Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 October 2014 to 09 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
various deviations with no negative impact on study (see Appendix A, attached)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
yes
Remarks:
various deviations with no negative impact on study (see Appendix A, attached)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance/physical state: Clear colourless liquid
- Storage conditions: Room temperature in the dark

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION PERIOD
- Sexually mature male and virgin female Crl:CD(SD) rats were used as the test system on this study. This species and strain of animal is recognized as appropriate for reproduction studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, WIL Research has reproductive historical control data for the Crl:CD(SD) rat.
- The number of animals selected for the study (25 rats/sex/group) was based on the United States EPA Health Effects Test Guidelines: OPPTS 870.3800, Reproduction and Fertility Effects, Aug-1998 and the OECD Guidelines for the Testing of Chemicals Guideline 416, Two-Generation Reproduction Toxicity Study, 22-Jan-2001, which recommend evaluation of approximately, and not less than, 20 pregnant females.
- Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality in each generation of the study, this was an appropriate number of animals to achieve the desired number of animals in each generation.
- Crl:CD(SD) rats (110 males and 110 females) were received in good health from Charles River Laboratories, Inc., Stone Ridge, NY, on 23-Oct-2014.
- The animals were approximately 32 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.
- Each rat was uniquely identified by a Monel metal ear tag displaying the animal number and housed for 12 days for acclimation purposes.
- During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior.

F0, F1 AND F2 ANIMAL HOUSING
- Following receipt (F0) or selection (F1) and until cohabitation, all F0 and F1 parental animals were housed 2-3 per cage by sex in clean, solid-bottom cages with bedding material (Bed-O'Cobs; The Andersons, Cob Products Division, Maumee, OH). - The bedding material is periodically analyzed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research.
- The rats were paired for mating in a clean, solid-bottom cages with bedding material. Following the breeding period, the F0 and F1 males were returned to their original social group of 2-3 males until the scheduled necropsy of the parental generations.
- Following positive evidence of mating, the F0 and F1 females were individually housed in plastic maternity cages with bedding material, ground corncob bedding.
- The dams were housed in these cages until weaning on lactation day 21. Following weaning of the litters in each generation (F1 and F2), the dams were returned to their original social group of 2-3 females until the scheduled necropsy of the parental generations, and 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 pairing.
- Females for which there was no evidence of mating were placed in solid-bottom cages with nesting material upon completion of a 14-day mating period.
- Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at WIL Research are accredited by AAALAC International.
- 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, DRINKING WATER AND MAINTENANCE OF F0 AND F1 ANIMALS
- 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 and provided to WIL Research. Feed lots used during the study were documented in the study records.
- Feeders were changed and sanitized once per week.
- Municipal water supplying the facility was sampled for contaminants according to WIL Research SOPs. Results of the diet and water analyses are maintained at WIL Research.
- No contaminants were present in animal feed or water at concentrations
sufficient to interfere with the objectives of this study.
- 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.

ENVIRONMENTAL CONDITIONS
- 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 68.2°F to 70.5°F (20.1°C to 21.4°C) and mean daily relative humidity ranged from 33.6% to 62.4% 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:
arachis oil
Details on exposure:
VEHICLE
The vehicle used in preparation of the test substance formulations and for administration to the control group was arachis (peanut) oil, NF (lot nos. 2DK0142, 1DB0384, 2DF0016, 2EA0218, and 2EA0347; exp. dates: 31-Oct-2015, 31-Jan-2015, 30-Apr-2015, 31-Jan-2016, and 31-Jan-2016, respectively).

PREPARATION
- The vehicle suspension was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light.
- The vehicle was mixed throughout the preparation, sampling, and dose administration procedures. Dosing formulations were prepared at the test substance concentrations indicated in the table below.
- The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
- The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

ORGANISATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- The test substance and vehicle control formulations were administered to the F0 and F1 males and females orally by gavage, via an appropriately sized flexible, Teflon-shafted, stainless steel ball-tipped dosing cannula, once daily for a minimum of 70 consecutive days prior to mating.
- Dose administration for the F0 and F1 males continued throughout mating and through the day prior to euthanasia.
- The F0 and F1 females continued to be dosed throughout mating, gestation, and lactation, through the day prior to euthanasia.
- The F0 males and females were dosed for 128-133 consecutive days, and F1 males and females were directly dosed for 136-147 consecutive days.
- A dosage volume of 5 mL/kg was used. 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.
- Dosage levels were selected by the Sponsor based on the results of previous studies with the test substance.
- In a previous developmental toxicity study (Herberth, 2015, WIL-168214), rats were administered the test substance from gestation days 6-19 at dosage levels of 250, 500, and 1000 mg/kg/day. All animals survived to the scheduled euthanasia on gestation day 20 and there were no significant clinical observations noted in any group during the study. Lower fetal body weights (male and female) were observed in all dose groups in the previous study; however, the reductions were most significant at 1000 mg/kg/day. As a result, dosage levels of 25, 100, and 400 mg/kg/day were chosen for use on the current study.
- The offspring of the F0 and F1 generations (F1 and F2 litters, respectively) in the present study were potentially exposed to the test substance in utero, as well as via the milk while nursing. The F1 pups selected for mating (25 sex/group) were directly administered the test substance following weaning (beginning on PND 21).
- The selected route of administration for this study was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Details on mating procedure:
BREEDING PROCEDURES
- The animals were paired on a 1:1 basis within each treatment group after a minimum of 70 days of treatment.
- All animals were randomly selected for cohabitation, avoiding sibling matings.
- A breeding record containing the male and female identification
numbers and the start date of cohabitation was maintained.
- Each female was cohabitated with 1 male in a solid-bottom cage containing bedding material.
- Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in 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. The animals were separated, and the female was housed in an individual solid-bottom cage with bedding material.
- When evidence of mating was not apparent after 14 days, the female was placed in solid-bottom cages with bedding material, with no further opportunity for mating.
- Each generation was mated once to produce 1 litter per dam (the F1 and F2 litters). – Prior to the F0 cohabitation (study day 70), male body weights ranged from 374 g to 744 g and female body weights ranged from 211 g to 322 g. The animals were approximately 16 weeks old. All animals were randomly selected for pairing.
- Prior to the F1 cohabitation (PND 98), male body weights ranged from 407 g to 638 g and female body weights ranged from 222 g to 381 g. The animals were approximately 14-15 weeks old.
- For the purpose of calculating pre-coital intervals, rats cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLING AND ANALYSIS
- Homogeneity of the test substance in the vehicle for a minimum of 7 days was previously demonstrated at concentrations spanning the range used in the current study (Haas, 2015, WIL-168211). In addition, stability of the test substance in the vehicle was demonstrated in a previous validation study (Van Hoven et al., 2014).
- Samples for homogeneity and/or concentration determinations were collected from the top, middle, and bottom strata of each test substance dosing formulation and from the middle stratum of the control group formulations prepared during the first month of the study, and once a month thereafter for the remainder of the study.
- In addition, due to changes in batch size from previous studies, samples for resuspension homogeneity determination were collected from the top and bottom strata of an aliquot taken from the first test substance dosing formulations prepared on study following room temperature storage for 8 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection.
- All samples were stored at room temperature, protected from light until shipped to Wildlife International, Easton, MD, for analysis. All analyses were conducted using a validated method.
Duration of treatment / exposure:
- The F0 males and females were dosed for 128-133 consecutive days.
- The F1 males and females were directly dosed for 136-147 consecutive days.
Frequency of treatment:
Daily
Details on study schedule:
- See diagram summarising study design (attached).
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 males and 25 females
Control animals:
yes, concurrent vehicle
Details on study design:
ASSIGNMENT OF F0 ANIMALS TO TREATMENT GROUPS
- At the conclusion of the acclimation period, all available F0 animals were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was selected for use in the computerized randomization procedure based on body weight stratification randomized in a block design.
- At that time, the individual body weights and corresponding animal identification numbers were entered into WTDMS. A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated and the animals were then arranged into groups according to the printout.
- Animals not assigned to study were transferred to the WIL Research colony.
- The experimental design for this study consisted of 3 test substance-treated groups and 1 control group, composed of 25 rats/sex each.
- The selected animals were approximately 6 weeks old at the initiation of test substance administration.
- Male body weights ranged from 174 g to 235 g and female body weights ranged from 126 g to 182 g on the initial day of test substance administration (study day 0).
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS AND SURVIVAL (F0 AND F1)
- All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.
- Detailed physical examinations were recorded weekly for all parental animals throughout the study period.
- In addition, animals were 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.
- Due to social housing, some observations could not be attributed to a single animal. In these instances, the observation was recorded in a separate computer protocol for the social group.

BODY WEIGHTS (F0 AND F1)
- Individual F0 and F1 male body weights were recorded weekly throughout the study and prior to the scheduled necropsy.
- Individual F0 and F1 female body weights were recorded weekly 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 and F1 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 0 (when possible), 1, 4, 7, 14, and 21. Body weight changes are presented for each of these intervals and for the entire gestation and lactation intervals (days 0-20 and 1-21, respectively).
- 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 are presented on the individual report tables until necropsy. 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. The time periods when body weight values were unavailable for a given animal were left blank or designated as “NA” on the individual report tables.

FOOD CONSUMPTION (F0 AND F1)
- F0 and F1 male and female food consumption was measured weekly 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, 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, 14, and 21. - Food intake was reported as g/animal/day and g/kg/day 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 and reported for these intervals.
- 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. The time periods when food consumption values were unavailable for a given animal were left blank or designated as“NA” on the individual report tables.
Oestrous cyclicity (parental animals):
ESTROUS CYCLES
- Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each F0 and F1 female for 21 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 estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P], beginning 21 days prior to initiation of the mating period and continuing until the detection of evidence of mating).
- Estrous 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. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation.
- Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.
Sperm parameters (parental animals):
SPERMATOGENIC ENDPOINT EVALUATIONS
- Immediately upon euthanasia, the reproductive tract of each F0 and F1 male was exposed via a ventral mid-line incision.
- The right epididymis was excised and weighed.
- 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 minimum 10-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 pipettes, 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 analyzer.
- The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported.
- Percent motile (or progressively motile) 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 (Linder et al., 1992).
- 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 and F1 males from all test
substance-treated groups were weighed, stored frozen, homogenized, and analyzed for determination of homogenization resistant spermatid count and calculation of sperm production rate (Blazak et al., 1985).
- 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.
- Sperm production rate = Number of sperm per gram of tissue / 6.1 days where 6.1 days = the rate of turnover of the germinal epithelium.
Litter observations:
F0 AND F1 PARTURITION
- All females were allowed to deliver naturally and rear their young to weaning (PND 21).
- During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia.
- Beginning 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 was first observed.

LITTER VIABILITY AND DEATHS
- Each litter was examined twice 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 or euthanized in extremis (by an intraperitoneal injection of sodium pentobarbital) from PND 0 to 4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984).
- Pups with external abnormalities that would warrant further skeletal examination were eviscerated and stained (Dawson, 1926) for subsequent skeletal evaluation. Findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt, or interfere with normal body function, or may be incompatible with life) as appropriate.
- 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.

LITTER REDUCTION
- To reduce variability among the litters, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4.
- Standardization of litter size was not performed on litters with fewer than 8 pups.
- All selections were performed by computerized randomization.
- The remaining offspring were weighed, euthanized by intraperitoneal injection of sodium pentobarbital, and discarded on PND 4.

CLINICAL OBSERVATIONS
- Litters were examined daily for survival and any adverse changes in appearance or behavior.
- A clinical observation was recorded for each pup on PND 1, 4, 7, 14, and 21.
- Any abnormalities in nesting and nursing behavior were recorded.

BODY WEIGHTS
- Pups were individually weighed on PND 1, 4, 7, 14, and 21. Mean pup weights were
presented by sex for each litter and by group.
- When body weight values were unavailable for a given animal (due to unscheduled death, weighing error, etc.) group mean values were calculated for that interval using the available data.
- The time periods when body weight values were unavailable for a given animal were designated as “NA”on the individual report tables.

SEX DETERMINATION
- Pups were individually sexed on PND 0, 4, 14, and 21.

WEANING AND SELECTION
- Each dam and litter remained housed together until weaning on lactation day 21. - Twenty-five male and 25 female F1 pups/litter from each group (control, 25, 100, and 400 mg/kg/day) were randomly selected prior to weaning (PND 21) to comprise the F1 generation. These pups (a minimum of 1 male and 1 female per litter, when available) were administered the test substance daily beginning on PND 21.
- Pups were selected for the F1 generation as follows. A computerized randomization
procedure was used to select a minimum of 1 male and 1 female per litter, when
available. An additional male and/or female were selected from a litter, if necessary, to obtain 25 males and 25 females for each group. Pups selected for the F1 generation retained the dam number, followed by a hyphen "-" and the digit tattoo marking (i.e., 99999-01). F2 pups retained the dam number. However, due to software spacing constraints, the -01, -02, etc. designation in the F1 dam number was replaced with the corresponding letter of the alphabet (i.e., -01 = A, -02 = B, etc.) on some report tables.
- Following weaning, F1 and F2 animals were uniquely identified by a Monel metal ear tag displaying the animal number.

DEVELOPMENT LANDMARKS – BALANOPREPUTIAL SEPARATION
- Each male pup was observed for balanopreputial separation beginning on PND 35
(Korenbrot et al., 1977).
- 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.

DEVELOPMENT LANDMARKS – VAGINAL PATENCY
- Each female pup was observed for vaginal perforation beginning on PND 25 (Adams
et al., 1985).
- 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 or an animal was paired for breeding.
- Body weights were recorded at the age of attainment of this landmark.
Postmortem examinations (parental animals):
SCHEDULED EUTHANASIA – F0 AND F1 GENERATIONS (ADULTS)
- All surviving F0 adults were euthanized by carbon dioxide inhalation following the selection of the F1 generation and completion of a detailed physical examination.
- All surviving F1 adults were euthanized by carbon dioxide inhalation following weaning of the F2 pups.
- A complete necropsy and selective histopathologic examination were conducted.

MACROSCOPIC EXAMINATION – F0 AND F1 ANIMALS
- A complete necropsy was conducted on all parental animals (F0 and F1) 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.
- Clinical findings that were verified at necropsy were designated CEO. For females that delivered or had macroscopic evidence of implantation, 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 with macroscopic evidence of implantation and 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 F0 and F1 parental tissues and organs shown in the table below were collected and were placed in 10% neutral-buffered formalin.

ORGAN WEIGHTS
- Except as noted in the table below, paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported.
- When organ weights could not be determined for an animal (due to weighing error, lost or damaged organ, etc.), group mean values were calculated using the available data.
- The organs for which weights could not be determined were designated as “NA”on the individual report tables.

HISTOPATHOLOGY AND MICROSCOPIC EXAMINATION
- Microscopic evaluations were performed on the tissues shown in the table below for all F0 and F1 parental animals from the control and high-dose groups and for all adult animals found dead and euthanized in extremis.
- After fixation, protocol-specified tissues were trimmed according to WIL Research SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to WIL Research’s SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin.
- After fixation, the ovaries were shipped to WIL Research Hillsborough for processing. The tissues were trimmed according to WIL Research SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to WIL Research SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin.
- 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 all parental animals that were found dead or euthanized in extremis. In addition, reproductive organs of all animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire, or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility, or morphology were affected, were subjected to a histopathologic evaluation.
- Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, or other designations as appropriate. Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc.

Postmortem examinations (offspring):
SCHEDULED EUTHANASIA – F1 AND F2 WEANLINGS
- All remaining nonselected F1 and F2 weanlings were euthanized by carbon dioxide
inhalation and necropsied on PND 21 (see Deviations from the Protocol).
- Necropsies were performed and/or organ weights were recorded for these animals.

MACROSCOPIC EXAMINATION – F1 AND F2 WEANLINGS
- Gross necropsies with emphasis on developmental morphology and organs of the
reproductive system were performed on nonselected F1 and F2 weanlings euthanized by carbon dioxide inhalation on PND 21.
- F1 and F2 organs (brain, spleen, and thymus) were collected from 1 pup/sex/litter that survived to the scheduled termination on PND 21.
- These tissues and all gross lesions from F1 and F2 weanlings were preserved in
10% neutral-buffered formalin for possible future histopathologic examination; all other tissues and the carcasses were discarded.

ORGAN WEIGHTS – F1 AND F2 WEANLINGS
- The brain, spleen, and thymus were weighed from 1 pup/sex/litter at the scheduled necropsies on PND 21.

Statistics:
- Details of statistical analysis are given in the section titled 'any other information on materials and methods incl. tables'.
Reproductive indices:
- Mating, fertility, copulation and conception indices were calculated.
- Male (female) mating index (%) = [Number of males (females) with evidence of mating (or females confirmed pregnant) / Total number of males (females) used for mating] x 100
- Male fertility index (%) = [Number of males siring a litter / Total number of males used for mating] x 100
- Male copulation index (%) = [Number of males siring a litter / Number of males with evidence of mating (or females confirmed pregnant)] x 100
- Female fertility index (%) = [Number of females with confirmed pregnancy / Total number of females used for mating] x 100
- Female conception index (%) = [Number of females with confirmed pregnancy / Number of females with evidence of mating (or females confirmed pregnant)] x 100
Offspring viability indices:
- Litter parameters were calculated.
- Mean live litter size = Total viable pups on PND 0 / Number litters with vaiable pups on PND 0
- Postnatal survival between birth and PND 0 or PND 4 (pre-selection) (% per litter) = [Sum of viable pups per litter on PND 0 or PND 4 (pre-selection) / Number of pups born per litter] 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) / Number of litters per group] x 100 where N = PND 0 to 1, 1 to 4 (pre-selection), 4 (post-selection) to 7, 7 to 14, 14 to 21, birth to PND 4 (pre-selection) or 4 (post-selection) to 21,
- Total litter loss was determined when the last pup in the litter was found dead prior to the scheduled euthanasia.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- No test substance-related clinical findings were noted at the detailed physical examinations.
- Findings noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
- Approximately 1 hour following test substance administration, increased incidences of clear material on various body surfaces, including the mouth, nose, forelimb(s), and/or ventral neck, were noted for F0 males in the 100 mg/kg/day group and F0 males and females in the 400 mg/kg/day group compared to the control group. The clear material findings were primarily observed around the mouth, which were noted for all 25 F0 males and females in the 400 mg/kg/day group, as well as 17 of 25 F0 males in the 100 mg/kg/day group throughout the respective treatment periods. The other clear material findings (nose, forelimb[s], and ventral neck) were noted predominantly in the 400 mg/kg/day group (7 to 16 F0 males and 3 to 13 F0 females each), and occurred intermittently during the respective treatment periods.
- In addition, test substance-related increased incidences of red material around the mouth were noted for F0 males and females in the 400 mg/kg/day group compared to the control group. Red material around the mouth was noted for 17 males and 19 females in the 400 mg/kg/day group as early as study day 7 and continued throughout the remainder of the respective treatment periods.
- The aforementioned clear and red material findings occurred at a similar frequency in F0 males and females. No other test substance-related clinical findings were noted approximately 1 hour following dose administration at any dosage level.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- Two F0 females in the 400 mg/kg/day group were euthanized in extremis during the study.
- Female no. 30740 was euthanized in extremis on lactation day 0. On the day of euthanasia, this female was noted with the clinical findings piloerection, unkempt appearance, red material around the nose, mouth, and ventral trunk, and red vaginal discharge; however, no noteworthy changes in body weight or food consumption were noted for this female prior to euthanasia. Based upon macroscopic and microscopic findings the cause of death was determined to be ulceration of the ileum, characterized by ulceration of the ileal mucosa with associated subacute inflammation. The relationship of this death to administration of the test substance was unclear.
- Female no. 30721 was euthanized in extremis on gestation day 22 following clinical findings of red material on various body surfaces, pale and cool extremities, a cool body, yellow material around the urogenital area, and decreased defecation at the detailed physical examinations. With the exception of 16 live fetuses retained in utero, no noteworthy macroscopic or microscopic findings were noted at necropsy, and therefore a cause of death was unable to be determined; however, the moribund condition of this female was likely related to a difficult delivery, although the relationship to administration of the test substance was unclear.
- No other possible treatment-related effects on survival were noted for F0 males and females at any dosage level.
- In the 100 mg/kg/day group, F0 male no. 30826 was found dead on study day 44 (prior to mating); no remarkable clinical findings or noteworthy changes in body weight or food consumption were noted for this male prior to death. However, based upon macroscopic and microscopic findings, the cause of death was determined to be an intubation error, and unrelated to administration of the test substance.
- All other F0 animals in the control, 25, 100, and 400 mg/kg/day groups survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
WEEKLY BODY WEIGHTS
- Test substance-related lower mean body weight gains were noted for the 400 mg/kg/day group F0 males compared to the control group generally throughout the entire generation; differences achieved significance (p<0.05 or p<0.01) during study day intervals 0-7, 28-35, 35-42, 42-49, 63-70, and 112-119. As a result, mean cumulative body weight gains for F0 males in the 400 mg/kg/day group were significantly (p<0.05) lower than the control group when the pre-mating period (study days 0-70) and the entire generation (study days 0-126) were evaluated. Consequently, mean F0 male body weights in this group were 4.7% to 10.7% lower than the control group during study days 21-126, with the differences achieving significance (p<0.05) during study days 49-126.
- No test substance-related effects on mean body weights and body weight gains were noted for F0 males in the 25 and 100 mg/kg/day groups and for F0 females at all dosage levels. 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), with the following exceptions. Significantly (p<0.05 or p<0.01) higher mean body weight gains were noted for F0 males during study days 49-56 (25 mg/kg/day group) and 91-98 (100 mg/kg/day group), and for F0 females in the 400 mg/kg/day group during study days 42-49 compared to the control group. However, the differences in mean body weight gain noted for F0 males and females in these groups were transient, did not occur in a dose-related manner, and/or were not of sufficient magnitude to affect mean body weights and overall mean body weight gains; therefore, they were not attributed to test substance administration.
GESTATION
- Mean F0 maternal body weights and body weight gains were unaffected by test substance administration during gestation. Differences between the control, 25, 100, and 400 mg/kg/day groups were slight and not statistically significant.
LACTATION
- Mean F0 maternal body weights and body weight gains were unaffected by test substance administration during lactation. Differences between the control, 25, 100, and 400 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):
- See discussion of food consumption and food efficiency (below).
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
WEEKLY FOOD CONSUMPTION AND FOOD EFFICIENCY
- Mean F0 food consumption, evaluated as g/animal/day, and food efficiency in the 25, 100, and 400 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 were slight and not statistically significant, with the following exceptions. Significantly (p<0.01) lower mean food efficiency was noted for F0 males in the 400 mg/kg/day group during study days 14-21, 28-35, 63-70, and 112-119, and for F0 females in this group during study days 42-49 compared to the control group. In addition, significantly (p<0.05) higher mean food efficiency was noted for F0 males in the 25 mg/kg/day group during study days 49-56. These differences were not considered test substance-related.
GESTATION
- Test substance-related higher mean food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 400 mg/kg/day group F0 females sporadically throughout gestation; differences were significant (p<0.05 or p<0.01) during gestation days 4-7 and 17-20. As a result, significantly (p<0.05 or p<0.01) higher mean food consumption was noted in this group when the overall gestation treatment period (gestation days 0-20) was evaluated. However, no test substance-related effects on mean gestational body weights were noted for F0 females in the 400 mg/kg/day group; therefore, the increased mean food consumption noted in this group was not considered adverse.
- Mean food efficiency for F0 females in the 400 mg/kg/day group was similar to the control group during gestation days 0-4, 4-7, and 7-11, but generally significantly (p<0.05 or p<0.01) lower during the remainder of gestation (days 11-14, 14-17, and 17-20), and when the overall gestation treatment period (gestation days 0-20) was evaluated. The differences in mean food efficiency at 400 mg/kg/day were not considered test substance-related due to the absence of corresponding effects on mean gestational body weights.
- Mean F0 maternal food consumption and food efficiency in the 25 and 100 mg/kg/day groups were unaffected by test substance administration during gestation. Differences from the control group were slight and not statistically significant, with the following exceptions. Significantly (p<0.05) lower mean g/animal/day food consumption during gestation days 0-4 and higher mean food efficiency during gestation days 4-7 were noted in the 100 mg/kg/day group compared to the control group. However, these transient differences were not of sufficient magnitude to affect mean food consumption or food efficiency when the overall gestation treatment period (gestation day 0-20) was evaluated, nor mean body weight gains during these intervals. Therefore, the differences were not attributed to test substance administration.
LACTATION
- Mean F0 maternal food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency were unaffected by test substance administration during lactation. - Differences between the control, 25, 100, and 400 mg/kg/day groups were slight and not statistically significant.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- There was test substance-related higher liver weights in the 400 mg/kg/day group males and females, and a test substance-related lower group mean final body weight in the 400 mg/kg/day group males.
- In males, there was a 10.4% lower (p<0.05) group mean final body weight in the 400 mg/kg/day group. There was also higher liver weight relative to final body weight (16.0%, p<0.01) in the 400 mg/kg/day group males, and this change exceeded the WIL Research historical control range. There was no microscopic correlation to the higher liver weights in males.
- There were several other statistically significant higher organ weights relative to final body weight in the 400 mg/kg/day group males, but these changes were attributable to the lower final body weight rather than to a direct test substance-related effect. These changes in organ weights included: higher right testis/final body weight (p<0.01), higher left testis/final body weight (p<0.01), higher brain/final body weight (p<0.01), and higher kidney/ final body weight (p<0.05).
- There was a lower absolute pituitary weight (13.3%, p<0.01) and a lower pituitary weight relative to brain weight (13.4%, p<0.01), with no change in the pituitary weight relative to final body weight, in the 400 mg/kg/day group males. Lower pituitary weights are commonly associated with lower group mean final body weights, and the changes in pituitary weights were not considered a direct effect of test substance.
- In females, there was a higher absolute (17.4%, p<0.01) liver weight; higher liver weight relative to group mean final body weight (16.9%, p<0.01); and higher liver weight relative to group mean brain weight (18.5%, p<0.01) in the 400 mg/kg/day group. The changes exceeded the WIL Research historical control ranges. There was no microscopic correlation to the higher liver weights.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- One F0 male and 2 F0 females in the 100 and 400 mg/kg/day groups, respectively, were found dead or euthanized in extremis during the generation. F0 female no. 30721 in the 400 mg/kg/day group was euthanized in extremis prior to parturition on gestation day 22. At necropsy, this female had a small thymus, as well as 16 live fetuses (with no apparent malformations) retained in utero.
- F0 female no. 30740 in the 400 mg/kg/day group was euthanized in extremis on lactation day 0. At necropsy, this female was noted with dark green and tan colored areas on the ileum, dark red areas on the glandular portion of the stomach, and dark red vaginal contents.
- In the 100 mg/kg/day group, F0 male no. 30826 was found dead on study day 44. Necropsy findings for this male consisted of a perforated esophagus and a depressed area on the kidney. All other F0 males and females survived to the scheduled necropsies.
- At the scheduled F0 male and female necropsies, no test substance-related internal findings were observed at any dosage level. Macroscopic findings observed in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
- No test substance-related effects were observed on the number of former implantation sites and the number of unaccounted-for sites. The differences between the control and test substance-treated groups were slight and not statistically significant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- Female nos. 30721 and 30740 in the 400 mg/kg/day group were euthanized in extremis on gestation day 22 (prior to parturition) and lactation day 0, respectively.
- The only significant histologic findings for female no. 30721 were severe necrosis and severe lymphoid depletion of the thymus. Because both of these findings were non-specific, a cause of death could not be determined; however, the moribund condition of this female was likely related to a difficult delivery. For female no. 30740, the cause of death was ulceration of the ileum, characterized by ulceration of the ileal mucosa with associated subacute inflammation. This animal also had non-inflammatory mucosal erosions of the glandular stomach consistent with stress. The relationship of this death to administration of the test substance was unclear.
- In the 100 mg/kg/day group, F0 male no. 30826 was found dead on study day 44. Microscopically, acute hemorrhage of the esophageal submucosa, consistent with acute trauma, was noted for this male. The cause of death was an intubation error and unrelated to test substance administration. All other F0 males and females survived to the scheduled necropsies.
- There were no test substance-related microscopic findings in males or females. All histologic changes were considered to be incidental findings unrelated to test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
- In one control group female (no. 30707), two 100 mg/kg/day group females (nos. 30761 and 30800), and one 400 mg/kg/day group female (no. 30783), there was an adenoma reported as a mass in the skin (no. 30761) or subcutaneous tissue (nos. 30707, 30800, and 30783). These adenomas were benign, well delineated, well differentiated, and consistent with mammary gland adenomas.
- In one 400 mg/kg/day group female (no. 30720), there was a gross finding of pale thyroid glands and a microscopic finding of mild hypertrophy of the follicular cells of the thyroid gland. This change in thyroid gland epithelium is commonly associated with induction of metabolic enzymes in the liver. The higher liver weights in the 400 mg/kg/day group females would be compatible with this microscopic, non-adverse change in the thyroid gland. Since the finding was in an isolated animal, and there were no group mean changes in thyroid weights or higher thyroid weight in this particular animal, the follicular epithelial hypertrophy was not considered test substance-related.
- One control group female (no. 30786) had a follicular cyst; one 400 mg/kg/day group female (no. 30776) had a luteal cyst; and one 400 mg/kg/day group female (no. 30805) had a paraovarian cyst in the ovary. Any variation in number or types of cysts were considered to represent normal biological variability unrelated to test substance.
- In one 100 mg/kg/day group male with reduced fertility (no. 30890), there were changes in the testis that would explain the clinical findings. There was Sertoli cell vacuolation, a focal infiltrate of lymphocytes, syncytial giant cells, and hypospermia in the epididymis. Since there was no dose-response profile, this isolated finding was considered spurious and unrelated to the test substance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
- See discussion of reproductive performance (below) and Table 2 (attached).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
SPERMATOGENIC ENDPOINT EVALUATIONS
- 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 concentration.
- Differences from the control group were slight and were not statistically significant.
Reproductive performance:
no effects observed
Description (incidence and severity):
REPRODUCTIVE PERFORMANCE
- F0 male and female reproductive parameters are presented in Table 2 (attached).
- No test substance-related effects on F0 reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. Two, 1, 2, and 2 male and female pairs in the control, 25, 100, and 400 mg/kg/day groups, respectively, had evidence of mating but did not produce a litter.
- There was reduced fertility in 8 female rats: 2 control group females (nos. 30697 and 30749), one 25 mg/kg/day group female (no. 30750), two 100 mg/kg/day group females (nos. 30702 and 30765), and three 400 mg/kg/day group females (nos. 30717, 30721, and 30793). The ovaries in these animals contained both current and old corpora lutea, suggesting these animals had been cycling. The uteri of these animals had relatively small circumferences, with condensed and eosinophilic endometrial stroma having little glandular development, when compared to animals without reduced fertility. At the time of necropsy, the vagina was microscopically consistent with diestrus in 6 of these animals (nos. 30697, 30750, 30702, 30765, 30717, and 30793). The microscopic appearance of the reproductive tissues in these 6 animals was compatible with a normal nulliparous female adult rat. There was no microscopic cause for reduced fertility noted clinically.
- One control group female (no. 30749) with reduced fertility had a retained antral follicles and old corpora lutea, a normal dilated uterus compatible with estrus in a nulliparous female, and a vagina in estrus. The ovary had a follicle indicative of proestrus, indicative of pending ovulation. There was no primary or apparent cause for the reduced fertility noted clinically or the absence of current corpora lutea.
- One 400 mg/kg/day group female (no. 30721) with reported reduced fertility was pregnant and was euthanized in extremis. This females was euthanized prior to parturition on gestation day 22, and had retained 16 live fetuses in utero.
- There were 2 control group males (nos. 30806 and 30852), one 25 mg/kg/day group male (no. 30856), two 100 mg/kg/day group males (nos. 30815 and 30890), and three 400 mg/kg/day group males (nos. 30840, 30849, and 30901) with reduced fertility. There were no microscopic correlates to the reduced fertility, except for male no. 30890 in the 100 mg/kg/day group. This animal had hypospermia and degenerative changes in the testis consisting of vacuolated Sertoli cells and syncytial giant cells. These changes in 1 mid-dose group animal were considered spurious and unrelated to the test substance. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.
GESTATION LENGTH AND PARTURITION
- No test substance-related effects were noted on mean gestation lengths or the process of parturition at any dosage level. The mean gestation lengths in the 25, 100, and 400 mg/kg/day groups were 21.7, 21.7, and 22.1 days, respectively, compared to a mean gestation length of 21.7 days in the concurrent control group.
- The slight difference in the 400 mg/kg/day group was significant (p<0.05) compared to the concurrent control group; however, the value was within the WIL Research historical control data range (21.5 to 22.3 days). Therefore, the slightly longer mean gestation length in this group was not considered test substance-related. No signs of dystocia were noted at any dosage level.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: no effects on F0 and F1 reproductive performance at any dosage level
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: parental systemic toxicity

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- No test substance-related clinical findings were noted at the detailed physical examinations. Findings noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
- Approximately 1 hour following test substance administration, increased incidences of clear material on various body surfaces, including the mouth, nose, forelimb(s), and/or ventral neck, were noted for F1 males in the 100 mg/kg/day group and F1 males and females in the 400 mg/kg/day group compared to the control group.
- The clear material findings were primarily observed around the mouth, and were noted for 24 of 25 F1 males and 23 of 25 F1 females in the 400 mg/kg/day group, as well as 15 of 25 F1 males in the 100 mg/kg/day group throughout the respective treatment periods.
- The other clear material findings (nose, forelimb[s], and/or ventral neck) were noted exclusively in the 400 mg/kg/day group (2 to 4 F1 males and 7 F1 females), and occurred intermittently during the respective treatment periods.
- In addition, test substance-related increased incidences of red material around the mouth were noted for F1 males and females in the 400 mg/kg/day group compared to the control group. Red material around the mouth was noted for 17 males and 18 females in the 400 mg/kg/day group as early as PND 35 and continued sporadically throughout the remainder of the respective treatment periods.
- No other test substance-related clinical findings were noted approximately 1 hour following dose administration at any dosage level.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- Male no. 30712-09 in the 25 mg/kg/day group was found dead on PND 27. Based on macroscopic and microscopic findings, the cause of mortality for this male was dilatation of ventricles of the brain, which was considered a congenital developmental defect unrelated to the test substance.
- In the control group, 1 male (no. 30734-06) and 1 female (no. 30734-16) were euthanized in extremis on PND 27 and gestation day 10, respectively. The moribund condition of male no. 30734-06 was determined to be dilatation of ventricles of the brain (congenital defect), while female no. 30734-16 was had a grossly visible nodule that correlated to an exostoses adjacent to the pituitary; this exostoses was considered the cause of morbidity and a spontaneous occurrence.
- All other F1 animals in the control, 25, 100, and 400 mg/kg/day groups survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
WEEKLY BODY WEIGHTS
- The effects on mean body weight and body weight gain for F1 males in the 400 mg/kg/day group during the pre-weaning period continued into the post-weaning period. Test substance-related lower weekly mean body weight gains were noted for F1 males in the 400 mg/kg/day group generally throughout the entire generation; the differences were significant (p<0.05 or p<0.01) during PND 21-42, 49-56, 70-77, 84-91, and 119-126. As a result, mean cumulative body weight gains for F1 males in this group were significantly (p<0.01) lower than the control group when the pre-mating period (PND 21-98) and the entire generation (PND 21-154) were evaluated. Consequently, mean F1 male body weights in this group were 10.6% to 16.1% lower than the control group during PND 21-154; the differences were significant (p<0.01).
- No test substance-related effects on mean body weight and body weight gain were noted for F1 males in the 25 and 100 mg/kg/day groups. The values in these test substance-treated groups were generally similar to the control group values throughout the entire generation (PND 21-154), with the exception of a significantly (p<0.05) higher mean body weight gain in the 100 mg/kg/day group during PND 126-133. This difference in mean body weight gain noted for F1 males in this group was transient, did not occur in a dose-related manner, and was not of sufficient magnitude to affect mean body weight and overall mean body weight gain; therefore, it was not attributed to test substance administration.
- Similarly to the F1 males, the effects on mean body weight and body weight gain for F1 females in the 400 mg/kg/day group during the pre-weaning period continued into the post-weaning period. Test substance-related, significantly (p<0.05 or p<0.01) lower mean body weight gains were noted for F1 females in the 400 mg/kg/day group during PND 21-28 and 28-35 compared to the control group. Thereafter, mean body weight gains in this group were similar to the control group throughout the remainder of the pre-mating period (PND 35-98) and when the overall pre-mating period (PND 21-98) was evaluated, with the exception of a transient, significantly (p<0.05) higher mean body weight gain during PND 56-63. However, mean body weights in the 400 mg/kg/day group F1 females were 5.4% and 16.3% lower than the control group during PND 21-98; the differences were generally significant (p<0.05 or p<0.01).
- No test substance-related effects on mean body weight and body weight gain were noted for F1 females in the 25 and 100 mg/kg/day groups. The values in these test substance-treated groups were generally similar to the control group values throughout the pre-mating period (PND 21-98), with the exception of significantly (p<0.05 or p<0.01) higher mean body weight gains in these groups during PND 56-63. These differences in mean body weight gain noted for F1 females in the 25 and 100 mg/kg/day groups were transient, did not occur in a dose-related manner, and was not of sufficient magnitude to affect mean body weight and overall mean body weight gain; therefore, they were not attributed to test substance administration.
GESTATION
- Mean F1 maternal body weight gains were unaffected by test substance administration during gestation at all dosage levels. Any statistically significant differences between the control, 25, 100, and 400 mg/kg/day groups were slight, transient, and/or occurred in a manner that was not dose-related. However, mean body weight in the 400 mg/kg/day group was significantly (p<0.05) lower (6.8%) than the control group on gestation day 0, the result of test substance-related effects on mean body weight noted in this group during the pre-mating period. Mean body weights in the 400 mg/kg/day group were generally similar to the control group during the remainder of gestation (days 4-20); differences were slight and not statistically significant.
LACTATION
- Mean F1 maternal body weights and body weight gains were unaffected by test substance administration during lactation. Differences between the control, 25, 100, and 400 mg/kg/day groups were slight and not statistically significant, with the following exceptions. Significantly (p<0.05 or p<0.01) higher mean body weight gains were noted in the 400 mg/kg/day group compared to the control group during lactation days 4-7 and 14-21. The statistical significance noted in this group during these intervals was attributed to the low values noted for control group animals, rather than the test substance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- See discussion of food consumption and food efficiency (below).
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
WEEKLY FOOD CONSUMPTION AND FOOD EFFICIENCY
- Mean weekly food consumption, evaluated as g/animal/day, in the 400 mg/kg/day group was significantly (p<0.05 or p<0.01) lower during PND 28-35, 35-42, 84-91, and 119-126 for F1 males and during PND 28-35 for F1 females. The lower food consumption was attributed to test substance administration and corresponded to lower mean body weight gains noted in this group during these intervals.
- Mean food consumption in the 400 mg/kg/day group F1 females was comparable to the control group during the remainder of the pre-mating period.
- Mean food efficiency was unaffected by test substance administration for F1 males and females.
- Significantly (p<0.05) higher mean food efficiency was noted for F1 males in the 100 mg/kg/day group during PND 126-133 compared to the control group. This difference was considered transient and not test substance-related, as there was no dose response or corresponding effect on mean body weight.
- Mean food consumption and food efficiency in the 25 and 100 mg/kg/day group F1 males and females were unaffected by test substance administration. The values in the test substance-treated groups were similar to the control group during the pre-mating period (females) and entire generation (males), with the following exception. Significantly (p<0.05) lower mean food consumption was noted for F1 males in the 25 mg/kg/day group compared to the control group during PND 35-42. This difference was considered transient and not test substance-related, as there was no corresponding effect on mean body weight or body weight gain.
GESTATION
- Test substance-related higher mean food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 400 mg/kg/day group F1 females sporadically throughout gestation; differences were generally significant (p<0.05 or p<0.01). As a result, significantly (p<0.05 or p<0.01) higher mean food consumption was noted in this group when the overall gestation treatment period (gestation days 0-20) was evaluated. However, mean body weights in the 400 mg/kg/day group were similar to or lower than the control group throughout gestation; therefore, the increased mean food consumption noted in this group was not considered adverse.
- Significantly (p<0.05 or p<0.01) higher mean food efficiency was noted in the 400 mg/kg/day group during gestation days 0-4 and 7-11 compared to the control group; however, these differences were not of sufficient magnitude to affect the overall gestation period (days 0-20).
- Mean maternal food consumption and food efficiency were unaffected by test substance administration in the 25 and 100 mg/kg/day groups during gestation.
- Differences were slight and not statistically significant, with the following exceptions. Significantly (p<0.01) lower mean food consumption was noted in the 100 mg/kg/day group during gestation days 14-17, and significantly (p<0.01) higher mean food efficiency was noted in the 25 and 100 mg/kg/day groups during gestation days 7-11 compared to the control group. These transient differences were not considered test substance-related.
LACTATION
- Mean F1 maternal food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency were unaffected by test substance administration during lactation. - Differences between the control, 25, 100, and 400 mg/kg/day groups were slight, not statistically significant, and/or did not occur in a dose-related manner.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
ORGAN WEIGHTS
- There was a test substance-related lower mean final body weight (15.2%, p<0.01) in the 400 mg/kg/day group males.
- This change in group mean final body weight was the reason for many additional statistically significant changes in group mean organ weights in the 400 mg/kg/day males, including lower adrenal gland weights (absolute and relative to brain weight); lower brain weights (absolute and relative to final body weight); lower left cauda epididymis weight (absolute); higher right cauda epididymis weight (relative to final body weight); lower left epididymis weight (absolute); higher right epididymis weights (relative to final body weight); higher kidney weight (relative to final body weight); lower pituitary weights (absolute and relative to final body and brain weights); lower spleen weight (absolute); higher seminal vesicle/coagulating gland/accessory gland fluid (relative to final body weight); higher left and right testes weights (relative to final body weight); higher liver weight (relative to final body weight); higher thyroid gland weight (relative to final body weight). These changes in group mean organ weight parameters were within the WIL Research historical control data ranges.
- Several statistically significant changes in group mean organ weight parameters were considered a result of slightly lower final body weights in the 25 and 100 mg/kg/day groups that were not considered test substance-related. These changes included lower left cauda epididymis weights (absolute and relative to brain weight) in the 100 mg/kg/day group males; lower left epididymis weights (absolute and relative to brain weight) in the 100 mg/kg/day group males; lower right epididymis weights (absolute and relative to brain weight) in the 100 mg/kg/day group males; lower left testis weight (absolute and relative to brain weight) in the 25 and 100 mg/kg/day group males; and lower pituitary weights (absolute and relative to brain weight) in the 25 and 100 mg/kg/day group males. The values were within the WIL Research historical control data ranges and were considered unrelated to test substance administration.
- There was a lower group mean right epididymis weight (absolute) in the 25 mg/kg/day group males. This lower weight was due to an outlier (male no. 30730-05) that had severe atrophy of the right testis and severe hypospermia of the right epididymis. The lesion was unilateral and considered spurious and unrelated to test substance administration.
- There was no test substance-related effect on group mean final body weights in F1 females. There were test substance-related higher liver weights (absolute, relative to final body weight, and relative to brain weight) in the 400 mg/kg/day group females. These values exceeded the values in the WIL Research historical control data ranges.
- There were higher group mean thymus weights (absolute and relative to body weight and brain weight) in the 400 mg/kg/day group females, but the values were within the WIL Research historical control data ranges, and therefore were not considered a test substance-related effect.
- Several statistically significant changes in group mean organ weight parameters were present in the 400 mg/kg/day group females, but these changes were considered a result of the slightly lower (2.1%) group mean final body weight in this group. These changes included: lower brain weight (absolute) and lower pituitary weights (absolute and relative to brain weight). These changes in organ weights were within the WIL Research historical control data ranges.
- There was a lower kidney weight relative to brain weight in the 25 mg/kg/day group females; however, this change did not show a dose-response relationship, did not affect absolute kidney weight or kidney weight relative to brain weight, was not associated with histologic findings, and was therefore considered spurious and unrelated to test substance administration.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
MACROSCOPIC EXAMINATION
- Three F1 animals in the 400 mg/kg/day group were found dead prior to the scheduled necropsy. Male no. 30751-01 was found dead on PND 34; at necropsy, gross findings for this male included pale lung lobes that were not fully collapsed and dark red discoloration of the thyroid glands.
- Female nos. 30755-08 and 30782-14 were found dead on PND 32; at necropsy, pale lung lobes that were not fully collapsed were noted for each of these females. Based on the macroscopic findings of lungs not fully collapsed, a gavage error during dosing was considered a possible cause of death for these animals; however, a relationship to treatment could not be dismissed. No other possible treatment-related effects on survival were noted in the test substance-treated groups.
- In the 25 mg/kg/day group, male no. 30712-09 was found dead on PND 27; at necropsy, gross findings for this male included dilatation of the ventricles of the brain, white areas on the liver, and dark red contents in the cranial cavity.
- In the control group, male no. 30734-06 was euthanized in extremis on PND 27 and female no. 30734-16 was euthanized in extremis on gestation day 10. At necropsy, male no. 30734-06 had dilatation of the ventricles of the brain, gas filled cecum, ileum, and jejunum, and a pale liver, and female no. 30734-16 had gas filled cecum, colon, duodenum, ileum, jejunum, and stomach, a small thymus, and a dark red nodule on the bone adjacent to the pituitary; female no. 30734-16 was also gravid with 17 normally developing implantations in utero.
- All other F1 animals survived until scheduled necropsy.
- At the scheduled necropsies, no test substance-related internal findings were observed for F1 males and females at any dosage level. Macroscopic findings observed in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
- No test substance-related effects were observed on the number of pups born, the number of former implantation sites, and the number of unaccounted-for sites. The differences between the control and test substance-treated groups were slight and not statistically significant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
MICROSCOPIC EXAMINATIONS
- Three F1 animals in the 400 mg/kg/day group were found dead prior to the scheduled necropsy; all 3 deaths were attributed to test substance administration. Male no. 30751-01 in this group was found dead on PND 34. The cause of death for this male was undetermined microscopically, as findings were limited to congestion of the thyroid glands.
- Female nos. 30755-08 and 30782-14 in this group were found dead on PND 32. Microscopically, findings were limited to mineralization in the kidneys for female no. 30755-08, and therefore the cause of death for these females was undetermined.
– No other test substance-related effects on survival were noted for F1 animals in the test substance-treated groups.
- In the 25 mg/kg/day group, male no. 30712-09 was found dead on PND 27; microscopically, findings for this male included dilatation of the ventricles of the brain and cystic kidneys. Dilatation of the ventricles was considered the cause of death, and is a congenital developmental defect unrelated to the test substance.
- In the control group, F1 male no. 30734-06 was euthanized in extremis on PND 27 and F1 female no. 30734-16 was euthanized in extremis on gestation day 10. Microscopically, male no. 30734-06 had dilatation of the ventricles of the brain, which was considered the cause of death, while female no. 30734-16 had an exostoses adjacent to the pituitary, which was considered the cause of morbidity and a spontaneous occurrence.
- All other F1 males and females survived to the scheduled necropsies.
- There were no test substance related histologic changes noted for F1 males at any dosage level.
- There was test substance-related hepatocellular hypertrophy in the 400 mg/kg/day group females. The change was minimal to mild, correlated to higher liver weights in this group, and was considered an adaptive, non-adverse change.
- Incidence of selected histopathologic findings at scheduled necropsy of F1 parental animals is presented in Table 4 (attached).
- There were no other test substance-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 substance. There was no test substance-related alteration in the prevalence, severity or histologic character of those incidental tissue alterations.
- There were no test substance-related alterations in ovarian primordial follicle counts among the ovaries examined.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
- See discussion of reproductive performance.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
SPERMATOGENIC ENDPOINT EVALUATIONS
- 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.
- Significantly (p<0.05) lower mean left cauda epididymis weights in the 100 and 400 mg/kg/day groups and significantly (p<0.05) lower left testis weights in the 25 and 100 mg/kg/day groups were noted compared to the control group. These findings were not considered test substance-related.
- No other statistically significant differences were noted at any dosage level.
Reproductive performance:
no effects observed
Description (incidence and severity):
REPRODUCTIVE PERFORMANCE
- F1 male and female reproductive parameters are presented in Table 3 (attached).
- No test substance-related effects on F1 reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. Males that did not sire a litter numbered 2, 0, 2, and 3 in the control, 25, 100, and 400 mg/kg/day groups, respectively. Females that had evidence of mating but were nongravid numbered 3, 0, 2, and 3 in the same respective groups.
- The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.
GESTATION LENGTH AND PARTURITION
- No test substance-related effects were noted on mean gestation lengths or the process of parturition at any dosage level.
- Mean F1 gestation lengths in the test substance-treated groups were similar to the control group value. Differences were slight and were not statistically significant.
- The mean gestation lengths in the 25, 100, and 400 mg/kg/day groups were 21.9, 21.7, and 22.0 days, respectively, compared to mean gestation lengths of 21.8 days in the concurrent control group and 21.9 days in the WIL Research historical control data.
- No signs of dystocia were noted at any dosage concentration.

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: no effects on F0 and F1 reproductive performance at any dosage level
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: parental systemic toxicity

Target system / organ toxicity (P1)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
GENERAL PHYSICAL CONDITION
- The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study were unaffected by test substance administration.
- Pups (litters) that were found dead numbered 18(9), 7(7), 6(5), and 56(12) in the control, 25, 100, and 400 mg/kg/day groups, respectively. Sixteen (7), 5(5), 9(4), and 34(12) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.
- A higher number of pups were found dead in the 400 mg/kg/day group compared to the control group. The increased mortality in this group was primarily attributed to 3 females (nos. 30757, 30740, and 30720) that combined for 41 of the 56 found dead pups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
- The mean number of pups born, live litter size, and percentage of males per litter at birth were unaffected by administration of the test substance to the F0 parental animals at all dosage levels.
- Test substance-related effects on F1 postnatal survival were noted in the 400 mg/kg/day group. Lower mean postnatal survival was noted in this group from PND 0-1, 1-4, and 4-7, which resulted in lower postnatal survival from birth to PND 4 and from PND 4-21. Although none of the differences were statistically significant when compared to the control group, the values from the aforementioned intervals were lower than the respective minimum mean values in the WIL Research historical control database.
- The lower values in the 400 mg/kg/day group were primarily attributed to female nos. 30720 and 30740 (total litter losses on PND 1 and 0, respectively) and no. 30757 (10 dead pups during PND 1-7), but viability was also reduced (≤80.0%) for 8 females during birth to PND 4 and for 4 females during PND 4-21.
- Mean postnatal survival between birth and PND 0 (relative to number born), PND 0-1, 1-4 (pre-selection), 4 (post-selection)-7, 7-14, 14-21, and from birth to PND 4 (pre-selection) and PND 4 (post-selection) to PND 21 were unaffected by administration of the test substance to the F0 parental animals at 25 and 100 mg/kg/day. Differences from the control group were slight, were not statistically significant, and/or did not occur in a dose-related manner.
CLINICAL OBSERVATIONS AND SURVIVAL
- Likely test substance-related effects on survival were noted for F1 animals in the 400 mg/kg/day group. One male (no. 30751-01) was found dead on PND 34 and 2 females (nos. 30755-08 and 30782-14) were found dead on PND 32. No clinical findings during the detailed physical examinations and post-dosing observations, and no noteworthy changes in mean body weight were noted for these animals prior to death. The cause of mortality was undetermined for these animals based on the gross and microscopic findings however, macroscopic findings of lungs not fully collapsed, indicative of a dosing error, was considered a possible cause of death for these animals; however, a relationship to treatment could not be dismissed.
- There were no other possible test substance-related effects on survivability in the F1 generation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
OFFSPRING BODYWEIGHTS
- Test substance-related effects on pup body weights were noted in the 400 mg/kg/day group during the pre-weaning period (PND 1-21).
- Mean F1 male and female pup birth weights (PND 1) in the 400 mg/kg/day group were generally similar to the control group. However, lower mean male and female pup body weight gains were noted in this group during the entire pre-weaning period (PND 1-21) compared to the control group (significant [p<0.05 or p<0.01] during PND 4-21). As a result, mean male and female pup body weights in this group were up to 12.9% and 15.0%, respectively, lower than the control group during PND 7-21; differences from the control group were significant (p<0.01) on PND 14 and 21.
- Mean male and female pup body weights and body weight changes in the 25 and 100 mg/kg/day groups were unaffected by test substance administration throughout the postnatal period. No statistically significant differences from the control group were noted.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- See discussion for second parental generation.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
- See discussion for second parental generation.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
BALANOPREPUTIAL SEPARATION
- Mean ages of attainment of balanopreputial separation were unaffected by test substance administration.
- The mean ages of attainment of balanopreputial separation were 46.1, 46.4, and 46.5 days in the 25, 100, and 400 mg/kg/day groups, respectively, when compared to 45.7 days in the control group and 45.6 days in the WIL Research historical control data.
- Mean body weights at the age of attainment were 234.5 g, 236.3 g, and 209.5 g in the same respective groups compared to 233.1 g in the control group and 231.7 g in the WIL Research historical control data; the difference in mean body weight in the 400 mg/kg/day group was significant (p<0.01) compared to the control group. This body weight deficit (10.1%) corresponded to the lower mean body weights and body weight gains in this group during the weekly body weight evaluations
VAGINAL PATENCY
- Mean ages of attainment of vaginal patency were unaffected by test substance administration.
- The mean ages of attainment of vaginal patency were 35.2, 34.7, and 35.1 days in the 25, 100, and 400 mg/kg/day groups, respectively, when compared to 34.3 days in the control group and 33.6 days in the WIL Research historical control data.
- Mean body weights at the age of attainment were 118.1 g, 118.4 g, and 106.6 g in the same respective groups when compared to 119.7 g in the control group and 111.0 g in the WIL Research historical control data; the difference in mean body weight in the 400 mg/kg/day group was significant (p<0.01) compared to the control group. This body weight deficit (10.9%) corresponded to the lower mean body weights and body weight gains in this group during the weekly body weight evaluations.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
ORGAN WEIGHTS
- During necropsies of weanlings selected for organ weights (PND 21), test substance-related, significantly (p<0.05 or p<0.01) lower mean final body weights were noted for the 400 mg/kg/day group F1 males (11.8%) and females (10.6%) compared to the control group.
- Lower mean absolute brain and absolute spleen weights were also noted for F1 males and females in the 400 mg/kg/day group compared to the control group; the differences were generally significant (p<0.05 or p<0.01). Although the mean absolute brain and spleen weights in this group were within the respective WIL Research historical control data ranges, the differences generally occurred in a dose related manner, and were considered test substance-related.
- No test substance-related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed at 25 and 100 mg/kg/day. None of the differences from the control group were statistically significant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
NECROPSIES OF PUPS FOUND DEAD
- The numbers of pups (litters) found dead from PND 0 through the selection of the F1 generation were 18(9), 7(7), 6(5), and 56(12) in the control, 25, 100, and 400 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration to 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, internal findings were limited to the 400 mg/kg/day group, and consisted of dilated renal pelves for pup no. 30735-01 and dark red tracheal contents and dark red discolored lungs (all lobes) for pup nos. 30757-04 and 30799-05. Because these findings were noted infrequently in single pups, they were not attributed to the test substance. No other internal findings were noted.
NECROPSIES OF WEANLINGS NOT SELECTED FOR ORGAN WEIGHTS (PND 21)
- No internal findings that could be attributed to parental administration with the test substance were noted at the necropsy of weanlings euthanized on PND 21. – Internal findings were limited to 2 pups in the 25 mg/kg/day group, and consisted of a missing tail (entire length) and cystic kidneys for pup no. 30804-01 and cystic kidneys for pup no. 30804-07. These findings were noted infrequently, in single pups, and in a manner that was not dose related, and therefore were not attributed to the test substance. No other internal findings were noted.
NECROPSIES OF WEANLINGS SELECTED FOR ORGAN WEIGHTS (PND 21)
- At the PND 21 necropsy of F1 weanlings selected for organ weights, no internal findings that could be attributed to parental administration with the test substance were noted.
- Internal findings were limited to dark red areas on the thymus for 1 pup (no. 30762-09) in the 100 mg/kg/day group. Because this finding was noted in a single pup and in a manner that was not dose related, it was not attributed to the test substance. No other internal findings were noted.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
- See discussion for second parental generation.
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Remarks:
maternal dose
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Remarks on result:
other:
Remarks:
neonatal toxicity

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
GENERAL PHYSICAL CONDITION
- The general physical condition (defined as the occurrence and severity of clinical findings) of all F2 pups in this study were unaffected by F1 parental test substance administration.
- Pups that were found dead or euthanized in extremis numbered 9(8), 12(6), 30(9), and 40(13) in the control, 25, 100, and 400 mg/kg/day groups, respectively. Eight (5), 2(2), 6(3), and 16(9) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.
- A higher number of pups were found dead in the 400 mg/kg/day group compared to the control group. The increased mortality in this group was primarily attributed to 2 female (nos. 30748-12 and 30748-09) that combined for 21 of the 40 found dead or euthanized in extremis pups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
- A lower live litter size on PND 0 was noted in the 400 mg/kg/day group (11.7 per dam) compared to the control group (13.5 per dam). The difference was not statistically significant, but the value was comparable to the minimum mean value in the WIL Research historical control data (11.6 per dam). As a result, a lower (not statistically significant) mean litter proportion (89.7% per litter) of postnatal survival was noted at birth (PND 0) in the 400 mg/kg/day group compared to the control group (98.4% per litter). A lower mean litter proportion of postnatal survival continued to be noted in the 400 mg/kg/day group during PND 0-1 (89.9% per litter), and consequently from birth to PND 4 (79.1% per litter) compared to the control group (95.7% and 90.9% per litter, respectively). The differences from the control group were not statistically significant, but the values in the 400 mg/kg/day group were below the minimum mean litter proportion values in the WIL Research historical control data (93.3% [PND 0], 94.6% [PND 0-1], and 83.8% [birth to PND 4]). The lower postnatal survival in the 400 mg/kg/day group was partially attributed to 2 females from a single litter (nos. 30748-09 and 30748-12), with total litter loss on PND 0 and a litter survival value of 21.4% from birth to PND 4, respectively. The mean litter proportions of postnatal survival in the 400 mg/kg/day group were similar to the control group during PND 4-21. No test substance-related effects on the percentage of males at birth and the mean number of pups born were noted at 400 mg/kg/day.
- There were no test substance-related effects on the mean number of pups born, live litter size, percentage of males per litter at birth, and postnatal survival in the 25 and 100 mg/kg/day groups. Differences from the control group were slight, not statistically significant, and/or did not occur in an dose-related manner.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
OFFSPRING BODY WEIGHTS
- Mean F2 male and female pup birth weights on PND 1 were similar between the control and 400 mg/kg/day group. Additionally, mean F2 male and female pup body weights and body weight gains in the 400 mg/kg/day group were generally similar to the control group during PND 1-7.
- However, significantly (p<0.01) lower mean F2 male and female pup body weight gains were noted in this group during the remainder of the pre-weaning period (PND 7-21) compared to the control group. As a result, mean F2 male and female pup body weights in this group were up to 15.4% and 14.6%, respectively, lower than the control group from PND 14-21; differences from the control group were significant (p<0.01).
- Mean F2 male and female pup body weights and body weight changes in the 25 and 100 mg/kg/day groups were unaffected by test substance administration throughout the postnatal period. No statistically significant differences from the control group were noted.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
ORGAN WEIGHTS
- Test substance-related, significantly (p<0.01) lower mean final body weights were noted for the 400 mg/kg/day group F2 males (14.3%) and females (15.4%) compared to the control group.
- Lower mean absolute and relative (to final body and/or brain weights) brain and spleen weights were also noted for F2 males and females in the 400 mg/kg/day group compared to the control group; the differences were generally significant (p<0.05 or p<0.01). Although the mean absolute brain and spleen weights in this group were within the respective WIL Research historical control data ranges, the differences occurred in a dose-related manner, and were considered test substance-related.
- No test substance-related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed at 25 and 100 mg/kg/day.
- None of the differences from the control group were statistically significant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
NECROPSIES OF PUPS FOUND DEAD
- The numbers of pups (litters) found dead or euthanized in extremis from PND 0 through the selection of the F2 generation were 9(8), 12(6), 30(9), and 40(13) in the control, 25, 100, and 400 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 or euthanized in extremis.
- Aside from the presence or absence of milk in the stomach, internal findings consisted of the following. In the 400 mg/kg/day group, pup no. 30751-04-01 had the variation small lungs (all lobes) and pup nos. 30748-09-05 and 30748-09-10 had the variation hemorrhagic ring around the iris (unilateral or bilateral). In the 100 mg/kg/day group, pup no. 30722-14-11 had the variation renal papilla(e) not developed and distended ureters and pup no. 30762-16-02 had a small thymus and a dark red discolored right eye.
- In the 25 mg/kg/day group, pup no. 30766-07-01 had the malformation cleft palate (palatine plates not joined; entire length) and the variations sternebrae malaligned (slight or moderate; nos. 2-5), thoracic vertebral centra not fully ossified (nos. 5 and 10-12), and 14th rudimentary ribs. These findings were not attributed to the test substance as they occurred in single pups (litters) and/or did not occur in a dose-related manner.
- No other internal findings were noted.
NECROPSIES OF WEANLINGS NOT SELECTED FOR ORGAN WEIGHTS (PND 21)
- No internal findings that could be attributed to parental administration of the test substance were noted at the necropsy of weanlings euthanized on PND 21.
- Internal findings, including distended ureter(s) and dilated renal pelves, were noted in single pups, similarly in the control group, and in a manner that was not dose-related.
NECROPSIES OF WEANLINGS SELECTED FOR ORGAN WEIGHTS (PND 21)
- At the PND 21 necropsy of F2 weanlings selected for organ weights, no internal findings were observed at any dosage level.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Remarks:
maternal dose
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Remarks on result:
other: neonatal toxicity

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

ANALYSIS OF DOSING FORMULATIONS

- Results of homogeneity and concentration analysis are summarised in Table 1 (attached).

- The mean concentrations of the 5 and 80 mg/mL formulations prepared on 03-Nov-2014 were 78.4% and 116% of the target concentration, and the mean concentrations of the 5 and 20 mg/mL formulations prepared on 16-Jun-2015 were 63.5% and 83.0% of the target concentration; all other samples were within WIL Research SOP range for suspensions (85% to 115%).

- The out-of-specification results noted in the 5 mg/mL group had no impact on the study because the NOAEL for parental systemic toxicity was determined to be a higher dosage level (20 mg/mL).

- The out-of-specification results noted in the 20 and 80 mg/mL groups had no impact on the study because the difference from the WIL Research SOP range was minimal (≤2%), and the formulations were prepared the same way on each day.

- Additionally, the results of the analyses on 16-Jun-2015 were consistent with the results obtained during the method validation 70-day storage assessment;i.e., formulations at lower prepared concentrations have lower mean percent recovery following prolonged storage (Haas, 2015, WIL-168211).

- For logistical reasons (i.e., sample shipment and method development times) sample analyses on 16-Jun-2015 in this study were conducted approximately 27 days after formulation and dosing. However, because all formulations were used for dosing within 8 days of preparation, it is believed that the animals on this study received the intended dose concentration/dosage levels.

- All analyzed dosing formulations were homogeneous and the test substance was not detected in the vehicle formulation that was administered to the control group (Group 1).

Applicant's summary and conclusion

Conclusions:
Based on the absence of effects on F0 and F1 reproductive performance at any dosage level, a dosage level of 400 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for parental reproductive toxicity of test item administered orally by gavage to Crl:CD(SD) rats. Based on the lower live litter size on PND 0 for F2 pups, decreased postnatal survival, and lower mean body weights and body weight gains during the pre-weaning period for F1 and F2 pups at 400 mg/kg/day, the NOAEL for neonatal toxicity was considered to be 100 mg/kg/day. Based on moribundity for F0 females and mortality for F1 males and females, as well as lower mean body weights, body weight gains, and/or food consumption for F0 males and F1 males and females at 400 mg/kg/day, the NOAEL for parental systemic toxicity was considered to be 100 mg/kg/day.
Executive summary:

GUIDELINE

The study was conducted to evaluate the potential adverse effects of the test item on reproduction in a two generation study. This included determining the effects of the test substance on male and female reproductive processes, including gonadal function, estrous cyclicity, mating behavior, conception, gestation, parturition, lactation, weaning, and on growth and development of the offspring. A minimum of 1 litter per dam was produced in each generation. The protocol was designed in general accordance with the United States EPA Health Effects Test Guidelines: OPPTS 870.3800, Reproduction and Fertility Effects, Aug-1998, and the OECD Guidelines for the Testing of Chemicals, Guideline 416, Two-Generation Reproduction Toxicity Study, 22-Jan-2001.

 

METHODS

Four groups of male and female Crl:CD(SD) rats (25/sex/group) were administered the test substance, daily by oral gavage, for at least 70 consecutive days prior to mating. Dosage levels were 25, 100, and 400 mg/kg/day for the F0 and F1 generations. A concurrent control group of 25 rats/sex/group received the vehicle (arachis [peanut] oil) on a comparable regimen. F0 animals were approximately 6 weeks of age at the initiation of test substance administration. The test substance was administered to offspring selected to become the F1 parental generation following weaning (beginning on PND 21). The F0 and F1 males continued to receive the test substance throughout mating and through the day prior to euthanasia. The F0 and F1 females continued to receive the test substance throughout mating, gestation, and lactation, and through the day prior to euthanasia. For both generations (F1 and F2), 8 pups per litter (4 per sex, when possible) were selected on PND 4 to reduce the variability among the litters. Offspring (25/sex/group) from the pairing of the F0 animals were selected on PND 21 to constitute the F1 generation. F0 males and females were dosed for 128-133 consecutive days and F1 males and females were exposed for 136-147 consecutive days, respectively.

 

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals for males and females throughout the study (including during gestation and lactation for females). Vaginal lavages were performed daily for determination of estrous cycles beginning 21 days prior to cohabitation. All F0 and F1 females were allowed to deliver and rear their pups until weaning on lactation day 21. Detailed physical examinations, body weights, and sexes for F1 and F2 pups were recorded at appropriate intervals. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the F1 rats selected to constitute the F1 generation. Nonselected F1 pups were necropsied on PND 21, and F2 pups were necropsied on PND 21. Selected organs were weighed for 1 pup/sex/litter from both F1 and F2 pups that were necropsied on PND 21. Each surviving F0 and F1 parental animal received a complete detailed gross necropsy following the completion of weaning of the F1 and F2 pups, respectively; selected organs were weighed. Spermatogenic endpoints (sperm motility [including progressive motility], morphology, and numbers) were recorded for all F0 and F1 males, and ovarian primordial follicle counts were recorded for all F1 females in the control and high-dose groups and for all F1 females suspected of reduced fertility. Designated tissues from F0 and F1 parental animals were examined microscopically.

 

RESULTS

Test substance-related effects on F0 and F1 parental survival occurred at 400 mg/kg/day. In the F0 generation, 1 female was euthanizedin extremison lactation day 0 following noteworthy clinical findings of piloerection, unkempt appearance, red material around the nose, mouth, and ventral trunk, and red vaginal discharge. Ulceration of the ileum, characterized by ulceration of the ileal mucosa with associated subacute inflammation,

was determined as the cause of the moribund condition for this female; the relationship of this death to administration of the test substance was unclear. An additional F0 female in this group was euthanizedin extremison gestation day 22 following noteworthy clinical findings of pale and cool extremities, a cool body, and red material on various body surfaces. Although a cause of death was undetermined microscopically, the aforementioned clinical signs, in conjunction with the retention of 16 live fetusesinutero, pointed to a difficult delivery as the likely cause of death; however, a relationship to administration of the test substance was possible. In the F1 generation, 1 male was found dead on PND 34 and 2 females were found dead on PND 32. The cause of death for these animals was undetermined based on macroscopic and microscopic findings. However, based on macroscopic lung findings, gavage error was considered a likely cause of death, although a relationship to treatment with the test substance could not be completely dismissed. No other possible test substance-related effects on survival were noted for F0 and F1 parental animals at any dosage level.

 

One F0 male in the 100 mg/kg/day group was found dead on study day 44 due to an intubation error and 1 F1 male in the 25 mg/kg/day group was found dead on PND 27; dilatation of the ventricles of the brain was determined to be the cause of moribundity for the F1 male in the 25 mg/kg/day group. In the control group, 1 F1 male was euthanizedinextremison PND 27 and 1 F1 female was euthanizedin extremisgestation day 10 due to clinical signs and effects on body weight; dilatation of the ventricles of the brain and an exostoses adjacent to the pituitary, respectively, were determined to be the causes for the moribund condition of these animals. All other F0 and F1 parental animals survived to the scheduled necropsies.

 

Test substance-related clinical findings, including clear material on various body surfaces and red material around the mouth, were noted for F0 and F1 parental animals in the 100 and/or 400 mg/kg/day groups approximately 1 hour following dose administration. Clear material findings, primarily observed around the mouth, were noted for the majority of F0 and F1 males and females in the 400 mg/kg/day group, as well as to a slightly lesser extent for F0 and F1 males in the 100 mg/kg/day group, throughout the respective treatment periods. Red material around the mouth was noted predominantly for F0 and F1 males and females in the 400 mg/kg/day group throughout the respective treatment periods. No test substance-related clinical findings were noted at the detailed physical examinations for F0 and F1 parental animals at any dosage level. Therefore, because these clear and red material findings noted during the post-dosing observations did not generally persist to the detailed physical examinations the following day, they were not considered adverse.

 

Test substance-related lower mean body weight gains, in the absence of corresponding reduced mean food consumption, were noted for F0 males in the 400 mg/kg/day group throughout the entire generation, resulting in a mean body weight on study day 126 that was 10.4% lower than the control group. Mean body weights, body weight gains, and food consumption for F0 males in the 25 and 100 mg/kg/day groups were similar to the control group throughout the entire generation. For F0 females, mean body weights and body weights gains were similar to the control group throughout the pre-mating period, as well as during gestation and lactation at all dosage levels. Test substance-related higher mean food consumption and food efficiency were noted for F0 females in the 400 mg/kg/day group during gestation; however, mean body weights and body weight gains were similar to the control group throughout gestation; therefore, the increases were not considered adverse. No test substance-related effects on mean food consumption and/or food efficiency were noted for F0 females in the 400 mg/kg/day group during the pre-mating period and lactation, as well as in the 25 and 100 mg/kg/day groups throughout pre-mating, gestation, and lactation.

 

In the F1 generation, lower mean body weights and body weight gains were noted for males and females in the 400 mg/kg/day group, which were continuations of effects that originated during the pre-weaning period. Lower mean body weight gains, with generally reduced mean food consumption, were noted for F1 males throughout the entire generation, resulting in a mean body weight on PND 154 that was 15.5% lower than the control group. Mean body weights, body weight gains, and food consumption for F1 males in the 25 and 100 mg/kg/day groups were similar to the control group throughout the entire generation. For the F1 females, lower mean body weight gains and/or reduced mean food consumption were noted in the 400 mg/kg/day group for the first 2 weeks following weaning (PND 21-35), but similar to the control group thereafter. However, mean body weights in this group were 5.4% to 16.3% lower than the control group throughout the pre-mating period (PND 21-98). Mean body weights and body weight changes for F1 females were similar to the control group at 25 and 100 mg/kg/day during the pre-mating period, and at all dosage levels during gestation and lactation. Test substance-related higher mean food consumption was noted for F1 females in the 400 mg/kg/day group during gestation; however, mean body weights and body weight gains were similar to the control group throughout gestation; therefore, the increases were not considered adverse. No test substance-related effects on mean food consumption and/or food efficiency were noted for F1 females in the 400 mg/kg/day group during lactation, as well as in the 25 and 100 mg/kg/day groups throughout pre-mating, gestation, and lactation.

 

No test substance-related effects were observed on F0 and F1 reproductive performance (estrous cycles, mating, fertility, copulation or conception indices, the mean number of days between pairing and coitus, and the mean length of gestation), parturition, or the mean numbers of implantation sites and unaccounted-for sites. Spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate, motility, progressive motility, and the percentage of morphologically normal sperm) were also unaffected by test substance administration.

 

There were no test substance-related macroscopic findings noted for F0 and F1 males or females at any dosage level. Test substance-related higher mean liver weights (absolute and relative to final body weight and/or brain weight) were noted in the 400 mg/kg/day group F0 males and females and F1 females. With the exception of the F1 females, there were no microscopic correlates; therefore, these organ weight changes were not considered adverse. Test substance-related hepatocellular hypertrophy was noted for F1 females in the 400 mg/kg/day group; the change was minimal to mild, correlated to higher liver weights in this group, and was considered an adaptive, non-adverse change. No test substance-related organ weight changes or microscopic findings were noted for F0 and F1 males and females at 25 and 100 mg/kg/day, organ weight changes for F1 males at 400 mg/kg/day, and microscopic findings for F0 males and females and F1 males at 400 mg/kg/day.

 

Total litter losses and increased numbers of pups found dead between PND 1-7 were noted for F0 females in the 400 mg/kg/day group. As a result, test substance-related lower mean postnatal survival was noted from birth to PND 4 and PND 4-21 for F1 pups in the 400 mg/kg/day group. The mean numbers of F1 pups born, percentage of males at birth, live litter size on PND 0, and general physical condition of pups at all dosage levels and postnatal survival at 25 and 100 mg/kg/day were unaffected by F0 parental test substance administration. A test substance-related lower mean F2 live litter size on PND 0 was noted in the 400 mg/kg/day group. Additionally, test substance-related lower postnatal survival was observed for F2 pups in the 400 mg/kg/day group during birth to PND 4, primarily due to a total litter loss for 1 F1 female on PND 0 and an increased numbers of pups found dead between PND 0-4 for an additional F1 female in this group. Postnatal survival for F2 pups at 400 mg/kg/day was similar to the control group during the remainder of the postnatal period (PND 4-21). The mean number of F2 pups born, live litter size on PND 0, and postnatal survival at 25 and 100 mg/kg/day, and the percentages of males at birth and general physical condition of pups at all dosage levels were unaffected by F1 parental test substance administration.

 

Test substance-related effects on mean F1 and F2 pup body weight were noted during the pre-weaning period. Lower mean pup body weight gains were noted for F1 males and females during PND 4-21 and for F2 males and females during PND 7-21. As a result, mean F1 and F2 pup body weights were up to 15.0% and 15.4% lower for males and females, respectively, during the pre-weaning period. Mean F1 and F2 male and female pup body weights and body weight gains in the 25 and 100 mg/kg/day groups were unaffected by parental test substance administration.

 

No test substance-related effects on the mean ages of attainment of balanopreputial separation or vaginal patency were noted in the F1 weanlings. However, mean body weights at the age of attainment of these developmental landmarks were lower in the 400 mg/kg/day group males and females due to the aforementioned decrements in mean body weights and body weight gains.

 

No macroscopic findings noted at any dosage level in the F1 and F2 pups that were found dead, euthanizedin extremis, or euthanized at the scheduled necropsy on PND 21 that was attributed to F0 or F1 parental test substance administration.

 

Test substance-related lower mean final body weights were noted for F1 and F2 males and females in the 400 mg/kg/day group on PND 21. In addition, test substance-related lower mean absolute brain and spleen weights for F1 male and female pups and lower mean absolute and relative (to final body weight and brain weight) brain and spleen weights for F2 males and females were noted on PND 21. No test substance-related effects on organ weights were observed for F1 and F2 male and female pups in the 25 and 100 mg/kg/day groups.

 

CONCLUSIONS

Based on the absence of effects on F0 and F1 reproductive performance at any dosage level, a dosage level of 400 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for parental reproductive toxicity of test item administered orally by gavage to Crl:CD(SD) rats. Based on the lower live litter size on PND 0 for F2 pups, decreased postnatal survival, and lower mean body weights and body weight gains during the pre-weaning period for F1 and F2 pups at 400 mg/kg/day, the NOAEL for neonatal toxicity was considered to be 100 mg/kg/day. Based on moribundity for F0 females and mortality for F1 males and females, as well as lower mean body weights, body weight gains, and/or food consumption for F0 males and F1 males and females at 400 mg/kg/day, the NOAEL for parental systemic toxicity was considered to be 100 mg/kg/day.