations. One male and 2 females at 750 mg/kg/day had necrosis in the glandular stomach. Thymic atrophy and or apoptosis were attributed to stress. Two early deaths (200 and 400 mg/kg/day group females) had severe tubular necrosis in the kidneys. These observations lacked an associated inflammatory response and were hence initially suggestive of autolysis except that the other tissues examined in these animals lacked sufficient concurrent autolysis.
Primary necropsy:
The only finding that was attributed directly to the test item in scheduled sacrifice animals was periportal to midzonal hepatocellular vacuolation in male rats at 100 mg/kg/day and above. Hepatocellular vacuolation was not considered to be a test item effect in female rats as it was minimal and occurred in only 2 animals (one at 100 mg/kg/day and one at 400 mg/kg/day).
Three 400 mg/kg/day group males had observations of hepatocellular necrosis; however, these observations were subcapsular or randomly located and minimal in severity suggesting no relationship to treatment.
Thymic atrophy was secondarily present in four 400 mg/kg/day group females.
There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test item. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Female no. 20644 had an unusual finding in an ovary of a benign sertoli’s cell tumor.
Female no. 20627 had an intrauterine mass composed of retained placental tissue.
There were no discernible test item-related reproductive effects. Two 400 mg/kg/day testes from 2 different rats had unilateral focal (1 tubule) degeneration. This was not related to the test item.
Recovery necropsy:
There were no test item-related effects in recovery animals. Hepatocellular vacuolation, although restricted to individual hepatocytes in one 0 mg/kg/day rat of either sex, had the same overall incidences between 0 mg/kg/day and 400 mg/kg/day recovery animals of each sex.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: lack of adverse test item-related effects in the 100, 200, and 400 mg/kg/day groups

2

Dose descriptor:

NOAEL

Remarks:

Reproductive effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test item-related effects on F0 male and female fertility and mating indices, F0 male copulation index, F0 female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

PND 0 LITTER DATA AND POSTNATAL SURVIVAL
The mean number of pups born, live litter size, and the percentage of males at birth in the 100, 200, and 400 mg/kg/day groups were similar to the control group values. Postnatal survival in the 100, 200, and 400 mg/kg/day groups were unaffected by test item administration.
GENERAL PHYSICAL CONDITION
The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by parental administration of the test item. Pups that were found dead numbered 3(2), 2(2), 8(4), and 8(6) pups (litters) in the control, 100, 200, and 400 mg/kg/day groups, respectively. One, 4, and 2 pups in the control, 100, and 200 mg/kg/day groups, respectively, was missing and presumed to have been cannibalized. In addition, 11 pups in the 200 mg/kg/day group were euthanized due to the death of dam no. 20626.
OFFSPRING BODY WEIGHTS
Mean male and female pup body weights and body weight changes during PND 1-4 in the 100, 200, and 400 mg/kg/day groups were unaffected by parental administration of the test item. Mean birth body weights (PND 1) in the 400 mg/kg/day group males and females were slightly lower (not statistically significant) than the control group values and the minimum mean values in the WIL Research historical control data. However, because these differences were small and did not reach statistical significance they were not considered to be test item-related.
NECROPSIES OF PUPS FOUND DEAD OR EUTHANIZED IN EXTREMIS
The numbers of pups (litters) found dead during PND 0-4 numbered 3(2), 2(2), 8(4), and 8(6) in the control, 100, 200, and 400 mg/kg/day groups, respectively. Aside from the presence or absence of milk in the stomach, no other internal findings were noted in the test item groups. One pup in the control group had a developmental variation of renal papilla(e) not developed.
NECROPSIES OF PUPS EUTHANIZED DUE TO DEATH OF DAM
In the 200 mg/kg/day group, 11 fetuses in litter no. 20626 were euthanized due to the death of the dam on lactation day 1. Aside from the presence of milk in the stomach, internal findings of renal papilla(e) not fully developed (Woo and Hoar grade 1) and a developmental variation of renal papilla(e) not developed were noted for 1 fetus each.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this screening study, no test item-related effects on F0 male and female fertility and mating indices, F0 male copulation index, F0 female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 400 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity, the NOAEL for systemic toxicity was considered to be 400 mg/kg/day based on the lack of adverse test item-related effects in the 100, 200, and 400 mg/kg/day groups and the NOAEL for neonatal toxicity was 400 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.

Executive summary:

Test Guidance

OECD Guideline No. 422

Method and materials

This study was designed to investigate the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development. The dosing regimen was followed by a 14-day recovery period to evaluate the recovery, persistence, or progression of any toxic effects.

The test item in the vehicle (100% polyethylene glycol 400) was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The 400 and 750 mg/kg/day groups (Groups 4 and 5, respectively) consisted of 15 rats/sex and the 100 and 200 mg/kg/day groups (Groups 2 and 3, respectively) consisted of 10 rats/sex. The dosage volume was 5 mL/kg. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 11 weeks of age at the beginning of test item administration. Ten males/group received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Ten females/group received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-43 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39-40 doses. The extra 5 rats/sex in the control and 400 mg/kg/day groups were assigned to the recovery phase and remained on study for an additional 14-day non-dosing recovery period following 28 and 39 doses, respectively. Due to mortality and excessive toxicity in the 750 mg/kg/day group, this group was terminated following 4 days of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group following approximately 28 days of dose administration and for 5 females/group on lactation day 4. All F0 females selected for pairing were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (hematology and coagulation and serum chemistry) were performed on 5 F0 animals/sex/group at the scheduled necropsy following the dosing period and from 5 F0 animals/sex in the control and 400 mg/kg/day groups at the recovery necropsy. F0 males were euthanized following completion of the mating period or 14-day recovery period and F0 females were euthanized on lactation day 4 for females that delivered, on post-mating day 25 for females that failed to deliver, or following the 14-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals at the primary and recovery necropsies.

Results

Test item-related moribundity and mortality was noted for F0 males and females in the 750 mg/kg/day group. Four males and 3 females in this group were found dead or euthanized in extremis on study day 3 or 4. Due to the excessive toxicity observed at 750 mg/kg/day, as evidenced by clinical findings that included slight to severe tremors, prostration, cool and pale body, labored respiration, unkempt appearance, and red, clear, and/or yellow material on various body surfaces at the detailed physical examinations and/or 1 hour post-dosing observations, the surviving males and females in this group was euthanized on study day 4. One female in the 400 mg/kg/day group was found dead on study day 12. The cause of death for this female was determined to be severe renal tubular necrosis. In addition, 1 female in the 200 mg/kg/day group was euthanized in extremis on lactation day 1. The cause of moribundity of this female was determined to be liver and kidney necrosis. Because the microscopic findings noted in the females that were found dead or euthanized in extremis were not observed at 750 mg/kg/day, the death at 400 mg/kg/day and moribundity at 200 mg/kg/day were not attributed to the test item. All other animals survived to the scheduled necropsies. Test item-related clinical findings of red material around the nose and red, yellow, and clear material around the mouth were noted in a dose-related manner for males and females in the 100, 200, and 400 mg/kg/day groups at approximately 1 hour following dose administration, generally throughout the treatment period. These findings generally did not persist to the following day and in the absence of any other signs of toxicity for F0 animals the aforementioned clinical findings were not considered to be adverse. No test item-related effects on F0 body weight or food consumption parameters were noted for males and females at any dosage level. There were no test item-related effects noted on functional observational battery parameters or motor activity for F0 animals at any dosage level. Mean male and female mating, fertility, copulation, and conception indices in the 100, 200, and 400 mg/kg/day groups were unaffected by test item administration. The mean numbers of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration. There were no test item-related gross necropsy observations noted for males and females at any dosage level.

The mean numbers of former implantation sites and F1 pups born, live litter size, percentage of males at birth, postnatal survival, the general physical condition of the F1 pups, and pup body weights and body weight gains in the 100, 200, and 400 mg/kg/day groups were unaffected by parental test item administration. Necropsy findings for F1 pups that died or were euthanized or due to death of dam were not suggestive of any association with maternal administration of the test item. Test item-related higher liver weights (absolute and/or relative to body weight and brain weight) were noted for males and females in all test item-treated groups following the dosing period and these effects persisted into the recovery period for the 400 mg/kg/day group males and females. Higher mean thyroid/parathyroid weights (absolute and relative to brain and body weight) were noted for the 400 mg/kg/day group males at the

end of the dosing period; these effects did not persist to the recovery necropsy and therefore the relationship to the test item is inconclusive. Test item-related higher reticulocyte counts were noted in the 400 mg/kg/day group males on study day 28 and at the recovery necropsy. In addition, higher serum protein levels (albumin, total protein, and/or globulin) were noted for the 200 and 400 mg/kg/day group males on study day 28 and higher mean phosphorus levels were noted for females in the 200 and 400 mg/kg/day groups on lactation day 4. Test item-related periportal to midzonal hepatocellular vacuolation was noted in males at all dosage levels on study day 28 and was no longer present for the 400 mg/kg/day group males at the recovery necropsy. This finding was generally minimal and difficult to attribute to the increase in liver weights, and was therefore not considered to be adverse.

Conclusion

Under the conditions of this screening study, no test item-related effects on F0 male and female fertility and mating indices, F0 male copulation index, F0 female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 400 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 400 mg/kg/day based on the lack of adverse test item-related effects in the 100, 200, and 400 mg/kg/day groups. The NOAEL for neonatal toxicity was 400 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.