[No public or meaningful name is available]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 November 2016 to 21 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

various (see Appendix 1, attached)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

- Appearance/physical state: Brown, opaque, viscous liquid
- Storage conditions: 18 to 24 °C protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION PERIOD
- Sexually mature male and virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. The animal model, the Crl:CD(SD) rat, is recognized as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, Charles River Ashland has reproductive historical control data in the Crl:CD(SD) rat.
- The number of animals selected for this study (15 [Groups 1 and 5] or 10 [Groups 2–4] rats/sex/group) was based on the OECD Guideline for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016, which recommends that evaluation of each group be initiated with at least 10 males and 12 to 13 females per group.
- Females were evaluated for estrous cyclicity during the pretest period and any females that failed to exhibit normal 4–5 day estrous cycles (e.g., EDDDE), repeatedly during the pretest period, were excluded from the study; therefore, the extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination.
- Crl:CD(SD) rats (66 males and 78 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 08 Nov 2016. The animals were approximately 51 days old upon receipt. Each animal was examined by a qualified biologist on the day of receipt. On the day following receipt, all animals were weighed and clinical observations were recorded. Each animal was uniquely identified using a programmable microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region during the acclimation period. The animals were housed for an acclimation period of 22 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior, and the testes were palpated at least once for all males.

ANIMAL HOUSING
- Following receipt and until pairing, all F0 animals were housed 2–3 rats/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 Charles River.
- During cohabitation, breeding phase rats (10/sex/group) were paired in a solid-bottom cage with bedding material. Following the breeding period, the males were individually housed until the scheduled necropsy. Following positive evidence of mating, the females were individually housed until euthanasia on Lactation Day 14. Females with no evidence of mating or that failed to deliver were individually housed until Postcohabitation or Postmating Day 25. The 5 rats/sex in the control and high-dose groups that were assigned to the recovery phase were not paired for mating and remained housed in groups of 2–3 in clean solid-bottom cages with bedding material until euthanasia.
- Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland 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.

DRINKING WATER AND MAINTENANCE
- 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 Charles River. 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 Charles River SOPs. The results of the diet and water analyses are maintained at Charles River. 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, with the following exception. All males and females (including those not selected for clinical pathology evaluation) were fasted prior to clinical pathology blood collection when food, but not water, was withheld.

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 68 °F to 78 °F (20 °C to 26 °C) and 30 % to 70 %, 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 72.8 °F to 75.2 °F (22.7 °C to 24.0 °C) and mean daily relative humidity ranged from 36.7 % to 53.6 % during the study.
- Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod (see Appendix 1 – Deviations to study protocol). 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

Remarks:

used in preparation of control and test substance formulations (Lot number 2FG0172; expiry date 30 June 2017)

Details on exposure:

CONTROL SUBSTANCE
- The control substance was mineral oil (expiry date 17 August 2018).

TEST ITEM PREPARATION
- Dosing formulations were prepared at the test substance concentrations indicated in the table below.
- The control and 1, 30, and 200 mg/mL 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 (18 °C to 24 °C), protected from light.
- The 75 mg/mL formulations were prepared daily as a dilution of the 200 mg/mL formulation (see Appendix 1 – Deviations to study protocol).
- Formulations were heated in a water bath at 50 °C ± 5 °C for a minimum of 60 minutes during preparation and aliquots were stirred and heated at 45 °C ± 5 °C for a minimum of 60 minutes prior to dispensing on each day of dosing (see Appendix 1 – Deviations to study protocol).
- The control and 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’s designee and were found to be visibly homogeneous and acceptable for administration.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- At the conclusion of the acclimation period, all available males and females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health, meeting acceptable body weight requirements, and exhibiting normal 4–5 day estrous cycles (females) was selected for use in the computerized randomization procedure based on body weight stratification 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. The animals then were arranged into groups according to the printout. Animals not assigned to study were transferred to the Charles River rat colony or euthanized by carbon dioxide inhalation and discarded.
- The experimental design consisted of 4 test substance-treated groups and 1 control group composed of 10 rats/sex/group. An additional 5 rats/sex in the control and high-dose group were selected to be evaluated following a 14-day recovery period; animals assigned to the recovery period were not evaluated for reproductive toxicity. At the initiation of dose administration (Study Day 0), the males and females were approximately 10 weeks old. Male body weights ranged from 314 g to 450 g and female body weights ranged from 203 g to 267 g on Study Day 0. The animals were approximately 12 weeks old when paired on Study Day 13; female body weights ranged from 220 g to 301 g on Gestation Day 0.

TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- Study group assignment is shown in the table below.
- The vehicle and test substance formulations were administered orally by gavage, via appropriately sized flexible, disposable, plastic feeding tubes (Instech Solomon, Plymouth Meeting, PA) once daily.
- The males selected for pairing were dosed during Study Days 0–29 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 30 doses.
- The females selected for pairing were dosed during Study Days 0 through the day prior to euthanasia (14 days prior to pairing through Lactation Day 13) for a total of 49-62 doses.
- Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (Postmating or Postcohabitation Day 25) for a total of 49–52 doses.
- The extra 5 rats/sex in the control and high-dose groups were not used for mating, but were treated on a comparable regimen beginning on Study Day 0; following 30 doses for males and 49 doses for females, these animals remained on study for a 15-day recovery period. The dose volume for all groups was 5 mL/kg.
- Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose.
- All animals were dosed at approximately the same time each day.
- Dosage levels were selected based on the results of a previous 14-day range-finding study in which male and female rats were administered the test substance at dosage levels of 25, 125, 500, and 1000 mg/kg/day. In the previous study, there were no significant clinical observations noted in any dosage group. In addition, mean body weights and food consumption were unaffected by the test substance in the males while mean body weight gains in the females were slightly lower than the control group in all test substance-treated groups. As a result, dosage levels of 5, 150, 375, and 1000 mg/kg/day were chosen for the current study.
- The selected route of administration for this study was oral (gavage) because this a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLING AND ANALYSIS
- Test substance homogeneity and resuspension homogeneity following 8 days of room temperature storage at concentrations of 1 and 200 mg/mL were established in a previous study. In addition, test substance stability following 23 hours and 8 days of room temperature storage at these same concentrations was established. Therefore, stability assessments were not conducted as part of the current study.
- Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the first and last 1, 30, 75, and 200 mg/mL dosing formulations and from the middle stratum of the first and last control group dosing formulations (see Appendix 1 – Deviations to study protocol).
- An additional sample from the 200 mg/mL formulation prepared on 30 Nov 2016 used to prepare the daily 75 mg/mL dosing formulation via dilution was collected and analyzed for homogeneity and concentration.
- One set of samples from each collection was subjected to the appropriate analyses. All remaining samples were stored at room temperature as back-up. All analyses were conducted by the Charles River Analytical Chemistry Department using a validated high performance liquid chromatography method with charged aerosol detection.

Details on mating procedure:

BREEDING PROCEDURES
- The 10 rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained.
- Each female was 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 following a vaginal lavage and verified by a second biologist.
- Each mating pair was examined daily. The day when evidence of mating was identified was termed Gestation Day 0 and the animals were separated.
- If evidence of copulation was not detected after 14 days of pairing, the animals were separated without further opportunity for mating.
- For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.

Duration of treatment / exposure:

- Males received 14 daily doses prior to mating and were dosed throughout the mating period through 1 day prior to euthanasia for a total of 30 doses.
- Females received 14 daily doses prior to pairing and were dosed through Lactation Day 13 for a total of 49–62 doses.
- Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (Postcohabitation Day 25 or Postmating Day 25, respectively) for a total of 49–52 doses.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

mineral oil formulation

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

375 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- 10 males and 10 females (5, 150 and 375/kg bw/day groups)
- 15 males and 15 females (vehicle control and 1000 mg/kg bw/day groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- An overview of study design is attached.

Examinations

Maternal examinations:

CLINICAL OBSERVATIONS AND SURVIVAL
- All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily and individual detailed physical examinations were conducted weekly (prior to dose administration during the treatment period) (see Appendix 1 – Deviations to study protocol).
- Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals.
- Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labour, delayed labour) or other difficulties. In addition, the social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal; only positive findings were recorded.

BODY WEIGHTS
- Individual male body weights were recorded weekly throughout the study and prior to the scheduled euthanasia. Individual female body weights were recorded weekly until evidence of copulation was observed or until euthanasia (for females assigned to recovery phase). Mean weekly body weights and body weight changes were presented for each interval. In addition, cumulative mean body weight changes were presented for the pre-mating dosing period (males and females) and for the entire dosing period (males and recovery phase females).
- 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, 10, 13, and 14 (fasted). Body weight changes were presented for each of these intervals and for the entire gestation and lactation
intervals (Days 0–20 and 1–13, respectively). Weekly body weights for females with no evidence of mating were presented on the individual report tables until necropsy.
- When body weights could not be determined for an animal during a given interval (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 designated as “NA” on the individual report tables.

FOOD CONSUMPTION
- Food consumption was recorded on the corresponding weekly body weight days until pairing (for animals paired for breeding) or euthanasia (for animals assigned to the recovery period).
- Food 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 for animals selected for pairing.
- Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 10, and 13.
- Following the breeding period, individual food consumption for females with no evidence of mating was measured on a weekly basis until the scheduled euthanasia. Weekly food consumption for females with no evidence of mating was presented in the individual report tables until necropsy.
- Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage.
- When food consumption could not be determined for a given interval (due to a weighing error, scheduled euthanasia, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable were left blank or designated as “NA” on the individual report tables.

PARTRUITION
- All females selected for pairing were allowed to deliver naturally and rear their young to PND 13 (see Appendix 1 – Deviations from study protocol).
- During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia.
- On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded.
- Individual gestation length was calculated using the date delivery started.

FOB ASSESSMENTS
- FOB assessments were recorded for 5 animals/sex/group following approximately 28 days of dose administration (Study Week 4, males selected for pairing) or on Lactation Day 13 (females). The FOB used at Charles River is based on previously developed protocols. FOB testing was performed by the same biologists, to the extent possible, without knowledge of the animal’s group assignment. The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB.
- All animals were observed for the parameters listed in the table below.
- Forelimb and hindlimb grip strength were measured using a device similar to the one described by Meyer. The animal was allowed to grip a T-shaped grip bar with its forepaws and was pulled back gently along a platform until its grip was broken. As the backward locomotion continues, the animal’s hindpaws reach a T-shaped rearlimb grip bar, which it is allowed to grasp and then forced to release by continued pulling. Mark-10 series EG digital force gauges (Mark-10 Corporation, Copiague, NY) were used to record the maximum strain required to break forelimb and hindlimb grip. The average of 3 valid measurements was taken as the animal’s score for each grip strength measure.

MOTOR ACTIVITY
- Motor activity was assessed for 5 animals/sex/group following approximately 28 days of dose administration (Study Week 4, males selected for pairing) or on Lactation Day 13 (females).
- Motor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilizes a series of infrared photobeams surrounding an amber plastic, rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams.
- The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 60 dB to 80 dB. Each animal was tested separately. Data were collected in 5-minute epochs (print intervals) and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation.
- Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).

CLINICAL PATHOLOGY
- Parameters listed in the tables below were evaluated.
- Blood samples for clinical pathology evaluations (hematology, coagulation, and serum chemistry) were collected from 5 animals/sex/group at the scheduled necropsies (Study Day 30 for males and Lactation Day 14 for females selected for pairing) and from 5 animals/sex in the control and high-dose groups at the recovery necropsy (following a 15-day recovery period; Study Day 44 for males and Study Day 62 for females).
- The animals were fasted overnight prior to blood collection. Blood for serum chemistry and hematology was collected from the retro-orbital sinus following isoflurane anesthesia. Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing K2EDTA (hematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).

MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all F0 parental animals at the scheduled termination.
- All F0 adults were euthanized by carbon dioxide inhalation.
- Males were euthanized following completion of the mating period or following the 15-day recovery period; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia.
- Females that delivered were euthanized on Lactation Day 14; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia.
- Females that failed to deliver were euthanized on Postmating Day 25 (females with evidence of mating) or Postcohabitation Day 25 (females with no evidence of mating); uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulphide solution for detection of early implantation loss.11 For females that delivered, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each female that delivered by subtracting the number of pups born from the number of former implantation sites observed.
- Females not selected for pairing were euthanized following the 15-day recovery period.
- Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. At the time of necropsy, tissues and organs listed in the table below were placed in 10 % neutral-buffered formalin (except as noted).

ORGANS WEIGHED AT NECROPSY
- 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.

HISTOLOGY AND MICROSCOPIC EXAMINATIONS
- After fixation, protocol-specified tissues were trimmed according to Charles River SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to Charles River SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.
- Microscopic examination was performed on all tissues listed in the table below from all animals in the control and 1000 mg/kg/day groups at the primary necropsy. Gross lesions were examined from all animals in all groups at the primary necropsy. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section, or other reasons 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.

THYROID HORMONE ANALYSIS
- Blood samples for thyroid hormone analysis were collected as follows:
(a) Blood (at least 1 mL/sample) was collected from the jugular vein of all F0 males and females on the day of scheduled euthanasia (Study Days 30 and 44 for males and Lactation Day 14 and Study Day 62 for females).
(b) On PND 4, blood (at least 1 mL; pooled by litter) was collected via cardiac puncture under isoflurane inhalation from 2 culled pups/litter.
(c) On PND 13, blood (at least 1 mL) was collected via cardiac puncture under isoflurane inhalation from 1 pup/sex/litter.
- Blood was collected into tubes without anticoagulant and allowed to clot at room temperature for at least 30 minutes. Serum was isolated in a refrigerated centrifuge and stored frozen (-65ºC to -85°C).
- The following thyroid hormone parameter was evaluated for F0 males at the primary necropsy and F1 PND 13 males and females: Thyroxine (T4)
- The samples from the F0 females and F1 culled pups (PND 4) were not analyzed, but stored frozen (-65ºC to -85°C).

Ovaries and uterine content:

OESTROUS CYCLES
- Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female for 10 days prior to randomization and continuing for females selected for the breeding phase until evidence of copulation was observed or until termination of the mating period for females with no evidence of mating.
- The average cycle length was calculated and reported for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] until the detection of evidence of mating), beginning with the first day of dose administration. 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. For breeding phase females, the cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation.

Fetal examinations:

F1 LITTER VIABILITY AND DEATHS
- Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained.
- Intact offspring that were found dead from PND 0 to 4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels.
- A detailed gross necropsy was performed on any pup found dead after PND 4. The carcass of each pup was then discarded.

F1 LITTER REDUCTION
- To reduce variability among the litters, 8 pups/litter, 4 pups/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.
- Blood samples for possible future thyroid hormone analysis were collected from 2 culled pups/litter (pooled by litter) on PND 4; pups were euthanized by an intraperitoneal injection of sodium pentobarbital following blood collection and discarded.
- Remaining culled pups (not used for blood collection) were weighed, euthanized by an intraperitoneal injection of sodium pentobarbital on PND 4 (see Appendix 1 – Deviations from study protocol), and discarded.

F1 CLINICAL OBSERVATIONS
- Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a clinical examination on PND 1, 4, 7, 10, and 13 (see Appendix 1 – Deviations from study protocol).
- Any abnormalities in nesting and nursing behavior were recorded.
- The anogenital distance of all F1 pups was measured on PND 1; the absolute distance and the absolute distance relative to the cube root of body weight were reported for each pup (see Appendix 1 – Deviations from study protocol). Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle.

F1 BODY WEIGHTS
- Pups were individually weighed on PND 1, 4, 7, 10, and 13 (see Appendix 1 – Deviations from study protocol). Mean pup weights were presented by sex for each litter and by dose group.
- When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods a given pup was not weighed were left blank or designated as “NA” on the individual report tables.

F1 SEX DETERMINATION
- Pups were individually sexed on PND 0, 4, and 13.

F1 ASSESSMENT OF AREOLAS / NIPPLE ANLAGEN
- On PND 13, all F1 male offspring were evaluated for the presence of thoracic nipples/areolae in accordance with established methods.
- The number of nipples were recorded if nipples were present, and zero was recorded if nipples were absent.

SCHEDULED EUTHANASIA
- On PND 13, surviving F1 rats were euthanised via an intraperitoneal injection of sodium pentobarbital. Blood samples were collected for thyroid hormone analysis immediately prior to euthanasia from 1 pup/sex/litter; gross necropsies were conducted and the thyroids (with parathyroids, if present) were collected and placed in 10 % neutral-buffered formalin for possible histopathological examination.
- Remaining pups (not used for blood collection) were discarded without examination.

Statistics:

See below

Indices:

MATING, FERTILITY AND COPULATION INDICES
- Mating fertility and copulation/conception indices were calculated using the following equations:
(i) Male (female) mating index (%) = [Number of males (females) with evidence of mating (or confirmed pregnant) / total number of males (females) used for mating] * 100
(ii) Male fertility index (%) = [Number of males siring a litter / total number of males used for mating] * 100.
(iii) Male copulation index (%) = [Number of males siring a litter / number of males with evidence of mating (or females with confirmed pregnancy] * 100.
(iv) Female fertility index (%) = [Number of females with confirmed pregnancy / total number of females used for mating] * 100.
(v) Female conception index (%) = [Number of females with confirmed pregnancy / number of females with evidence or mating (or confirmed pregnancy)] * 100.

CALCULATION OF LITTER PARAMETERS
- Litter parameters were defined as shown in the equations below:
(i) Mean live litter size = Total number of viable pups on PND 0 / number of litters with viable pups on PND 0.
(ii) Postnatal survival between birth and PND 0 or PND 4 (% per litter) = Sum of (viable pups per litter on PND 0 or PND 4 / Number of pups born per litter) / Number of litters per group * 100.
(iii) 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 * 100 where N = PND 0–1, 1–4 (pre-selection), 4 (post-selection)–7, 7–10, 10–13, and 4 (post-selection)–13.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Clinical observations of red and clear material around the mouth were noted for F0 females in the 1000 mg/kg/day group approximately 1 hour following dose administration generally throughout the dosing period. These observations did not persist to the daily examinations on the following day, were not observed in males, and therefore were not considered adverse.
- Other clinical observations noted in the 5, 150, 375, and 1000 mg/kg/day groups at the daily examinations, weekly detailed physical examinations, and approximately 1 hour following dose administration, including hair loss on the forelimbs, red material around the nose, and scabbing on the facial area, were noted infrequently, similarly in the control group, and/or in a manner that was not dose-related.

Mortality:

no mortality observed

Description (incidence):

- All F0 males and females survived to the scheduled necropsies

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Males: Mean body weights and body weight gains in the 5, 150, 375, and 1000 mg/kg/day group F0 males were unaffected by test substance administration throughout the study. None of the differences from the control group were statistically significant. In addition, mean body weights and body weight gains in the 1000 mg/kg/day group were comparable to the control group during the recovery period.
- Females (weekly): Mean body weights and body weight gains in the 5, 150, 375, and 1000 mg/kg/day group F0 females were unaffected by test substance administration during the pre-mating period (Study Days 0–13). None of the differences from the control group were statistically significant. Mean body weight gain for the 1000 mg/kg/day group F0 females not paired for mating was similar to the control group for the remainder of the dosing period (Study Days 13–48), with the exception of a significantly (p < 0.01) higher mean body weight gain compared to the control group during Study Days 13–21. This transient increase in body weight gain was not of sufficient magnitude to effect mean absolute body weights or the body weight gain for the overall dosing period (Study Days 0–48) in this group and was not attributed to test substance administration. During the recovery period (Study Days 49–62), mean body weights and body weight gains in the 1000 mg/kg/day group F0 females were similar to the control group; differences from the control group were slight and not statistically significant.
- Females (gestation): Mean body weight gains in the 1000 mg/kg/day group were similar to the control group during Gestation Days 0–17. However, a lower mean body weight gain was noted in the 1000 mg/kg/day group F0 females compared to the control group during Gestation Days 17–20 and as a result when the overall gestation period (Days 0–20) was evaluated; the differences were significant (p < 0.05 or p < 0.01). These decrements were not of sufficient magnitude to affect mean absolute body weights in the 1000 mg/kg/day group. Mean body weights and body weight gains in the 5, 150, and 375 mg/kg/day group F0 females were unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.
- Females (lactation): Mean body weights and body weight gains in the 5, 150, 375, and 1000 mg/kg/day group F0 females were unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Males: Mean food consumption, evaluated as g/animal/day, in the 5, 150, 375, and 1000 mg/kg/day group F0 males was similar to that in the control group throughout the study. No statistically significant differences were observed.
- Females (weekly): Mean food consumption, evaluated as g/animal/day, in the 5, 150, 375, and 1000 mg/kg/day group F0 females was unaffected by test substance administration during the premating period (Study Days 0–13). None of the differences from the control group were statistically significant. Mean food consumption for the 1000 mg/kg/day group F0 females not paired for mating was similar to the control group for the remainder of the dosing period (Study Days 13–48). During the recovery period (Study Days 49–62), mean food consumption in the 1000 mg/kg/day group F0 females was similar to that in the control group.
- Females (gestation): Mean maternal food consumption, evaluated as g/animal/day, in the 5, 150, 375, and 1000 mg/kg/day groups was unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.
- Females (lactation): Mean maternal food consumption, evaluated as g/animal/day, in the 375 and 1000 mg/kg/day groups was similar to the control group during Lactation Days 1–10. However, lower mean food consumption was noted in these groups compared to the control group during Lactation Days 10–13 and as a result when the overall lactation period (Days 1–13) was evaluated; the differences were significant (p < 0.05 or p < 0.01). Although the lower mean food consumption in the 375 and 1000 mg/kg/day groups was attributed to test substance administration, in the absence of corresponding effects on absolute mean body weights or body weight gains in these groups the differences were not considered adverse. Mean maternal food consumption in the 5 and 150 mg/kg/day groups was unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no effects on hematology and coagulation parameters. Significantly (p < 0.05) higher mean reticulocyte values (absolute and percent) were noted in the 1000 mg/kg/day group F0 males compared to the control group at the primary necropsy. In the absence of other effects on red cell parameters or histopathologic correlates, these changes were not considered test substance-related.
- Other significant (p < 0.05) differences from the control group values did not occur in a dose-related manner, were only observed in 1 sex, were of minimal magnitude, were within the Charles River Ashland historical control ranges, and/or there were no histopathologic correlates and therefore these differences were not considered test substance-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Significantly (p < 0.05) higher mean ALT levels were noted in the 375 and 1000 mg/kg/day group F0 males at the primary necropsy. Significantly (p < 0.05 or p < 0.01) lower mean cholesterol levels were also noted in the 150, 375, and 1000 mg/kg/day group F0 males and 375 and 1000 mg/kg/day group F0 females at the primary necropsies.
- In addition, higher mean phosphorus levels were noted in the 1000 mg/kg/day group F0 males and females and lower mean bile acid values were noted in F0 females at all dosage levels at the primary necropsies; the differences were significant (p < 0.05 or p < 0.01) compared to the control group.
- The changes in ALT, cholesterol, phosphorus, and bile acid levels were not considered to be adverse because no histologic correlates were observed in these groups. Other significant (p < 0.05 or p < 0.01) differences from the control group were not observed in a dose-related manner, no similar occurrence were noted in the opposite sex, the changes were of minimal magnitude, and/or the values were within the Charles River Ashland historical control data ranges. Therefore, these differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Home cage parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 4 (males) or on Lactation Day 14 (females), with the following exception. A significantly (p < 0.05) lower number of males sitting or standing normally was noted for the 150 mg/kg/day group during Study Week 4. In the absence of a dose response, this difference was not attributed to test substance administration.
- Handling parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 4 (males) or on Lactation Day 14 (females).
- Open field parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 4 (males) or on Lactation Day 14 (females), with the following exceptions. Significantly (p < 0.01) lower urination counts were noted for F0 males in the 150, 375, and 1000 mg/kg/day groups during the Study Week 4 evaluation. These differences were attributed to the high value in the concurrent control group compared to the mean value in the Charles River Ashland historical control data version 2016.02 and based on the lack of any other signs of toxicity or effects on FOB parameters in these groups, no relationship to the test substance was apparent.
- Sensory parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 4 (males) or on Lactation Day 14 (females).
- Neuromuscular parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 4 (males) or on Lactation Day 14 (females).
- Physiological parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 4 (males) or on Lactation Day 14 (females).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Statistically significantly higher mean adrenal gland weights (absolute and relative to final body weight) were noted in the 375 and 1000 mg/kg/day group females at the primary necropsy, with no microscopic correlates, the finding was considered to be adaptive and nonadverse.
- There were no other alterations in organ weights. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. Higher mean thyroid/parathyroid gland (absolute and relative to final body and brain weights) and kidney (relative to final body weight) weights were noted in the 1000 mg/kg/day group males at the primary necropsy. There were no microscopic or clinical pathology correlates, dose-response relationships were lacking, and mean and individual weights were within the historical control database range; therefore, the findings were not considered to be related to administration of the test substance.
- No alterations were noted at the recovery necropsy. However, higher right mean testis weight relative to final body weight was noted in the 1000 mg/kg/day group males at the RN. Mean left testis (absolute and relative to final body and brain weights) and absolute right testis weights were similar to the control group and no differences in testis weights were noted at the PN; therefore, the difference was attributed to biological variability and not related to administration of the test substance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance.
- The mean numbers of unaccounted-for sites and implantation sites in the 5, 150, 375, and 1000 mg/kg/day groups were similar to the control group values.

Neuropathological findings:

no effects observed

Description (incidence and severity):

- Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test substance administration at all concentrations when evaluated during Study Week 4 (males) or on Lactation Day 14 (females). Values obtained from the 6 subintervals evaluated (0–10, 11–20, 21–30, 31–40, 41–50 and 51–60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the Charles River Ashland historical control data. Differences from the control group were slight, not statistically significant when analysed by a repeated measures analysis, within the Charles River Ashland historical control data ranges and/or did not occur in a dose-related manner. No remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated during Study Week 4 (males) or on Lactation Day 14 (females).

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- No noteworthy microscopic findings were noted in the 1000 mg/kg/day group males or females at the primary necropsy. One finding in a single 1000 mg/kg/day group female (No. 3686) deserves mention, however. Fibrosarcoma was noted in the pericardium, characterised by an unencapsulated, infiltrative mass composed of streams and bundles of spindle cells separated by moderate matrix. No other proliferative spindle cell findings were noted in this or other 1000 mg/kg/day group animals. Spontaneous mesenchymal tumors are reported uncommonly in rats. The relationship of fibrosarcoma in this animal to administration of test item is considered unlikely.
- 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 alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

THYROID HORMONES
- There were no effects on thyroid hormone values in the F0 males at any dosage level. A significantly (p < 0.05) lower mean T4 value was noted for the 1000 mg/kg/day group F0 males at the recovery necropsy. The value was within the Charles River Ashland historical control data version 2017.01 range, there were no similar effects noted at the primary necropsy, and therefore this decrease in mean T4 was considered to be the result of normal biological variation and was not considered to be of toxicological significance.

Maternal developmental toxicity

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

- Mean gestation lengths in the 5, 150, 375, and 1000 mg/kg/day groups were similar to those in the control group.
- No statistically significant differences were noted.
- No signs of dystocia were noted in these groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

- The F0 male and female reproductive parameters are presented in the attached table.
- No effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. One male each in the 150 and 375 mg/kg/day groups did not sire a litter. One female each in these same respective groups were determined to be nongravid.
- 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.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

maternal abnormalities

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): - Lower mean F1 male and female pup body weights (up to 19.9 % and 21.5 %, respectively) and body weight gains were noted in the 1000 mg/kg/day group compared to the control group throughout the postnatal period (PND 1–13); the differences were generally significant (p < 0.05 or p < 0.01).
- In the 375 mg/kg/day group, mean F1 male and female pup body weight gains were slightly lower (not statistically significant) than the control group during the first week of the postnatal period (PND 1–7) followed by significantly (p < 0.01) lower mean body weight gains during PND 7–10. Mean F1 pup body weight gains in this group remained slightly lower than the control group during PND 10–13. As a result of the lower mean F1 pup body weights observed in the 375 mg/kg/day group, mean F1 male and female pup body weights in this group were significantly (p < 0.01) lower (up to 20.4 % and 23.0 %, respectively) than the control group on PND 10 and 13.
- Mean F1 male and female pup body weights and body weight changes during PND 1–13 in the 5 and 150 mg/kg/day groups were unaffected by parental administration of the test substance. No statistically significant differences from the control group were noted.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

- Postnatal survival in the 5, 150, and 375 mg/kg/day groups was unaffected by test substance administration.
- Postnatal survival in the 1000 mg/kg/day group was slightly lower (not statistically significant) than the control group during PND 0–1, 1–4, and birth to PND 4. The value in the 1000 mg/kg/day group during birth to PND 4 (85.0 % per litter) was lower than the minimum mean value in the Charles River Ashland historical control data (84.5 % per litter). The slightly lower postnatal survival observed in the 1000 mg/kg/day group correlated to the increase in the number of pups found dead and missing.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- The percentage of males at birth in the 5, 150, 375, and 1000 mg/kg/day groups were similar to the control group values.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

- The mean number of pups born and live litter size in the 5, 150, 375, and 1000 mg/kg/day groups were similar to the control group values.

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

- The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by parental test substance administration.
- An increase in the number of pups found dead (17 pups from 6 litters; found dead during PND 0–8) and missing (presumed cannibalized; 11 pups from 6 litters) was noted in the 1000 mg/kg/day group compared to the control group (3 pups from 3 litters and 2 pups from 2 litters, respectively).

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

F1 NECROPSIES OF PUPS FOUND DEAD
- The numbers of pup (litters) found dead during PND 0–13 numbered 3(3), 4(3), 3(3), 6(4), and 17(6) in the control, 5, 150, 375, and 1000 mg/kg/day groups, respectively.
- Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

F1 SCHEDULED PUP NECROPSIES (PND 13)
- No internal findings that could be attributed to parental test substance administration were noted at the necropsy of pups euthanised on PND 13.
- Internal findings were limited to the 375 mg/kg/day group and included a small right testis in Pup No. 3745-03 and dark red discoloration of the salivary glands in Pup No. 3688-15. No other internal findings were noted.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

F1 ANOGENITAL DISTANCE (effects observed – not substance related)
- The anogenital distances (absolute, relative to pup body weight, and relative to the cube root of pup body weight) in the 5, 150, 375, and 1000 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant, with the following exception.
- A significantly (p < 0.05) longer anogenital distance relative to the cube root of pup body weight was noted for F1 females in 1000 mg/kg/day group compared to the control group. However, this difference was considered secondary to the lower F1 pup body weights in this group, and therefore was not considered a direct effect of parental test substance administration.

F1 AREOLAE / NIPPLE ANLAGEN (no effects observed)
- Areolae/nipple anlagen in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13.
- There were no retained nipples in any animals.

F1 THYROID HORMONE ANALYSIS (PND 13) (effects observed – not substance related)
- There were no noteworthy effects on thyroid hormone values in the F1 males and females at any dosage level on PND 13.
- A significantly (p < 0.01) lower mean T4 value was noted in the 1000 mg/kg/day group F1 females compared to the control group on PND 13. However, this value was within the Charles River Ashland historical control data range.
- Other differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in postnatal survival

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

ANALYSES OF DOSING FORMULATIONS

- Results of the analyses of dosing formulations are summarised in the table attached.

- The analysed dosing formulations were within the protocol-specified requirements for homogeneity and the SOP requirement for suspensions (85 % to 115 %), with the following exception. The RSD for the mean concentration of the 200 mg/mL formulation prepared on 29 Nov 2016 was 56 %; this formulation was not used for dose administration. A new 200 mg/mL formulation was prepared on 30 Nov 2016 and the results (3.6 % RSD) met the acceptance criteria. The test substance was not detected in the analysed vehicle formulation that was administered to the control group (Group 1).

Applicant's summary and conclusion

Conclusions:

Under the conditions of this screening study, no effects on F0 male and female fertility and mating indices, F0 male copulation index, F0 female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. The NOAEL for F0 systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse effects in the 5, 150, 375, and 1000 mg/kg/day groups. The NOAEL for F1 neonatal toxicity was 150 mg/kg/day based on the lower postnatal survival (not statistically significant) in the 1000 mg/kg/day group and lower pup body weights (within historical control ranges) and body weight gains in the 375 and 1000 mg/kg/day groups.

Executive summary:

GUIDELINE

The objectives of the study were to investigate the potential toxic effects of the test substance when administered to rats for a minimum of 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behaviour, and conception through day 13 of postnatal life. The protocol was designed in general accordance with the OECD Guideline for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016.

 

METHODS

The test substance, in the vehicle (arachis [peanut] oil) was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and mid-dose groups (Groups 2–4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 5, 150, 375, and 1000 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle (an arachis and mineral oil mixture; the percentage of mineral oil matched that in the high-dose group) on a comparable regimen. The dose volume was 5 mL/kg for all groups. Males and females were approximately 10 weeks of age at the beginning of test substance administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 30 doses.Females received 14 daily doses prior to pairing and were dosed through Lactation Day 13 for a total of 49–62 doses; females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (Postcohabitation Day 25 or Postmating Day 25, respectively) for a total of 49–52 doses.The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on Study Day 0; following 30 doses for males and49 doses for females, these animals were assigned to a 15-day nondosing recovery period.

 

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group following approximately 28 days of dose administration and for 5 females/group on Lactation Day 13. All F0 females selected for pairing were allowed to deliver and rear their pups until Lactation Day 13. F1 clinical observations, body weights, and sexes were recorded at appropriate intervals and anogenital distance was recorded on PND 1. To reduce variability among the litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4; blood samples for possible thyroid hormone analysis were collected from the culled pups (2/litter). All F1 male pups were evaluated for areolae/nipple anlagen on PND 13. Remaining F1 pups were euthanized on PND 13; blood samples for thyroid hormone analysis were collected and the thyroid with parathyroids were collected from 1 pup/sex/litter. Clinical pathology evaluations (hematology, coagulation, and serum chemistry) were performed on 5 F0 animals/sex/group at the primary necropsy and 5 animals/sex in the control and high-dose groups at the recovery necropsy. Blood samples for thyroid hormone analysis were collected from F0 males and females at the primary and recovery necropsies; only male samples were analyzed. F0 males were euthanized following completion of the mating period or 15-day recovery period and F0 females were euthanized on Lactation Day 13 for females that delivered, Postcohabitation or Postmating Day 25 for females that failed to deliver, or following the 15-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups at the primary necropsy.

 

RESULTS

All F0 males and female in the control, 5, 150, 375, and 1000 mg/kg/day groups survived to the scheduled necropsies. Clinical observations noted for F0 females in the 1000 mg/kg/day group consisted of red and clear material around the mouth approximately 1 hour following dose administration. These clinical observations did not persist to the daily examinations on the following days, were not observed in males, and therefore were not considered adverse. No effects were noted on F0 male body weights, body weight gains, or food consumption at any dosage level throughout the study.

 

Mean body weights, body weight gains, and food consumption in the 5, 150, 375, and 1000 mg/kg/day group F0 females were unaffected by test substance administration during the pre-mating period. During gestation, a lower overall body weight gain (Gestation Days 0–20) was noted for the 1000 mg/kg/day group F0 females compared to the control group due to lower body weight gains in this group during the last 3 days of gestation (Gestation Days 17–20). This difference had no effect on mean absolute body weights and there was no corresponding effects on mean food consumption during this time period. Thus, the lower mean maternal body weights during this period were attributed to the lower mean F1 pup body weights observed in this group, and therefore were not considered a direct systemic effect of maternal toxicity or adverse. Mean body weights, body weight gains, and food consumption in the 5, 150, and 375 mg/kg/day groups were unaffected by test substance administration during gestation. Lower mean food consumption was noted in the 375 and 1000 mg/kg/day group F0 females when the overall lactation period (Days 1–13) was evaluated due to lower mean food consumption in these groups during the last 3 days of lactation. These decrements did not affect mean body weights or body weight gains in the 375 or 1000 mg/kg/day groups during lactation, and therefore were not considered adverse. Mean body weights, body weight gains, and food consumption in the 5 and 150 mg/kg/day groups were unaffected by test substance administration during lactation. During the recovery period (Study Days 49–62), mean body weights, body weight gains, and food consumption in the 1000 mg/kg/day group F0 females were similar to the control group.

 

Mean F0 male and female reproductive performance (mating, fertility, and copulation/conception indices) and the process of parturition in the 5, 150, 375, and 1000 mg/kg/day groups were unaffected by test substance administration. The mean number of days between pairing and coitus, gestation length, and estrous cycle length were unaffected by test substance administration. There were no effects noted on the mean numbers of implantation sites and unaccounted-for sites in F0 females at any dosage level.

 

There were no effects noted during the FOB or motor activity evaluations for F0 animals at any dosage level.

 

Higher mean adrenal gland weights were noted in the 375 and 1000 mg/kg/day group females at the primary necropsy. Adrenal gland weights were similar to the control group at the recovery necropsy and no microscopic correlates were noted; therefore, the increased adrenal gland weights were considered to be adaptive and nonadverse. There were no other effects on organ weights, and no noteworthy gross necropsy observations or microscopic findings were noted F0 males and females at any dosage level during the dosing and recovery periods. Effects on serum chemistry parameters at the primary necropsy included higher mean alanine transferase (ALT) values for F0 males in the 375 and 1000 mg/kg/day groups and lower meancholesterol values for males in the 150, 375, and 1000 mg/kg/day groups and females in the 375 and 1000 mg/kg/day groups. In the absence of histologic correlates for these changes, the differences in ALT and cholesterol observed in the test substance-treated groups were not considered adverse. No noteworthy alterations in hematology and coagulation parameters were noted for F0 males and females in the 5, 150, 375, and 1000 mg/kg/day groups during the dosing and recovery evaluations. Mean serum T4 levels in the F0 males were unaffected by test substance administration. Mean numbers of F1 pups born, live litter size, and percentage of males at birth were unaffected by parental test substance administration. A higher number of pups that were found deadand missing (presumed cannibalized) was noted in the 1000 mg/kg/day group compared to the control group resulting in slightly lower (not statistically significant) postnatal survival in this group from birth to PND 4. There were no effects on F1 survival in the 5, 150, and 375 mg/kg/day groups and no noteworthy clinical findings for F1 pups at any dosage level.

 

Lower mean F1 pup body weight gains were noted for the 1000 mg/kg/day group males and females throughout the postnatal period resulting in mean F1 pup body weights in this group that were up to 19.9 % and 21.5 % lower, respectively, than the control group during PND 1–13. Mean F1 pup birth weights (PND 1) in the 375 mg/kg/day group were similar to the control group. However, mean pup body weight gains in this group were slightly lower than the control group during the first week of the postnatal period and markedly lower than the control group during PND 10–13. As a result of the lower mean F1 pup body weight gains in the 375 mg/kg/day group, mean F1 male and female body weights were lower (up to 20.4 % and 23 %, respectively) than the control group on PND 10 and 13. F1 pup body weights and body weight gains in the 5 and 150 mg/kg/day groups were unaffected by parental test substance administration. There were no effects on F1 anogenital distance or areolae/nipple anlagen (males only) in the 5, 150, 375, and 1000 mg/kg/day groups. No necropsy findings were noted for F1 pups in the test substance-treated groups on PND 13. There were no noteworthy changes in mean serum T4 levels in F1 males and females at any dosage level on PND 13.

 

CONCLUSION

 

Under the conditions of this screening study, no effects on F0 male and female fertility and mating indices, F0 male copulation index, F0 female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. The NOAEL for F0 systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse effects in the 5, 150, 375, and 1000 mg/kg/day groups. The NOAEL for F1 neonatal toxicity was 150 mg/kg/day based on the lower postnatal survival (not statistically significant) in the 1000 mg/kg/day group and lower pup body weights (within historical control ranges) and body weight gains in the 375 and 1000 mg/kg/day groups.