Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A dosage level of 200 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for female reproductive toxicity when the test item was administered orally by gavage to Crl:CD(SD) rats because dystocia was noted for F0 females at 500 mg/kg/day. There were no test substance-related effects on reproductive parameters for F0 males at any dosage level and a dosage level of 500 mg/kg/day, the highest dosage level tested, was considered to be the NOAEL for male reproductive toxicity. The NOAEL for neonatal toxicity was 200 mg/kg/day based on the lower number of F1 pups born, live litter size, lower postnatal survival and lower mean F1 pup body weights and body weight gains in the 500 mg/kg/day group (OECD 422).

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 July 2016 to 22 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
various (see attachment)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION PERIOD
- Overall design of the study is shown in the diagram attached.
- 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 recognised 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 rats/sex/group [Groups 2–4]) 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 oestrous cyclicity during the pretest period and any females that fail to exhibit normal 4–5 day oestrous 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. However, based on excessive toxicity observed at 500 mg/kg/day, only 7 females in this group delivered and only 3 litters survived to completion of evaluation of the F1 generation.
- Crl:CD(SD) rats (66 males and 78 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 28 Jul 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 21 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 behaviour, 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/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 analysed 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 in solid-bottom cages until the scheduled necropsy.
- Following positive evidence of mating, the females were individually housed in solid-bottom cages with bedding material. The dams and their litters were housed in these cages until euthanasia on Lactation Day 13.
- Females that failed to deliver were housed in solid-bottom cages with bedding material until post-mating 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 until euthanasia.
- Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining oral health of animals, and were
sanitised 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 sanitised 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 F0 males and females (including animals 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 to 78 °F (20 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 71.7 °F to 72.6 °F (22.1 °C to 22.6 °C) and mean daily relative humidity ranged from 39.5 % to 52.5 % 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.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- During 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 to 5 day oestrous cycles (females) was selected for use in the computerised 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. Replacement animals were arbitrarily assigned based on body weight. Animals not assigned to study were transferred to the Charles River rat colony.
- 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 15-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 304 g to 378 g and female body weights ranged from 201 g to 254 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 284 g on Gestation Day 0.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
Lot: 2EL0356; Expiry date: 31 December 2016
Details on exposure:
TEST ITEM PREPARATION
- The vehicle 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 (18 to 24 °C), protected from light.
- The vehicle was mixed throughout the sampling and dose administration procedures.
- Dosing formulations were prepared as indicated in the dose formulation concentration table (below).
- 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 to 24 °C), 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.

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. 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.
Analytical verification of doses or concentrations:
yes
Remarks:
investigation conducted for a previous study
Details on analytical verification of doses or concentrations:
SAMPLING AND ANALYSIS
- Homogeneity of the test substance in the vehicle following 8 days of room temperature storage and stability following 20 hours and 8 days of room temperature storage at concentrations ranging from 1 to 200 mg/mL were established in a previous study. Therefore, stability assessments were not conducted in the current study.
- Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the first and last test substance dosing formulations and from the middle stratum of the first and last control group dosing formulations. 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.
Duration of treatment / exposure:
- Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses.
- Females received 14 daily doses prior to pairing and were dosed through Lactation Day 13 for a total of 49 to 53 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 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 28 doses for males and 49 doses for females,
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
35 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
- Control group: 15 males and 15 females
- Test item 35 mg/kg bw/day group: 10 males and 10 females
- Test item 100 mg/kg bw/day group: 10 males and 10 females
- Test item 200 mg/kg bw/day group: 10 males and 10 females
- Test item 500 mg/kg bw/day group: 15 males and 15 females
Control animals:
yes, concurrent vehicle
Details on study design:
TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- 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 0–27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses.
- The females selected for pairing were dosed during Study Days 0 through the day prior to euthanasia (14 days prior to pairing through Lactation Day 13) for a total of 49–53 doses.
- Females that failed to deliver were dosed through the day prior to euthanasia (Post-Mating Day 25) for a total of 40 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 28 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.
- Study group assignment is shown in the table below.
- 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 100, 350, 500, and 1000 mg/kg/day. Two females in the 1000 mg/kg/day group were euthanised in extremis on Study Day 10 following significant body weight losses and clinical observations that included thin body, unkempt appearance, and dehydration. All other animals survived to the scheduled necropsy on Study Day 14. Slight reductions in overall body weight gains were noted in males at 350, 500, and 1000 mg/kg/day and females in all dosage groups. As a result, dosage levels of 35, 100, 200, and 500 mg/kg/day were chosen for the current study. The high-dosage level was expected to produce signs of toxicity without causing mortality in these animals. Lower dosage levels were selected to assess the dose response over a wide range of dosage levels and to establish a no-observed-adverse-effect level.
- 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.
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS AND SURVIVAL
- All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.
- Individual clinical observations were recorded daily and individual detailed physical examinations were recorded weekly (prior to dose administration during the treatment period).
- Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals.
- Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged 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 are presented for each interval. In addition, cumulative mean body weight changes are 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). 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
- 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 normalised 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; food consumption was reported as g/animal/day during gestation and lactation. Following the breeding period, food consumption for females with no evidence of mating and for all males was measured on a weekly basis until the scheduled euthanasia. Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage.

FOB ASSESSMENTS
- FOB assessments were recorded for 5 animals/sex/group on Study Day 24 (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 following 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 rear limb 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 on Study Day 24 (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
- Blood samples for clinical pathology evaluations (haematology, coagulation, and serum chemistry) were collected from 5 animals/sex/group at the scheduled/primary necropsies (Study Day 28 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 42 for males and Study Day 63 for females).
- The animals were fasted overnight prior to blood collection. Blood for serum chemistry and haematology was collected via the jugular vein. Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing K2EDTA (haematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).Parameters evaluated were as shown in the tables below.

THYROID HORMONE ANALYSIS
- Blood samples for thyroid hormone analysis were collected as follows.
(a) Blood (at least 1 mL) was collected via the jugular vein from all F0 males and females on the day of scheduled euthanasia (Study Days 28 and 42 for males and Lactation Day 14 and Study Day 63 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.
- Serum was isolated in a refrigerated centrifuge and stored frozen (approximately -70 °C).
- The following thyroid hormone parameters were evaluated for F0 males (primary necropsy only)
and F1 PND 13 males and females: Thyroxine (Total T4).
- The samples from the recovery phase F0 males, all F0 females, and F1 culled pups (PND 4) were
not analysed, but stored frozen (-65 to -85 °C).
Oestrous cyclicity (parental animals):
OESTROUS CYCLES
- Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the oestrous cycle of each female for 10 days prior to test substance administration 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 oestrous cycles (i.e., the total number of returns to metoestrus [M] or dioestrus [D] from oestrus [E] or prooestrus [P] until the detection of evidence of mating), beginning with the first day of dose administration.
- Oestrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. For breeding phase females, the cycle during which evidence of mating was observed for a given animal was not included in the mean individual oestrous cycle length calculation.
Litter observations:
LITTER VIABILITY AND DEATHS
- Each litter was examined daily for survival, and all deaths were recorded.
- All pups were individually identified by application of tattoo markings on the digits following completion of parturition.
- A daily record of litter size was maintained. Intact offspring that were found dead or euthanised in extremis (by 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.
- Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. The carcass of each pup was then discarded.

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 computerised randomization.
- Blood samples for possible future thyroid hormone analysis were collected from 2 culled pups/litter (pooled by litter) on PND 4; pup were euthanised by decapitation following blood collection and discarded. Remaining culled pups (not used for blood collection) were weighed, euthanised by an intraperitoneal injection of sodium pentobarbital on PND 4, and discarded.

CLINICAL OBSERVATIONS
- Litters were examined daily for survival and any adverse changes in appearance or behaviour.
- Each pup received a clinical examination on PND 1, 4, 7, 10, and 13.
- Any abnormalities in nesting and nursing behaviour 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. Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle.

BODY WEIGHTS
- Pups were individually weighed on PND 1, 4, 7, 10, and 13. 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.

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

ASSESSMENT OF AREOLAS / NIPPLE ANLAGEN
- On PND 13, all F1 male offspring were evaluated for the presence of thoracic nipples/areolae.
- A finding of “yes” and the number of nipples were recorded if nipples were present, and a finding of “no” was recorded if nipples were absent.
Postmortem examinations (parental animals):
MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all F0 parental animals found dead, euthanised in extremis or due to total litter loss, or at the scheduled termination. All surviving F0 adults were euthanised by carbon dioxide inhalation.
- Males were euthanised 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 euthanised on Lactation Day 14; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia.
- Females that failed to deliver were euthanised on Post-Mating Day 25 (females with 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. The female with a total litter loss was euthanised on the day of litter loss.
- 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. Numbers of corpora lutea were also recorded for females with macroscopic evidence of implantation and for females necropsied during gestation through Lactation Day 4. Females not selected for pairing were euthanised 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. Clinical findings that were verified at necropsy were designated CEO. At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin (except as noted in the table below).

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 haematoxylin and eosin. In
addition, PAS staining was used for the testes and epididymides.
- Microscopic examination was performed on all tissues listed from all animals in the control and 500 mg/kg/day groups at the primary necropsy and from all animals found dead or euthanised in extremis prior to the scheduled 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.
Postmortem examinations (offspring):
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; the thyroids (with parathyroids, if present) were weighed (following fixation) 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
Reproductive 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

PARTRUITION
- All females selected for pairing were allowed to deliver naturally and rear their young to PND 13.
- 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.
Offspring viability indices:
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. Pups that were euthanised due to death of the dam were excluded from pup viability calculations.
- Total litter loss was determined when the last pup in the litter was found dead or euthanised in extremis prior to the scheduled euthanasia. Litters that were euthanised prior to scheduled euthanasia due to reasons unrelated to test substance administration (e.g., death of the dam) were not considered to be a total litter loss on the data tables and were not included in the pup viability calculations.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related clinical observations noted for F0 males and females in the 200 and 500 mg/kg/day groups included rales, clear and red material around the mouth, and red material around the nose. These findings were noted at the daily examinations and/or approximately 1 hour following dose administration generally throughout the dosing period. Although these observations were considered test substance-related, they did not persist or the incidence was greatly decreased at the daily examinations on the following day and therefore these observations were not considered adverse. Other clinical observations noted in the test substance-treated groups at daily examinations, weekly detailed physical examinations, and 1 hour following dose administration, including yellow material and hair loss on various body surfaces, were noted infrequently and/or in a manner that was not dose-related.
Mortality:
mortality observed, treatment-related
Description (incidence):
- Six females (Nos. 5743, 5754, 5767, 5771, 5772, and 5779) in the 500 mg/kg/day group were euthanised in extremis due to dystocia (hunched posture, unkempt appearance, cool body, pale body, and/or pale extremities) or found dead on Gestation Day 22 or Lactation Day 0. Microscopic findings in 5 of these females were limited to vaginal and stress-related changes while former implantation sites with no foetuses were noted for the remaining female. In addition, Female No. 5794 in the 500 mg/kg/day group was euthanised due to a total litter loss on Lactation Day 0. Male No. 5677 in the 500 mg/kg/day group was found dead on Study Day 22 following a body weight loss (19 g) during Study Days 13 to 21. The cause of death for this male was pyelonephritis which was secondary to urinary calculi and therefore not attributed to the test substance. All other F0 males and females survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Males: Test substance-related lower mean body weight gains were noted for F0 males in the 500 mg/kg/day group throughout the entire study (Study Days 0 to 27); the differences were generally significant (p < 0.01) compared to the control group. Consequently, mean body weights in this group were 4.3% to 7.3% lower than the control group from Study Days 13 through 27; the differences were not statistically significant. During the recovery period (Study Days 28 to 41), mean body weight gains in the 500 mg/kg/day group F0 males were similar to or higher than the control group. However, mean body weights in this group remained 6.9 % to 10.2 % lower than the control group during the recovery period; the differences were significant (p < 0.05 or p < 0.01) on Study Days 28 and 35. Mean body weight gains in the 100 and 200 mg/kg/day group F0 males were similar to the control group during the pre-mating period (Study Days 0 to 13). However, slightly lower (not statistically significant) mean body weight gains were noted in these groups compared to the control group for the remainder of the dosing period (Study Days 13 to 21) and when the entire dosing period (Study Days 0 to 27) was evaluated; only the difference in the 100 mg/kg/day group was significant (p < 0.05). The decrements in body weight gain observed in the 100 and 200 mg/kg/day group F0 males were not of sufficient magnitude to affect mean absolute body weights in these groups and therefore these differences were not considered to be adverse. Mean body weights and body weight gains in the 35 mg/kg/day group F0 males were unaffected by test substance administration throughout the study. Differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner.
- Females (weekly): Mean body weights and body weight gains in the 35, 100, 200, and 500 mg/kg/day group F0 females were unaffected by test substance administration during the pre-mating period. Differences from the control group were slight and not statistically significant, with the following exception. A significantly (p < 0.05) lower mean body weight gain was noted for F0 females in the 200 mg/kg/day group when the overall pre-mating period (Study Days 0 to 13) was evaluated. In the absence of a dose response, this difference was not considered test substance-related. Mean body weight gains for F0 females in the 500 mg/kg/day group that were not paired for mating were similar to the control group for the remainder of the dosing period (Study Days 13 to 48); differences were slight and not statistically significant with the following exception. A statistically (p < 0.05) higher mean body weight gain was noted for the 500 mg/kg/day group F0 females compared to the control group during Study Days 27 to 35. The magnitude of this increase was not of sufficient magnitude to affect the overall dosing period (Study Days 0 to 48) and therefore this difference was not considered to be test substance-related. During the recovery period (Study Days 49 to 62), mean body weights and body weight gains in the 500 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 500 mg/kg/day group F0 females were similar to the control group during Gestation Days 0 to 14. Test substance-related lower mean body weight gains were noted for this group during the remainder of the gestation dosing period (Days 14 to 20); the difference from the control group was significant (p < 0.01) during Gestation Days 17 to 20. As a result, a significantly (p < 0.01) lower mean body weight gain was noted for the 500 mg/kg/day group F0 females compared to the control group when the overall gestation dosing period (Days 0 to 20) was evaluated and mean body weights in this group were 5.3% to 15.8% lower than the control group during Gestation Days 11 to 20; the difference was significant (p < 0.01) on Gestation Day 20. The lower body weight changes noted in the latter half of the gestation period in this group were likely attributed to the increased number of foetal resorptions noted in the animals that were found dead or euthanised in extremis. Mean body weight gains in the 200 mg/kg/day group F0 females were similar to the control group during Gestation Days 0 to 17. A test substance-related lower mean body weight gain was noted in this group compared to the control group during Gestation Days 17 to 20 which resulted in a lower mean body weight gain for the overall gestation period (Days 0 to 20); none of the differences were statistically significant. Mean body weights in the 200 mg/kg/day group F0 females were 5.9% to 9.0% lower (not statistically significant) than the control group during Gestation Days 11 to 20. Mean F0 female body weights and body weight gains in the 35 and 100 mg/kg/day groups were unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.
- Females (lactation): Mean body weight gains in the 500 mg/kg/day group F0 females were similar to or higher than the control group during Lactation Days 1 to 10. However, a test substance-related, significant (p < 0.01) mean body weight loss (18 g) was noted in this group compared to a body weight gain (9 g) in the control group during Lactation Days 10 to 13. As a result a lower mean body weight gain was noted for the 500 mg/kg/day group F0 females compared to the control group when the overall lactation period (Days 1 to 13) was evaluated and mean body weight was 8.7% lower than the control group on Lactation Day 13; the differences were not statistically significant. Mean body weights and body weight gains in the 35, 100, and 200 mg/kg/day groups 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: Test substance-related slightly lower mean food consumption, evaluated as g/animal/day, was noted for F0 males in the 500 mg/kg/day group during the pre-mating period (Study Days 0 to 13); the difference from the control group for Study Days 7 to 13 was significant (p < 0.01). Mean food consumption in this group was similar to the control group for the remainder of the dosing period and during the recovery period. Mean food consumption in the 35, 100, and 200 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): Test substance-related, significantly (p < 0.05) lower mean food consumption, evaluated as g/animal/day, was noted in the 500 mg/kg/day group F0 females compared to the control group during Study Days 0 to 7. Mean food consumption in this group was similar to the control group during the remainder of the pre-mating period (Study Days 7 to 13). Mean food consumption in the 500 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 to 48); the differences were slight and not statistically significant. During the recovery period (Study Days 56 to 62), mean food consumption in the 500 mg/kg/day group F0 females was similar to the control group. Mean food consumption in the 200 mg/kg/day group F0 females was similar to the control group during Study Days 0 to 7. During Study Days 7 to 13, significantly (p < 0.01) lower mean food consumption was noted for this group compared to the control group. However, this difference was not of sufficient magnitude to affect mean body weights in this group and therefore, the decreased food consumption was not considered test substance-related. Mean food consumption in the 35 and 100 mg/kg/day group F0 females was unaffected by test substance administration during the pre-mating period. None of the differences from the control group were statistically significant.
- Females (gestation): Mean F0 maternal food consumption, evaluated as g/animal/day, in the 35, 100, 200, and 500 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 food consumption in the 500 mg/kg/day group F0 females were similar to the control group during Lactation Days 1 to 10. However, significantly (p < 0.01) lower mean food consumption, corresponding to a mean body weight loss, was noted in this group compared to the control group during Lactation Days 10 to 13. When the overall lactation period was evaluated, mean food consumption in the 500 mg/kg/day group F0 females was similar to the control group. Mean maternal food consumption in the 35, 100, and 200 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, treatment-related
Description (incidence and severity):
- Test substance-related, statistically significantly lower mean absolute and percentage reticulocytes were noted in the 500 mg/kg/day group females at the primary necropsy. There were no associated changes in remaining red blood cell parameters; however, the finding correlated with minimally decreased cellularity within sternal bone marrow. There were no other test substance-related alterations in haematology and coagulation parameters at the primary necropsy.
- Reticulocyte parameters in the 500 mg/kg/day group females were similar to the control group at the recovery necropsy. Lower mean corpuscular haemoglobin concentration (MCHC) and higher red cell distribution width (RDW) values were noted at the recovery necropsy, consistent with maturation of the higher reticulocyte population noted at the primary necropsy. There were no test substance-related differences in the 500 mg/kg/day group males at the recovery necropsy; however, some statistically significant differences were noted, consisting of higher mean reticulocyte parameters and lower mean corpuscular haemoglobin (MCH) values. The differences were not considered to be related to administration of the test substance because values were similar to the control group at the primary necropsy.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
- There were no test substance-related alterations in serum chemistry parameters at the scheduled necropsies. However, lower mean serum potassium value was noted in the 500 mg/kg/day group males at the primary necropsy. There was no dose-response relationship, other electrolyte values were similar to the control group, and the mean value was within the historical control database range; the difference was attributed to biological variability.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
- 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 Day 24 (males) or on Lactation Day 13 (females).
- 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 Day 24 (males) or on Lactation Day 13 (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 Day 24 (males) or on Lactation Day 13 (females).
- 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 Day 24 (males) or on Lactation Day 13 (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 Day 24 (males) or on Lactation Day 13 (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 Day 24 (males) or on Lactation Day 13 (females).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
MOTOR ACTIVITY
- 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 on Study Day 24 (males) and Lactation Day 13 (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, with the following exceptions. Significantly (p ≤ 0.041) higher mean ambulatory counts were noted for F0 females in the 500 mg/kg/day group compared to the control group during the 11-20 and 31-40 minute intervals on Lactation Day 13. Because of the small sample size in this group (N=3) and the absence of an effect on the cumulative ambulatory count value, these differences were not considered to be test substance-related. No remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated on Study Day 24 (males) or Lactation Day 13 (females).
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Primary necropsy: Test substance-related decreased thymic cortical lymphocytes and sternal bone marrow cellularity were noted in 2 of the three 500 mg/kg/day group F0 females evaluated at the primary necropsy; the thymic finding correlated with lower mean thymus weight, and the bone marrow finding correlated with lower reticulocyte parameters. Both findings were considered to represent test substance-related stress-responses.
- Fail To Deliver (Post-Mating Day 25/Study Day 40) Necropsy: Two control group F0 females which failed to deliver pups were evaluated. Microscopic findings were similar to the control group females that delivered pups.
- Total Litter Loss (Study Day 37) Necropsy: One 500 mg/kg/day group F0 female with total litter loss was evaluated. Microscopic findings considered to be secondary to test substance-related stress-response consisted of decreased lymphocytes within the spleen and thymus; adrenal cortical congestion/haemorrhage; reduced exocrine pancreatic zymogen granules; and decreased sternal bone marrow cellularity. Within the vagina there was mucosal atrophy and increased prominence of mucification. Findings were similar to those noted in the 500 mg/kg/day group unscheduled death F0 females. 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.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
THYROID HORMONES
- Test substance-related, statistically significantly lower mean serum T4 values were noted in the 500 mg/kg/day group males with a dose-response relationship. Lower T4 values correlated with higher liver weights, consistent with increased T4 metabolism secondary to hepatocellular microsomal enzyme induction. There were no microscopic correlates.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
- Mean lengths of oestrous cycles in these groups were also similar to the control group value
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
REPRODUCTIVE PERFORMANCE
- The F0 male and female reproductive parameters are presented in the attached table.
- No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. Two males in the control group did not sire a litter and 2 females in this same group 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 oestrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.

GESTATION LENGTH AND PARTURITION
- Mean gestation lengths in the 35, 100, 200, and 500 mg/kg/day groups were similar to those in the control group. No statistically significant differences were noted. Dystocia (clinical observations of hunched posture, unkempt appearance, cool body, pale body, and/or pale extremities) was noted at 500 mg/kg/day resulting in 5 females in this group that were euthanised in extremis on Gestation Day 22 or Lactation Day 0. No signs of dystocia were noted at 35, 100, or 200 mg/kg/day.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no test substance-related effects on reproductive parameters at any dose level
Key result
Critical effects observed:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
GENERAL PHYSICAL CONDITION
- A test substance-related increase in the number of pups found dead was noted in the 500 mg/kg/day group compared to the control group; these pups were found dead on PND 0. The number of pups found dead and/or missing in the 35, 100, and 200 mg/kg/day groups was unaffected by maternal test substance administration.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- A lower number of pups born, corresponding lower number of implantation sites, and consequently mean live litter size on PND 0 were observed in the 500 mg/kg/day group (11.4 and 6.4 pups, respectively) when compared to the control group (15.1 and 14.8 pups, respectively); the differences were significant (p<0.05 or p<0.01). Lower (not statistically significant) postnatal survival was noted for the 500 mg/kg/day group compared to the control group from birth to PND 4 due to the lower survival on PND 0 (relative to the number of pups born). Postnatal survival in this group was similar to the control group for the remainder of the postnatal period (PND 4 through PND 13). The mean number of pups born, live litter size, and postnatal survival in the 35, 100, and 200 mg/kg/day groups were similar to the control group values. The percentage of males at birth was unaffected by test substance administration at all dosage levels.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Mean male and female F1 pup body weight gains in the 500 mg/kg/day group were generally lower than the control group throughout the postnatal period (PND 1–13); only the difference for females during PND 10–13 was significant (p<0.01). Lower (not statistically significant) mean F1 male and female body weights were noted compared to the control group from PND 1 to PND 13.
- Mean male and female F1 pup body weights and body weight gains during PND 1 to 13 in the 35, 100, and 200 mg/kg/day groups were unaffected by parental administration of the test substance. 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:
no effects observed
Description (incidence and severity):
- There were no test substance-related effects on thyroid weights in the F1 males and females at any dosage level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
NECROPSIES OF PUPS FOUND DEAD OR EUTHANISED DUE TO DEATH OF DAM
- The numbers of pups (litters) found dead or euthanised due to death of the dam during PND 0-13 numbered 3(1), 0(0), 2(2), 4(2), and 38(5) in the control, 35, 100, 200, and 500 mg/kg/day groups, respectively. Aside from the presence or absence of milk in the stomach, internal findings were limited to 3 pups in Litter No. 5769 in the 200 mg/kg/day group and consisted of an enlarged kidney, absent kidney and ureter, renal papillae(e) not fully developed and distended ureter(s), and/or a nodule on the kidney.

SCHEDULED PUP NECROPSIES (PND 13)
- No internal findings were noted at the necropsy of pups euthanised on PND 13.
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
ANOGENITAL DISTANCE
- The anogenital distances (absolute, relative to pup body weight, and relative to the cube root of pup body weight) in the 35, 100, 200, and 500 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

AREOLAE / NIPPLE ANLAGEN
- Areolae/nipple anlagen in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. The test substance-treated group values were not statistically different from the control group values.

THYROID HORMONE ANALYSIS (PND 13)
- Test substance-related, statistically significantly lower mean serum T4 values were noted in the 500 mg/kg/day group pups; a dose-response relationship was absent.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
matrnal dose
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

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 range for suspensions (85% to 115%) and were homogeneous.

- Test substance was not detected in the analysed vehicle formulation that was administered to the control group (Group 1).

Conclusions:
Under the conditions of this screening study, dystocia was noted for F0 females at 500 mg/kg/day. Therefore, a dosage level of 200 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for female reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. There were no test substance-related effects on reproductive parameters for F0 males at any dosage level. Therefore, a dosage level of 500 mg/kg/day, the highest dosage level tested, was considered to be the NOAEL for male reproductive toxicity. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 200 mg/kg/day based on adverse lower body weights, body weight gains, and reduced food consumption in the 500 mg/kg/day group males and females (during lactation). During the recovery period, the effects on body weight and food consumption were no longer observed in the 500 mg/kg/day group males and females with the exception of lower body weights which continued to be observed in males. The NOAEL for neonatal toxicity was 200 mg/kg/day based on the lower number of F1 pups born, live litter size, lower postnatal survival and lower mean F1 pup body weights and body weight gains in the 500 mg/kg/day group.
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 to 4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 35, 100, 200, and 500 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle 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 28 doses. Females received 14 daily doses prior to pairing and were dosed through Lactation Day 13 for a total of 49 to 53 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 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 28 doses for males and 49 doses for females, these animals were assigned to a 15-day non-dosing 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 on Study Day 24 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 euthanised on PND 13; blood samples for thyroid hormone analysis were collected and selected organs were weighed from 1 pup/sex/litter. Clinical pathology evaluations (haematology, 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 (Lactation Day 14 for females) and recovery necropsies; only primary necropsy male samples were analysed. F0 males were euthanised following completion of the mating period or 15-day recovery period and F0 females were euthanised on Lactation Day 14 for females that delivered, post-mating 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 found dead or euthanised in extremis and all F0 animals in the control and high-dose groups at the primary necropsy.

 

RESULTS

Test substance-related mortality and moribundity were noted at 500 mg/kg/day. One F0 female in the 500 mg/kg/day group was found dead and 5 F0 females were euthanised in extremis following signs of dystocia including hunched posture, unkempt appearance, cool body, pale body, and/or pale extremities on Gestation Day 22 or Lactation Day 0. Females with dystocia had foetal autolysis/resorption and macroscopic and microscopic findings consistent with stress response, including small spleen and thymus which correlated microscopically with decreased lymphocytes; adrenal cortical hypertrophy/hyperplasia and congestion/haemorrhage; reduced pancreatic zymogen granules; gastric ulceration; and reduced sternal bone marrow cellularity. Vaginal atrophy and mucification were additionally noted. Similar microscopic findings were noted in a single 500 mg/kg/day group female with total litter loss. In the 500 mg/kg/day group, 1 F0 male was found dead on Study Day 22; the cause of death was attributed to pyelonephritis secondary to urinary calculi and not considered test substance-related. All other F0 and F1 males and females survived to the scheduled necropsies. Test substance-related clinical observations of rales, clear and red material around the mouth, and red material around the nose were noted for F0 males and females in the 200 and 500 mg/kg/day groups approximately 1 hour following dose administration generally throughout the dosing period. These clinical observations were generally not observed or the incidence was considerably decreased at the daily examinations on the following day and therefore these observations were not considered adverse.

 

Test substance-related lower mean body weight gains were noted for F0 males in the 500 mg/kg/day group throughout the dosing period (Study Days 0 to 27) which corresponded to decreased food consumption in this group during the pre-mating period (Study Days 0 to 13) and resulted in mean body weights in the 500 mg/kg/day group F0 males that were up to 7.3% lower than the control group. Mean food consumption for the 5 males in the 500 mg/kg/day group that were not paired for breeding was similar to that in the control group during Study Days 13 to 27. Mean body weights in the 500 mg/kg/day group F0 males remained lower than the control group during the recovery period (Study Days 28 to 41) despite mean body weight gains that were similar to or higher than the control group. Test substance-related, slightly lower mean body weight gains were noted for F0 males in the 100 and 200 mg/kg/day groups compared to the control group when the overall dosing period (Study Days 0 to 27) was evaluated due to lower mean body weight gains in these groups during Study Days 13 to 21. However, the magnitude of the differences were not of sufficient magnitude to affect absolute mean body weights and there were no corresponding decreases in mean food consumption in these groups; therefore, the decrements in body weight observed for the 100 and 200 mg/kg/day group F0 males were not considered adverse.

Test substance-related lower mean food consumption was noted for F0 females in the 200 and 500 mg/kg/day groups during the pre-mating period (Study Days 0 to 13). Mean body weights and body weight gains in these groups were similar to the control group during the pre-mating period (Study Days 0 to 13) and when the entire treatment period (Study Days 0 to 48) for F0 females in the 500 mg/kg/day group that were not paired for mating. Therefore, the lower mean food consumption noted in the 200 and 500 mg/kg/day groups during the pre-mating period was not considered adverse. A lower mean body weight gain was noted for F0 females in the 500 mg/kg/day group compared to the control group when the entire gestation period (Days 0 to 20) was evaluated due to lower mean body weight gains during Gestation Days 14 to 20. As a result, mean body weights for F0 females in this group were 15.8% lower than the control group on Gestation Day 20. Mean food consumption in the 500 mg/kg/day group F0 females was similar to the control group during gestation. The changes in mean gestational body weights were considered secondary to the lower live litter sizes and reduced mean pup body weights noted at 500 mg/kg/day. A mean body weight loss and corresponding decreased food consumption were noted for F0 females in the 500 mg/kg/day group during Lactation Days 10 to 13 resulting in a lower mean body weight gain for this group when the overall lactation period (Days 1 to 13) was evaluated and mean body weights that were 8.7% lower than the control group on Lactation Day 13. Mean body weight gains, body weights, and food consumption in the 500 mg/kg/day group F0 females were similar to the control group during the recovery period (Study Days 49 to 62). In the 200 mg/kg/day group F0 females, mean body weights, body weight gains, and food consumption during gestation and lactation were similar to that in the control group. Mean body weights, body weight gains, and food consumption in the 35 mg/kg/day group F0 males and 35 and 100 mg/kg/day group F0 females and were unaffected by test substance administration throughout the study. No test substance-related effects were noted during the FOB or motor activity evaluations at any dosage level in the F0 generation.

 

F0 gestation length, oestrous cycle length, and reproductive performance (mating, fertility, and copulation/conception indices) in all test substance-treated groups and the process of parturition in the 35, 100, and 200 mg/kg/day groups were unaffected by test substance administration. A higher number of unaccounted-for sites (attributed to a single female) and a test substance-related lower number of implantation sites were noted in the 500 mg/kg/day group compared to the control group. As a result, a lower number of F1 pups born and live litter size on PND 0, with a corresponding greater number of pups that were found dead, were observed in this group. In addition, lower postnatal survival was noted for this group from birth to PND 4 due to 1 female with a total litter loss and 6 females in this group that had entirely resorbed litters. There were no test substance-related effects on unaccounted-for sites, implantation sites, or F1 survival and clinical condition in the 35, 100, and 200 mg/kg/day groups.

 

Test substance-related lower mean F1 pup body weight gains were noted in the 500 mg/kg/day group compared to the control group generally throughout the postnatal period resulting in mean F1 male and female body weights that were up to 13.1% and 14.7% lower, respectively. Mean F1 pup body weights and body weight gains in the 35, 100, and 200 mg/kg/day groups were similar to the control group. There were no test substance-related effects noted on F1 anogenital distance or areolae/nipple anlagen (males only) in the 35, 100, 200, and 500 mg/kg/day groups. No remarkable necropsy findings were noted for F1 pups that were euthanised on PND 13. There were no test substance-related effects on thyroid/parathyroid weights noted for F1 pups on PND 13. Test substance-related findings at the primary necropsy consisted of lower thymus weights which correlated with decreased thymic lymphocytes; decreased sternal bone marrow cellularity which correlated with lower reticulocyte parameters in the 500 mg/kg/day group F0 females; lower seminal vesicle weights in the 100 and 500 mg/kg/day group F0 males with no microscopic correlate; higher kidney weights in the 200 and 500 mg/kg/day group males with no microscopic correlate; and higher liver weights in the 500 mg/kg/day group F0 males with no microscopic correlate. Thymus, bone marrow, reticulocyte, and seminal vesicle changes were consistent with a test substance-related stress-response. Higher liver weights are a sensitive indication of hepatocellular microsomal enzyme induction, while microscopic evidence of hepatocellular hypertrophy is generally apparent only in livers with greater than 20% higher weights. Serum thyroxine (T4) values were lower in the 500 mg/kg/day group F0 males and F1 pups, with no thyroid weight changes or microscopic correlates in the F0 males; lower T4 values were attributed to enhanced metabolism of T4 secondary to test substance-related microsomal enzyme induction.

 

CONCLUSION

 

Under the conditions of this screening study, dystocia was noted for F0 females at 500 mg/kg/day. Therefore, a dosage level of 200 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for female reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. There were no test substance-related effects on reproductive parameters for F0 males at any dosage level. Therefore, a dosage level of 500 mg/kg/day, the highest dosage level tested, was considered to be the NOAEL for male reproductive toxicity. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 200 mg/kg/day based on adverse lower body weights, body weight gains, and reduced food consumption in the 500 mg/kg/day group males and females (during lactation). During the recovery period, the effects on body weight and food consumption were no longer observed in the 500 mg/kg/day group males and females with the exception of lower body weights which continued to be observed in males. The NOAEL for neonatal toxicity was 200 mg/kg/day based on the lower number of F1 pups born, live litter size, lower postnatal survival and lower mean F1 pup body weights and body weight gains in the 500 mg/kg/day group.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

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.

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 to 4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 35, 100, 200, and 500 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle 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 28 doses. Females received 14 daily doses prior to pairing and were dosed through Lactation Day 13 for a total of 49 to 53 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 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 28 doses for males and 49 doses for females, these animals were assigned to a 15-day non-dosing 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 on Study Day 24 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 euthanised on PND 13; blood samples for thyroid hormone analysis were collected and selected organs were weighed from 1 pup/sex/litter. Clinical pathology evaluations (haematology, 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 (Lactation Day 14 for females) and recovery necropsies;only primary necropsy male samples were analysed. F0 males were euthanised following completion of the mating period or 15-day recovery period and F0 females were euthanised on Lactation Day 14 for females that delivered, post-mating 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 found dead or euthanised in extremis and all F0 animals in the control and high-dose groups at the primary necropsy.

Test substance-related mortality and moribundity were noted at 500 mg/kg/day. One F0 female in the 500 mg/kg/day group was found dead and 5 F0 females were euthanised in extremis following signs of dystocia including hunched posture, unkempt appearance, cool body, pale body, and/or pale extremities on Gestation Day 22 or Lactation Day 0. Females with dystocia had foetal autolysis/resorption and macroscopic and microscopic findings consistent with stress response, including small spleen and thymus which correlated microscopically with decreased lymphocytes; adrenal cortical hypertrophy/hyperplasia and congestion/haemorrhage; reduced pancreatic zymogen granules; gastric ulceration; and reduced sternal bone marrow cellularity. Vaginal atrophy and mucification were additionally noted. Similar microscopic findings were noted in a single 500 mg/kg/day group female with total litter loss. In the 500 mg/kg/day group, 1 F0 male was found dead on Study Day 22; the cause of death was attributed to pyelonephritis secondary to urinary calculi and not considered test substance-related. All other F0 and F1 males and females survived to the scheduled necropsies. Test substance-related clinical observations of rales, clear and red material around the mouth, and red material around the nose were noted for F0 males and females in the 200 and 500 mg/kg/day groups approximately 1 hour following dose administration generally throughout the dosing period. These clinical observations were generally not observed or the incidence was considerably decreased at the daily examinations on the following day and therefore these observations were not considered adverse.

Test substance-related lower mean body weight gains were noted for F0 males in the 500 mg/kg/day group throughout the dosing period (Study Days 0 to 27) which corresponded to decreased food consumption in thisgroup during the pre-mating period (Study Days 0 to 13) and resulted in mean body weights in the 500 mg/kg/day group F0 males that were up to 7.3% lower than the control group. Mean food consumption for the 5 males in the 500 mg/kg/day group that were not paired for breeding was similar to that in the control group during Study Days 13 to 27. Mean body weights in the 500 mg/kg/day group F0 males remained lower than the control group during the recovery period (Study Days 28 to 41) despite mean body weight gains that were similar to or higher than the control group. Test substance-related, slightly lower mean body weight gains were noted for F0 males in the 100 and 200 mg/kg/day groups compared to the control group when the overall dosing period (Study Days 0 to 27) was evaluated due to lower mean body weight gains in these groups during Study Days 13 to 21. However, the magnitude of the differences were not of sufficient magnitude to affect absolute mean body weights and there were no corresponding decreases in mean food consumption in these groups; therefore, the decrements in body weight observed for the 100 and 200 mg/kg/day group F0 males were not considered adverse.

Test substance-related lower mean food consumption was noted for F0 females in the 200 and 500 mg/kg/day groups during the pre-mating period (Study Days 0 to 13). Mean body weights and body weight gains in these groups were similar to the control group during the pre-mating period (Study Days 0 to 13) and when the entire treatment period (Study Days 0 to 48) for F0 females in the 500 mg/kg/day group that were not paired for mating. Therefore, the lower mean food consumption noted in the 200 and 500 mg/kg/day groups during the pre-mating period was not considered adverse. A lower mean body weight gain was noted for F0 females in the 500 mg/kg/day group compared to the control group when the entire gestation period (Days 0 to 20) was evaluated due to lower mean body weight gains during Gestation Days 14 to 20. As a result, mean body weights for F0 females in this group were 15.8% lower than the control group on Gestation Day 20. Mean food consumption in the 500 mg/kg/day group F0 females was similar to the control group during gestation. The changes in mean gestational body weights were considered secondary to the lower live litter sizes and reduced mean pup body weights noted at 500 mg/kg/day. A mean body weight loss and corresponding decreased food consumption were noted for F0 females inthe 500 mg/kg/day group during Lactation Days 10 to 13 resulting in a lower mean body weight gain for this group when the overall lactation period (Days 1 to 13) was evaluated and mean body weights that were 8.7% lower than the control group on Lactation Day 13. Mean body weight gains, body weights, and food consumption in the 500 mg/kg/day group F0 females were similar to the control group during the recovery period (Study Days 49 to 62). In the 200 mg/kg/day group F0 females, mean body weights, body weight gains, and food consumption during gestation and lactation were similar to that in the control group. Mean body weights, body weight gains, and food consumption in the 35 mg/kg/day group F0 males and 35 and 100 mg/kg/day group F0 females and were unaffected by test substance administration throughout the study. No test substance-related effects were noted during the FOB or motor activity evaluations at any dosage level in the F0 generation.

F0 gestation length, oestrous cycle length, and reproductive performance (mating, fertility, and copulation/conception indices) in all test substance-treated groups and the process of parturition in the 35, 100, and 200 mg/kg/day groups were unaffected by test substance administration. A higher number of unaccounted-for sites (attributed to a single female) and a test substance-related lower number of implantation sites were noted in the 500 mg/kg/day group compared to the control group. As a result, a lower number of F1 pups born and live litter size on PND 0, with a corresponding greater number of pups that were found dead, were observed in this group. In addition, lower postnatal survival was noted for this group from birth to PND 4 due to 1 female with a total litter loss and 6 females in this group that had entirely resorbed litters. There were no test substance-related effects on unaccounted-for sites, implantation sites, or F1 survival and clinical condition in the 35, 100, and 200 mg/kg/day groups.Test substance-related lower mean F1 pup body weight gains were noted in the 500 mg/kg/day group compared to the control group generally throughout the postnatal period resulting in mean F1 male and female body weights that were up to 13.1% and 14.7% lower, respectively. Mean F1 pup body weights and body weight gains in the 35, 100, and 200 mg/kg/day groups were similar to the control group. There were no test substance-related effects noted on F1 anogenital distance orareolae/nipple anlagen (males only) in the 35, 100, 200, and 500 mg/kg/day groups. No remarkable necropsy findings were noted for F1 pups that were euthanised on PND 13. There were no test substance-related effects on thyroid/parathyroid weights noted for F1 pups on PND 13. Test substance-related findings at the primary necropsy consisted of lower thymus weights which correlated with decreased thymic lymphocytes; decreased sternal bone marrow cellularity which correlated with lower reticulocyte parameters in the 500 mg/kg/day group F0 females; lower seminal vesicle weights in the 100 and 500 mg/kg/day group F0 males with no microscopic correlate; higher kidney weights in the 200 and 500 mg/kg/day group males with no microscopic correlate; and higher liver weights in the 500 mg/kg/day group F0 males with no microscopic correlate. Thymus, bone marrow, reticulocyte, and seminal vesicle changes were consistent with a test substance-related stress-response. Higher liver weights are a sensitive indication of hepatocellular microsomal enzyme induction, while microscopic evidence of hepatocellular hypertrophy is generally apparent only in livers with greater than 20% higher weights. Serum thyroxine (T4) values were lower in the 500 mg/kg/day group F0 males and F1 pups, with no thyroid weight changes or microscopic correlates in the F0 males; lower T4 values were attributed to enhanced metabolism of T4 secondary to test substance-related microsomal enzyme induction.

Under the conditions of this screening study, dystocia was noted for F0 females at 500 mg/kg/day. Therefore, a dosage level of 200 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for female reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. There were no test substance-related effects on reproductive parameters for F0 males at any dosage level. Therefore, a dosage level of 500 mg/kg/day, the highest dosage level tested, was considered to be the NOAEL for male reproductive toxicity. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 200 mg/kg/day based on adverse lower body weights, body weight gains, and reduced food consumption in the 500 mg/kg/day group males and females (during lactation). During the recovery period, the effects on body weight and food consumption were no longer observed in the 500 mg/kg/day group males and females with the exception of lower body weights whichcontinued to be observed in males. The NOAEL for neonatal toxicity was 200 mg/kg/day based on the lower number of F1 pups born, live litter size, lower postnatal survival and lower mean F1 pup body weights and body weight gains in the 500 mg/kg/day group.

Effects on developmental toxicity

Description of key information

A dosage level of 200 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for female reproductive toxicity when the test item was administered orally by gavage to Crl:CD(SD) rats because dystocia was noted for F0 females at 500 mg/kg/day. There were no test substance-related effects on reproductive parameters for F0 males at any dosage level and a dosage level of 500 mg/kg/day, the highest dosage level tested, was considered to be the NOAEL for male reproductive toxicity. The NOAEL for neonatal toxicity was 200 mg/kg/day based on the lower number of F1 pups born, live litter size, lower postnatal survival and lower mean F1 pup body weights and body weight gains in the 500 mg/kg/day group (OECD 422).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 July 2016 to 22 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
various (see attachment)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION PERIOD
- Overall design of the study is shown in the diagram attached.
- 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 recognised 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 rats/sex/group [Groups 2–4]) 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 oestrous cyclicity during the pretest period and any females that fail to exhibit normal 4–5 day oestrous 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. However, based on excessive toxicity observed at 500 mg/kg/day, only 7 females in this group delivered and only 3 litters survived to completion of evaluation of the F1 generation.
- Crl:CD(SD) rats (66 males and 78 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 28 Jul 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 21 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 behaviour, 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/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 analysed 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 in solid-bottom cages until the scheduled necropsy.
- Following positive evidence of mating, the females were individually housed in solid-bottom cages with bedding material. The dams and their litters were housed in these cages until euthanasia on Lactation Day 13.
- Females that failed to deliver were housed in solid-bottom cages with bedding material until post-mating 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 until euthanasia.
- Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining oral health of animals, and were
sanitised 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 sanitised 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 F0 males and females (including animals 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 to 78 °F (20 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 71.7 °F to 72.6 °F (22.1 °C to 22.6 °C) and mean daily relative humidity ranged from 39.5 % to 52.5 % 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.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- During 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 to 5 day oestrous cycles (females) was selected for use in the computerised 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. Replacement animals were arbitrarily assigned based on body weight. Animals not assigned to study were transferred to the Charles River rat colony.
- 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 15-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 304 g to 378 g and female body weights ranged from 201 g to 254 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 284 g on Gestation Day 0.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
Lot: 2EL0356; Expiry date: 31 December 2016
Details on exposure:
TEST ITEM PREPARATION
- The vehicle 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 (18 to 24 °C), protected from light.
- The vehicle was mixed throughout the sampling and dose administration procedures.
- Dosing formulations were prepared as indicated in the dose formulation concentration table (below).
- 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 to 24 °C), 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.
Analytical verification of doses or concentrations:
yes
Remarks:
investigation conducted for a previous study
Details on analytical verification of doses or concentrations:
SAMPLING AND ANALYSIS
- Homogeneity of the test substance in the vehicle following 8 days of room temperature storage and stability following 20 hours and 8 days of room temperature storage at concentrations ranging from 1 to 200 mg/mL were established in a previous study. Therefore, stability assessments were not conducted in the current study.
- Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the first and last test substance dosing formulations and from the middle stratum of the first and last control group dosing formulations. 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. 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 were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses.
- Females received 14 daily doses prior to pairing and were dosed through Lactation Day 13 for a total of 49 to 53 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 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 28 doses for males and 49 doses for females,
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
35 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
- Control group: 15 males and 15 females
- Test item 35 mg/kg bw/day group: 10 males and 10 females
- Test item 100 mg/kg bw/day group: 10 males and 10 females
- Test item 200 mg/kg bw/day group: 10 males and 10 females
- Test item 500 mg/kg bw/day group: 15 males and 15 females
Control animals:
yes, concurrent vehicle
Details on study design:
TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- 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 0–27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses.
- The females selected for pairing were dosed during Study Days 0 through the day prior to euthanasia (14 days prior to pairing through Lactation Day 13) for a total of 49–53 doses.
- Females that failed to deliver were dosed through the day prior to euthanasia (Post-Mating Day 25) for a total of 40 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 28 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.
- Study group assignment is shown in the table below.
- 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 100, 350, 500, and 1000 mg/kg/day. Two females in the 1000 mg/kg/day group were euthanised in extremis on Study Day 10 following significant body weight losses and clinical observations that included thin body, unkempt appearance, and dehydration. All other animals survived to the scheduled necropsy on Study Day 14. Slight reductions in overall body weight gains were noted in males at 350, 500, and 1000 mg/kg/day and females in all dosage groups. As a result, dosage levels of 35, 100, 200, and 500 mg/kg/day were chosen for the current study. The high-dosage level was expected to produce signs of toxicity without causing mortality in these animals. Lower dosage levels were selected to assess the dose response over a wide range of dosage levels and to establish a no-observed-adverse-effect level.
- 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.
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 recorded weekly (prior to dose administration during the treatment period).
- Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals.
- Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged 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 are presented for each interval. In addition, cumulative mean body weight changes are 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). 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
- 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 normalised 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; food consumption was reported as g/animal/day during gestation and lactation. Following the breeding period, food consumption for females with no evidence of mating and for all males was measured on a weekly basis until the scheduled euthanasia. Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage.

OESTROUS CYCLES
- Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the oestrous cycle of each female for 10 days prior to test substance administration 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 oestrous cycles (i.e., the total number of returns to metoestrus [M] or dioestrus [D] from oestrus [E] or prooestrus [P] until the detection of evidence of mating), beginning with the first day of dose administration.
- Oestrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. For breeding phase females, the cycle during which evidence of mating was observed for a given animal was not included in the mean individual oestrous cycle length calculation.

FOB ASSESSMENTS
- FOB assessments were recorded for 5 animals/sex/group on Study Day 24 (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 following 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 rear limb 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 on Study Day 24 (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
- Blood samples for clinical pathology evaluations (haematology, coagulation, and serum chemistry) were collected from 5 animals/sex/group at the scheduled/primary necropsies (Study Day 28 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 42 for males and Study Day 63 for females).
- The animals were fasted overnight prior to blood collection. Blood for serum chemistry and haematology was collected via the jugular vein. Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing K2EDTA (haematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).Parameters evaluated were as shown in the tables below.

MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all F0 parental animals found dead, euthanised in extremis or due to total litter loss, or at the scheduled termination. All surviving F0 adults were euthanised by carbon dioxide inhalation.
- Males were euthanised 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 euthanised on Lactation Day 14; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia.
- Females that failed to deliver were euthanised on Post-Mating Day 25 (females with 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. The female with a total litter loss was euthanised on the day of litter loss.
- 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. Numbers of corpora lutea were also recorded for females with macroscopic evidence of implantation and for females necropsied during gestation through Lactation Day 4. Females not selected for pairing were euthanised 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. Clinical findings that were verified at necropsy were designated CEO. At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin (except as noted in the table below).

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 haematoxylin and eosin. In
addition, PAS staining was used for the testes and epididymides.
- Microscopic examination was performed on all tissues listed from all animals in the control and 500 mg/kg/day groups at the primary necropsy and from all animals found dead or euthanised in extremis prior to the scheduled 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) was collected via the jugular vein from all F0 males and females on the day of scheduled euthanasia (Study Days 28 and 42 for males and Lactation Day 14 and Study Day 63 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.
- Serum was isolated in a refrigerated centrifuge and stored frozen (approximately -70 °C).
- The following thyroid hormone parameters were evaluated for F0 males (primary necropsy only) and F1 PND 13 males and females: Thyroxine (Total T4).
- The samples from the recovery phase F0 males, all F0 females, and F1 culled pups (PND 4) were not analysed, but stored frozen (-65 to -85 °C).

Fetal examinations:
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 or euthanised in extremis (by 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.
- Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. The carcass of each pup was then discarded.

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 computerised randomization.
- Blood samples for possible future thyroid hormone analysis were collected from 2 culled pups/litter (pooled by litter) on PND 4; pup were euthanised by decapitation following blood collection and discarded. Remaining culled pups (not used for blood collection) were weighed, euthanised by an intraperitoneal injection of sodium pentobarbital on PND 4, and discarded.

CLINICAL OBSERVATIONS
- Litters were examined daily for survival and any adverse changes in appearance or behaviour.
- Each pup received a clinical examination on PND 1, 4, 7, 10, and 13.
- Any abnormalities in nesting and nursing behaviour 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. Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle.

BODY WEIGHTS
- Pups were individually weighed on PND 1, 4, 7, 10, and 13. 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.

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

ASSESSMENT OF AREOLAS / NIPPLE ANLAGEN
- On PND 13, all F1 male offspring were evaluated for the presence of thoracic nipples/areolae.
- A finding of “yes” and the number of nipples were recorded if nipples were present, and a finding of “no” was recorded if nipples were absent.

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. Pups that were euthanised due to death of the dam were excluded from pup viability calculations.
- Total litter loss was determined when the last pup in the litter was found dead or euthanised in extremis prior to the scheduled euthanasia. Litters that were euthanised prior to scheduled euthanasia due to reasons unrelated to test substance administration (e.g., death of the dam) were not considered to be a total litter loss on the data tables and were not included in the pup viability calculations.

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; the thyroids (with parathyroids, if present) were weighed (following fixation) 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

PARTRUITION
- All females selected for pairing were allowed to deliver naturally and rear their young to PND 13.
- 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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related clinical observations noted for F0 males and females in the 200 and 500 mg/kg/day groups included rales, clear and red material around the mouth, and red material around the nose. These findings were noted at the daily examinations and/or approximately 1 hour following dose administration generally throughout the dosing period. Although these observations were considered test substance-related, they did not persist or the incidence was greatly decreased at the daily examinations on the following day and therefore these observations were not considered adverse. Other clinical observations noted in the test substance-treated groups at daily examinations, weekly detailed physical examinations, and 1 hour following dose administration, including yellow material and hair loss on various body surfaces, were noted infrequently and/or in a manner that was not dose-related.
Mortality:
mortality observed, treatment-related
Description (incidence):
- Six females (Nos. 5743, 5754, 5767, 5771, 5772, and 5779) in the 500 mg/kg/day group were euthanised in extremis due to dystocia (hunched posture, unkempt appearance, cool body, pale body, and/or pale extremities) or found dead on Gestation Day 22 or Lactation Day 0. Microscopic findings in 5 of these females were limited to vaginal and stress-related changes while former implantation sites with no foetuses were noted for the remaining female. In addition, Female No. 5794 in the 500 mg/kg/day group was euthanised due to a total litter loss on Lactation Day 0. Male No. 5677 in the 500 mg/kg/day group was found dead on Study Day 22 following a body weight loss (19 g) during Study Days 13 to 21. The cause of death for this male was pyelonephritis which was secondary to urinary calculi and therefore not attributed to the test substance. All other F0 males and females survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Males: Test substance-related lower mean body weight gains were noted for F0 males in the 500 mg/kg/day group throughout the entire study (Study Days 0 to 27); the differences were generally significant (p < 0.01) compared to the control group. Consequently, mean body weights in this group were 4.3% to 7.3% lower than the control group from Study Days 13 through 27; the differences were not statistically significant. During the recovery period (Study Days 28 to 41), mean body weight gains in the 500 mg/kg/day group F0 males were similar to or higher than the control group. However, mean body weights in this group remained 6.9 % to 10.2 % lower than the control group during the recovery period; the differences were significant (p < 0.05 or p < 0.01) on Study Days 28 and 35. Mean body weight gains in the 100 and 200 mg/kg/day group F0 males were similar to the control group during the pre-mating period (Study Days 0 to 13). However, slightly lower (not statistically significant) mean body weight gains were noted in these groups compared to the control group for the remainder of the dosing period (Study Days 13 to 21) and when the entire dosing period (Study Days 0 to 27) was evaluated; only the difference in the 100 mg/kg/day group was significant (p < 0.05). The decrements in body weight gain observed in the 100 and 200 mg/kg/day group F0 males were not of sufficient magnitude to affect mean absolute body weights in these groups and therefore these differences were not considered to be adverse. Mean body weights and body weight gains in the 35 mg/kg/day group F0 males were unaffected by test substance administration throughout the study. Differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner.
- Females (weekly): Mean body weights and body weight gains in the 35, 100, 200, and 500 mg/kg/day group F0 females were unaffected by test substance administration during the pre-mating period. Differences from the control group were slight and not statistically significant, with the following exception. A significantly (p < 0.05) lower mean body weight gain was noted for F0 females in the 200 mg/kg/day group when the overall pre-mating period (Study Days 0 to 13) was evaluated. In the absence of a dose response, this difference was not considered test substance-related. Mean body weight gains for F0 females in the 500 mg/kg/day group that were not paired for mating were similar to the control group for the remainder of the dosing period (Study Days 13 to 48); differences were slight and not statistically significant with the following exception. A statistically (p < 0.05) higher mean body weight gain was noted for the 500 mg/kg/day group F0 females compared to the control group during Study Days 27 to 35. The magnitude of this increase was not of sufficient magnitude to affect the overall dosing period (Study Days 0 to 48) and therefore this difference was not considered to be test substance-related. During the recovery period (Study Days 49 to 62), mean body weights and body weight gains in the 500 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 500 mg/kg/day group F0 females were similar to the control group during Gestation Days 0 to 14. Test substance-related lower mean body weight gains were noted for this group during the remainder of the gestation dosing period (Days 14 to 20); the difference from the control group was significant (p < 0.01) during Gestation Days 17 to 20. As a result, a significantly (p < 0.01) lower mean body weight gain was noted for the 500 mg/kg/day group F0 females compared to the control group when the overall gestation dosing period (Days 0 to 20) was evaluated and mean body weights in this group were 5.3% to 15.8% lower than the control group during Gestation Days 11 to 20; the difference was significant (p < 0.01) on Gestation Day 20. The lower body weight changes noted in the latter half of the gestation period in this group were likely attributed to the increased number of foetal resorptions noted in the animals that were found dead or euthanised in extremis. Mean body weight gains in the 200 mg/kg/day group F0 females were similar to the control group during Gestation Days 0 to 17. A test substance-related lower mean body weight gain was noted in this group compared to the control group during Gestation Days 17 to 20 which resulted in a lower mean body weight gain for the overall gestation period (Days 0 to 20); none of the differences were statistically significant. Mean body weights in the 200 mg/kg/day group F0 females were 5.9% to 9.0% lower (not statistically significant) than the control group during Gestation Days 11 to 20. Mean F0 female body weights and body weight gains in the 35 and 100 mg/kg/day groups were unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.
- Females (lactation): Mean body weight gains in the 500 mg/kg/day group F0 females were similar to or higher than the control group during Lactation Days 1 to 10. However, a test substance-related, significant (p < 0.01) mean body weight loss (18 g) was noted in this group compared to a body weight gain (9 g) in the control group during Lactation Days 10 to 13. As a result a lower mean body weight gain was noted for the 500 mg/kg/day group F0 females compared to the control group when the overall lactation period (Days 1 to 13) was evaluated and mean body weight was 8.7% lower than the control group on Lactation Day 13; the differences were not statistically significant. Mean body weights and body weight gains in the 35, 100, and 200 mg/kg/day groups 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: Test substance-related slightly lower mean food consumption, evaluated as g/animal/day, was noted for F0 males in the 500 mg/kg/day group during the pre-mating period (Study Days 0 to 13); the difference from the control group for Study Days 7 to 13 was significant (p < 0.01). Mean food consumption in this group was similar to the control group for the remainder of the dosing period and during the recovery period. Mean food consumption in the 35, 100, and 200 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): Test substance-related, significantly (p < 0.05) lower mean food consumption, evaluated as g/animal/day, was noted in the 500 mg/kg/day group F0 females compared to the control group during Study Days 0 to 7. Mean food consumption in this group was similar to the control group during the remainder of the pre-mating period (Study Days 7 to 13). Mean food consumption in the 500 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 to 48); the differences were slight and not statistically significant. During the recovery period (Study Days 56 to 62), mean food consumption in the 500 mg/kg/day group F0 females was similar to the control group. Mean food consumption in the 200 mg/kg/day group F0 females was similar to the control group during Study Days 0 to 7. During Study Days 7 to 13, significantly (p < 0.01) lower mean food consumption was noted for this group compared to the control group. However, this difference was not of sufficient magnitude to affect mean body weights in this group and therefore, the decreased food consumption was not considered test substance-related. Mean food consumption in the 35 and 100 mg/kg/day group F0 females was unaffected by test substance administration during the pre-mating period. None of the differences from the control group were statistically significant.
- Females (gestation): Mean F0 maternal food consumption, evaluated as g/animal/day, in the 35, 100, 200, and 500 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 food consumption in the 500 mg/kg/day group F0 females were similar to the control group during Lactation Days 1 to 10. However, significantly (p < 0.01) lower mean food consumption, corresponding to a mean body weight loss, was noted in this group compared to the control group during Lactation Days 10 to 13. When the overall lactation period was evaluated, mean food consumption in the 500 mg/kg/day group F0 females was similar to the control group. Mean maternal food consumption in the 35, 100, and 200 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, treatment-related
Description (incidence and severity):
- Test substance-related, statistically significantly lower mean absolute and percentage reticulocytes were noted in the 500 mg/kg/day group females at the primary necropsy. There were no associated changes in remaining red blood cell parameters; however, the finding correlated with minimally decreased cellularity within sternal bone marrow. There were no other test substance-related alterations in haematology and coagulation parameters at the primary necropsy.
- Reticulocyte parameters in the 500 mg/kg/day group females were similar to the control group at the recovery necropsy. Lower mean corpuscular haemoglobin concentration (MCHC) and higher red cell distribution width (RDW) values were noted at the recovery necropsy, consistent with maturation of the higher reticulocyte population noted at the primary necropsy. There were no test substance-related differences in the 500 mg/kg/day group males at the recovery necropsy; however, some statistically significant differences were noted, consisting of higher mean reticulocyte parameters and lower mean corpuscular haemoglobin (MCH) values. The differences were not considered to be related to administration of the test substance because values were similar to the control group at the primary necropsy.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
- There were no test substance-related alterations in serum chemistry parameters at the scheduled necropsies. However, lower mean serum potassium value was noted in the 500 mg/kg/day group males at the primary necropsy. There was no dose-response relationship, other electrolyte values were similar to the control group, and the mean value was within the historical control database range; the difference was attributed to biological variability.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
- 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 Day 24 (males) or on Lactation Day 13 (females).
- 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 Day 24 (males) or on Lactation Day 13 (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 Day 24 (males) or on Lactation Day 13 (females).
- 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 Day 24 (males) or on Lactation Day 13 (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 Day 24 (males) or on Lactation Day 13 (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 Day 24 (males) or on Lactation Day 13 (females).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related, statistically significantly higher mean liver weights (absolute and relative to final body and brain weights) were noted in the 500 mg/kg/day group F0 males, and higher mean kidney weights relative to final body weight were noted in the 200 and 500 mg/kg/day group F0 males at the primary necropsy; there were no microscopic or clinical pathology correlates, although lower serum T4 values were considered likely related to higher liver weights through the mechanism of increased T4 metabolism associated with hepatocellular microsomal enzyme induction. Lower mean thymus weight (non-statistically significant) was noted in the 500 mg/kg/day group F0 females; lower weight correlated microscopically with minimal to mild decreased cortical lymphocytes. Lower mean seminal vesicle weights were noted in the 100 (absolute) and 500 (absolute and relative to brain weight) mg/kg/day group F0 males. There were no microscopic correlates, and weights were within the historical control database range; the finding was attributed to test substance-related stress-response.
- There were no organ weight differences at the recovery necropsy considered to be test substance-related. Mean liver and kidney weights in the 500 mg/kg/day group F0 males and thymus weights in the 500 mg/kg/day group F0 females were similar to the control group at the recovery necropsy, consistent with recovery. Statistically significant, non-test substance-related higher heart and thymus weights relative to body weight were noted in the 500 mg/kg/day group F0 males, and higher kidney (absolute and relative to final body and brain weights) and liver (relative to final body and brain weights) weights were noted in the 500 mg/kg/day group F0 females. There were no differences in these organ weights at the primary necropsy and weights were within the historical control database range; the differences were attributed to biological variability.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- There were 7 unscheduled deaths in the 500 mg/kg/day groups: 6 F0 females and 1 F0 male. The cause moribundity for 5 F0 females (Nos. 5743, 5754, 5767, 5771, and 5772) in the 500 mg/kg/day group euthanised on Gestation Day 22 or Lactation Day 0 was dystocia, although microscopic findings were restricted to vaginal and stress-response-related changes. Female No. 5779 in the 500 mg/kg/day group was found dead on Gestation Day 22; former implantation sites with no foetuses were noted for this female. Male No. 5677 in the 500 mg/kg/day group was found dead on Study Day 22. The cause of death for this male was attributed to pyelonephritis secondary to urinary calculi and therefore not considered to be test substance-related.
- Macroscopic observations in the 500 mg/kg/day group F0 females attributed to administration of the test substance were secondary findings, consisting of small spleen which correlated with decreased lymphocytes; small thymus which correlated with decreased cortical lymphocytes; and red matting of skin around the nose, mouth, and forelimbs and/or paws. In addition to decreased thymic and splenic lymphocytes, remaining test substance-related microscopic findings were consistent with stress-response, including adrenal cortical hypertrophy/hyperplasia; adrenal cortical congestion/haemorrhage; reduced zymogen granules within the exocrine pancreas; gastric ulceration; and decreased cellularity of sternal bone marrow. Female reproductive tract changes consisted of necrosis of implantation sites and vaginal mucosal atrophy and increased mucification.
- Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance in F0 females that failed to deliver, the F0 female with a total litter loss, or F0 males and females at the scheduled necropsies. A test substance-related lower mean number of implantation sites was noted in the 500 mg/kg/day group compared to the control group. Although the difference was not statistically significant, this was consistent with lower mean numbers of pups born and live litter size (see Section 6.3.1.) noted in the 500 mg/kg/day group. A greater (not statistically significant number) of mean unaccounted-for sites was noted in the 500 mg/kg/day group F0 females (2.3 per dam) compared to the concurrent control group (0.6 per dam). However, this difference was attributed to a single female (No. 5754) in this group with 8 unaccounted-for sites. The mean numbers of unaccounted-for sites and implantation sites in the 35, 100, and 200 mg/kg/day groups were similar to the control group values. Differences were slight and not statistically significant.
Neuropathological findings:
no effects observed
Description (incidence and severity):
MOTOR ACTIVITY
- 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 on Study Day 24 (males) and Lactation Day 13 (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, with the following exceptions. Significantly (p ≤ 0.041) higher mean ambulatory counts were noted for F0 females in the 500 mg/kg/day group compared to the control group during the 11-20 and 31-40 minute intervals on Lactation Day 13. Because of the small sample size in this group (N=3) and the absence of an effect on the cumulative ambulatory count value, these differences were not considered to be test substance-related. No remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated on Study Day 24 (males) or Lactation Day 13 (females).
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Primary necropsy: Test substance-related decreased thymic cortical lymphocytes and sternal bone marrow cellularity were noted in 2 of the three 500 mg/kg/day group F0 females evaluated at the primary necropsy; the thymic finding correlated with lower mean thymus weight, and the bone marrow finding correlated with lower reticulocyte parameters. Both findings were considered to represent test substance-related stress-responses.
- Fail To Deliver (Post-Mating Day 25/Study Day 40) Necropsy: Two control group F0 females which failed to deliver pups were evaluated. Microscopic findings were similar to the control group females that delivered pups.
- Total Litter Loss (Study Day 37) Necropsy: One 500 mg/kg/day group F0 female with total litter loss was evaluated. Microscopic findings considered to be secondary to test substance-related stress-response consisted of decreased lymphocytes within the spleen and thymus; adrenal cortical congestion/haemorrhage; reduced exocrine pancreatic zymogen granules; and decreased sternal bone marrow cellularity. Within the vagina there was mucosal atrophy and increased prominence of mucification. Findings were similar to those noted in the 500 mg/kg/day group unscheduled death F0 females. 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.
Histopathological findings: neoplastic:
not examined
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
- Mean gestation lengths in the 35, 100, 200, and 500 mg/kg/day groups were similar to those in the control group. No statistically significant differences were noted. Dystocia (clinical observations of hunched posture, unkempt appearance, cool body, pale body, and/or pale extremities) was noted at 500 mg/kg/day resulting in 5 females in this group that were euthanised in extremis on Gestation Day 22 or Lactation Day 0. No signs of dystocia were noted at 35, 100, or 200 mg/kg/day.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
THYROID HORMONES
- Test substance-related, statistically significantly lower mean serum T4 values were noted in the 500 mg/kg/day group males with a dose-response relationship. Lower T4 values correlated with higher liver weights, consistent with increased T4 metabolism secondary to hepatocellular microsomal enzyme induction. There were no microscopic correlates.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: dystocia
Key result
Abnormalities:
no effects observed
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): GENERAL PHYSICAL CONDITION
- A test substance-related increase in the number of pups found dead was noted in the 500 mg/kg/day group compared to the control group; these pups were found dead on PND 0. The number of pups found dead and/or missing in the 35, 100, and 200 mg/kg/day groups was unaffected by maternal test substance administration.

OFFSPRING BODY WEIGHTS
- Mean male and female F1 pup body weight gains in the 500 mg/kg/day group were generally lower than the control group throughout the postnatal period (PND 1–13); only the difference for females during PND 10–13 was significant (p<0.01). Lower (not statistically significant) mean F1 male and female body weights were noted compared to the control group from PND 1 to PND 13.
- Mean male and female F1 pup body weights and body weight gains during PND 1 to 13 in the 35, 100, and 200 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):
- A lower number of pups born, corresponding lower number of implantation sites, and consequently mean live litter size on PND 0 were observed in the 500 mg/kg/day group (11.4 and 6.4 pups, respectively) when compared to the control group (15.1 and 14.8 pups, respectively); the differences were significant (p<0.05 or p<0.01).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
- The percentage of males at birth was unaffected by test substance administration at all dosage levels.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
- The mean number of pups born, live litter size, and postnatal survival in the 35, 100, and 200 mg/kg/day groups were similar to the control group values.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
- Lower (not statistically significant) postnatal survival was noted for the 500 mg/kg/day group compared to the control group from birth to PND 4 due to the lower survival on PND 0 (relative to the number of pups born).
- Postnatal survival in this group was similar to the control group for the remainder of the postnatal period (PND 4 through PND 13).
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
NECROPSIES OF PUPS FOUND DEAD OR EUTHANISED DUE TO DEATH OF DAM
- The numbers of pups (litters) found dead or euthanised due to death of the dam during PND 0-13 numbered 3(1), 0(0), 2(2), 4(2), and 38(5) in the control, 35, 100, 200, and 500 mg/kg/day groups, respectively. Aside from the presence or absence of milk in the stomach, internal findings were limited to 3 pups in Litter No. 5769 in the 200 mg/kg/day group and consisted of an enlarged kidney, absent kidney and ureter, renal papillae(e) not fully developed and distended ureter(s), and/or a nodule on the kidney.

SCHEDULED PUP NECROPSIES (PND 13)
- No internal findings were noted at the necropsy of pups euthanised on PND 13.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
ANOGENITAL DISTANCE
- The anogenital distances (absolute, relative to pup body weight, and relative to the cube root of pup body weight) in the 35, 100, 200, and 500 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

AREOLAE / NIPPLE ANLAGEN
- Areolae/nipple anlagen in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. The test substance-treated group values were not statistically different from the control group values.

THYROID HORMONE ANALYSIS (PND 13)
- Test substance-related, statistically significantly lower mean serum T4 values were noted in the 500 mg/kg/day group pups; a dose-response relationship was absent.

ORGAN WEIGHTS (PND 13)
- There were no test substance-related effects on thyroid weights in the F1 males and females at any dosage level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
maternal dose
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

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 range for suspensions (85% to 115%) and were homogeneous.

- Test substance was not detected in the analysed vehicle formulation that was administered to the control group (Group 1).

Conclusions:
Under the conditions of this screening study, dystocia was noted for F0 females at 500 mg/kg/day. Therefore, a dosage level of 200 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for female reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. There were no test substance-related effects on reproductive parameters for F0 males at any dosage level. Therefore, a dosage level of 500 mg/kg/day, the highest dosage level tested, was considered to be the NOAEL for male reproductive toxicity. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 200 mg/kg/day based on adverse lower body weights, body weight gains, and reduced food consumption in the 500 mg/kg/day group males and females (during lactation). During the recovery period, the effects on body weight and food consumption were no longer observed in the 500 mg/kg/day group males and females with the exception of lower body weights which continued to be observed in males. The NOAEL for neonatal toxicity was 200 mg/kg/day based on the lower number of F1 pups born, live litter size, lower postnatal survival and lower mean F1 pup body weights and body weight gains in the 500 mg/kg/day group.
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 to 4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 35, 100, 200, and 500 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle 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 28 doses. Females received 14 daily doses prior to pairing and were dosed through Lactation Day 13 for a total of 49 to 53 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 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 28 doses for males and 49 doses for females, these animals were assigned to a 15-day non-dosing 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 on Study Day 24 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 euthanised on PND 13; blood samples for thyroid hormone analysis were collected and selected organs were weighed from 1 pup/sex/litter. Clinical pathology evaluations (haematology, 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 (Lactation Day 14 for females) and recovery necropsies; only primary necropsy male samples were analysed. F0 males were euthanised following completion of the mating period or 15-day recovery period and F0 females were euthanised on Lactation Day 14 for females that delivered, post-mating 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 found dead or euthanised in extremis and all F0 animals in the control and high-dose groups at the primary necropsy.

 

RESULTS

Test substance-related mortality and moribundity were noted at 500 mg/kg/day. One F0 female in the 500 mg/kg/day group was found dead and 5 F0 females were euthanised in extremis following signs of dystocia including hunched posture, unkempt appearance, cool body, pale body, and/or pale extremities on Gestation Day 22 or Lactation Day 0. Females with dystocia had foetal autolysis/resorption and macroscopic and microscopic findings consistent with stress response, including small spleen and thymus which correlated microscopically with decreased lymphocytes; adrenal cortical hypertrophy/hyperplasia and congestion/haemorrhage; reduced pancreatic zymogen granules; gastric ulceration; and reduced sternal bone marrow cellularity. Vaginal atrophy and mucification were additionally noted. Similar microscopic findings were noted in a single 500 mg/kg/day group female with total litter loss. In the 500 mg/kg/day group, 1 F0 male was found dead on Study Day 22; the cause of death was attributed to pyelonephritis secondary to urinary calculi and not considered test substance-related. All other F0 and F1 males and females survived to the scheduled necropsies. Test substance-related clinical observations of rales, clear and red material around the mouth, and red material around the nose were noted for F0 males and females in the 200 and 500 mg/kg/day groups approximately 1 hour following dose administration generally throughout the dosing period. These clinical observations were generally not observed or the incidence was considerably decreased at the daily examinations on the following day and therefore these observations were not considered adverse.

 

Test substance-related lower mean body weight gains were noted for F0 males in the 500 mg/kg/day group throughout the dosing period (Study Days 0 to 27) which corresponded to decreased food consumption in this group during the pre-mating period (Study Days 0 to 13) and resulted in mean body weights in the 500 mg/kg/day group F0 males that were up to 7.3% lower than the control group. Mean food consumption for the 5 males in the 500 mg/kg/day group that were not paired for breeding was similar to that in the control group during Study Days 13 to 27. Mean body weights in the 500 mg/kg/day group F0 males remained lower than the control group during the recovery period (Study Days 28 to 41) despite mean body weight gains that were similar to or higher than the control group. Test substance-related, slightly lower mean body weight gains were noted for F0 males in the 100 and 200 mg/kg/day groups compared to the control group when the overall dosing period (Study Days 0 to 27) was evaluated due to lower mean body weight gains in these groups during Study Days 13 to 21. However, the magnitude of the differences were not of sufficient magnitude to affect absolute mean body weights and there were no corresponding decreases in mean food consumption in these groups; therefore, the decrements in body weight observed for the 100 and 200 mg/kg/day group F0 males were not considered adverse.

Test substance-related lower mean food consumption was noted for F0 females in the 200 and 500 mg/kg/day groups during the pre-mating period (Study Days 0 to 13). Mean body weights and body weight gains in these groups were similar to the control group during the pre-mating period (Study Days 0 to 13) and when the entire treatment period (Study Days 0 to 48) for F0 females in the 500 mg/kg/day group that were not paired for mating. Therefore, the lower mean food consumption noted in the 200 and 500 mg/kg/day groups during the pre-mating period was not considered adverse. A lower mean body weight gain was noted for F0 females in the 500 mg/kg/day group compared to the control group when the entire gestation period (Days 0 to 20) was evaluated due to lower mean body weight gains during Gestation Days 14 to 20. As a result, mean body weights for F0 females in this group were 15.8% lower than the control group on Gestation Day 20. Mean food consumption in the 500 mg/kg/day group F0 females was similar to the control group during gestation. The changes in mean gestational body weights were considered secondary to the lower live litter sizes and reduced mean pup body weights noted at 500 mg/kg/day. A mean body weight loss and corresponding decreased food consumption were noted for F0 females in the 500 mg/kg/day group during Lactation Days 10 to 13 resulting in a lower mean body weight gain for this group when the overall lactation period (Days 1 to 13) was evaluated and mean body weights that were 8.7% lower than the control group on Lactation Day 13. Mean body weight gains, body weights, and food consumption in the 500 mg/kg/day group F0 females were similar to the control group during the recovery period (Study Days 49 to 62). In the 200 mg/kg/day group F0 females, mean body weights, body weight gains, and food consumption during gestation and lactation were similar to that in the control group. Mean body weights, body weight gains, and food consumption in the 35 mg/kg/day group F0 males and 35 and 100 mg/kg/day group F0 females and were unaffected by test substance administration throughout the study. No test substance-related effects were noted during the FOB or motor activity evaluations at any dosage level in the F0 generation.

 

F0 gestation length, oestrous cycle length, and reproductive performance (mating, fertility, and copulation/conception indices) in all test substance-treated groups and the process of parturition in the 35, 100, and 200 mg/kg/day groups were unaffected by test substance administration. A higher number of unaccounted-for sites (attributed to a single female) and a test substance-related lower number of implantation sites were noted in the 500 mg/kg/day group compared to the control group. As a result, a lower number of F1 pups born and live litter size on PND 0, with a corresponding greater number of pups that were found dead, were observed in this group. In addition, lower postnatal survival was noted for this group from birth to PND 4 due to 1 female with a total litter loss and 6 females in this group that had entirely resorbed litters. There were no test substance-related effects on unaccounted-for sites, implantation sites, or F1 survival and clinical condition in the 35, 100, and 200 mg/kg/day groups.

 

Test substance-related lower mean F1 pup body weight gains were noted in the 500 mg/kg/day group compared to the control group generally throughout the postnatal period resulting in mean F1 male and female body weights that were up to 13.1% and 14.7% lower, respectively. Mean F1 pup body weights and body weight gains in the 35, 100, and 200 mg/kg/day groups were similar to the control group. There were no test substance-related effects noted on F1 anogenital distance or areolae/nipple anlagen (males only) in the 35, 100, 200, and 500 mg/kg/day groups. No remarkable necropsy findings were noted for F1 pups that were euthanised on PND 13. There were no test substance-related effects on thyroid/parathyroid weights noted for F1 pups on PND 13. Test substance-related findings at the primary necropsy consisted of lower thymus weights which correlated with decreased thymic lymphocytes; decreased sternal bone marrow cellularity which correlated with lower reticulocyte parameters in the 500 mg/kg/day group F0 females; lower seminal vesicle weights in the 100 and 500 mg/kg/day group F0 males with no microscopic correlate; higher kidney weights in the 200 and 500 mg/kg/day group males with no microscopic correlate; and higher liver weights in the 500 mg/kg/day group F0 males with no microscopic correlate. Thymus, bone marrow, reticulocyte, and seminal vesicle changes were consistent with a test substance-related stress-response. Higher liver weights are a sensitive indication of hepatocellular microsomal enzyme induction, while microscopic evidence of hepatocellular hypertrophy is generally apparent only in livers with greater than 20% higher weights. Serum thyroxine (T4) values were lower in the 500 mg/kg/day group F0 males and F1 pups, with no thyroid weight changes or microscopic correlates in the F0 males; lower T4 values were attributed to enhanced metabolism of T4 secondary to test substance-related microsomal enzyme induction.

 

CONCLUSION

 

Under the conditions of this screening study, dystocia was noted for F0 females at 500 mg/kg/day. Therefore, a dosage level of 200 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for female reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. There were no test substance-related effects on reproductive parameters for F0 males at any dosage level. Therefore, a dosage level of 500 mg/kg/day, the highest dosage level tested, was considered to be the NOAEL for male reproductive toxicity. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 200 mg/kg/day based on adverse lower body weights, body weight gains, and reduced food consumption in the 500 mg/kg/day group males and females (during lactation). During the recovery period, the effects on body weight and food consumption were no longer observed in the 500 mg/kg/day group males and females with the exception of lower body weights which continued to be observed in males. The NOAEL for neonatal toxicity was 200 mg/kg/day based on the lower number of F1 pups born, live litter size, lower postnatal survival and lower mean F1 pup body weights and body weight gains in the 500 mg/kg/day group.

Endpoint:
developmental toxicity
Type of information:
experimental study planned
Study period:
As soon as possible after permission to proceed is received.
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Phosphoric acid, C11-14 (C13-rich)-isoalkyl mono- and di-esters, partially salted with 1-Propanamine, 3-(tridecyloxy)-, branched
- Name of the substance for which the testing proposal will be used: same as above

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies: The substance is not a known reproductive or developmental toxin, has been determined to be non-toxic via the oral and dermal routes under acute conditions and, based on available data, is not carcinogenic or mutagenic. Nevertheless, effects were reported following short-term repeated dose oral administration of the test item and these data do not provide sufficient information to allow reliable conclusions on developmental toxicity to be drawn from the available screening study.
- Available non-GLP studies: Non-GLP studies addressing developmental toxicity are not available for this substance.
- Historical human data: There are no appropriate historical human data available addressing the endpoint developmental toxicity.
- (Q)SAR: Tools to adequately address the developmental toxicity endpoint via (Q)SAR are not currently available.
- In vitro methods: No validated or regulatory accepted alternative methods are available for replacing animal testing with respect to developmental toxicity.
- Weight of evidence: There are reliable GLP studies available for the registered substance. Therefore, no weight of evidence approach needs to be considered.
- Grouping and read-across: Data describing developmental toxicity for similar substances is not currently available. However, data generated from the proposed study will also be used in classification decisions for a close analogue and for read-across when tonnage based registration obligations require update of the analogue dossier to Annex VIII. This will allow development toxicity to be understood without use of additional animals in separate studies.
- Testing is not technically possible: A screening study was successfully performed via the oral route in accordance with OECD 422. Consequently, there is no reason to believe that prenatal development cannot be investigated via oral (gavage) administration of the test material.
- Substance-tailored exposure driven testing: Exposure scenarios developed for Annex VIII risk assessment indicate that some exposure of humans to the test substance is to be expected under the defined conditions of use. Omission of data based on lack of human exposure is therefore not appropriate for this substance.
- Approaches in addition to above: Not applicable
- Other reasons: Not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- The substance is currently manufactured/used in volumes of 10-100 tonnes per annum and screening data were generated to address the mandatory reproductive/developmental toxicity endpoints via OECD 422. Since the screening study raised unforeseen issues that cannot be adequately explained by existing data, it is considered necessary to conduct a pre-natal developmental toxicity study (OECD 414) on one species in accordance with REACH Annex IX, Section 8.7.2.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: The need to investigate pre-natal development in a second species is not currently envisaged.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not applicable
Principles of method if other than guideline:
Not applicable
Species:
rat
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

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.

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 to 4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 35, 100, 200, and 500 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle 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 28 doses. Females received 14 daily doses prior to pairing and were dosed through Lactation Day 13 for a total of 49 to 53 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 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 28 doses for males and 49 doses for females, these animals were assigned to a 15-day non-dosing 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 on Study Day 24 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 euthanised on PND 13; blood samples for thyroid hormone analysis were collected and selected organs were weighed from 1 pup/sex/litter. Clinical pathology evaluations (haematology, 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 (Lactation Day 14 for females) and recovery necropsies;only primary necropsy male samples were analysed. F0 males were euthanised following completion of the mating period or 15-day recovery period and F0 females were euthanised on Lactation Day 14 for females that delivered, post-mating 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 found dead or euthanised in extremis and all F0 animals in the control and high-dose groups at the primary necropsy.

Test substance-related mortality and moribundity were noted at 500 mg/kg/day. One F0 female in the 500 mg/kg/day group was found dead and 5 F0 females were euthanised in extremis following signs of dystocia including hunched posture, unkempt appearance, cool body, pale body, and/or pale extremities on Gestation Day 22 or Lactation Day 0. Females with dystocia had foetal autolysis/resorption and macroscopic and microscopic findings consistent with stress response, including small spleen and thymus which correlated microscopically with decreased lymphocytes; adrenal cortical hypertrophy/hyperplasia and congestion/haemorrhage; reduced pancreatic zymogen granules; gastric ulceration; and reduced sternal bone marrow cellularity. Vaginal atrophy and mucification were additionally noted. Similar microscopic findings were noted in a single 500 mg/kg/day group female with total litter loss. In the 500 mg/kg/day group, 1 F0 male was found dead on Study Day 22; the cause of death was attributed to pyelonephritis secondary to urinary calculi and not considered test substance-related. All other F0 and F1 males and females survived to the scheduled necropsies. Test substance-related clinical observations of rales, clear and red material around the mouth, and red material around the nose were noted for F0 males and females in the 200 and 500 mg/kg/day groups approximately 1 hour following dose administration generally throughout the dosing period. These clinical observations were generally not observed or the incidence was considerably decreased at the daily examinations on the following day and therefore these observations were not considered adverse.

Test substance-related lower mean body weight gains were noted for F0 males in the 500 mg/kg/day group throughout the dosing period (Study Days 0 to 27) which corresponded to decreased food consumption in thisgroup during the pre-mating period (Study Days 0 to 13) and resulted in mean body weights in the 500 mg/kg/day group F0 males that were up to 7.3% lower than the control group. Mean food consumption for the 5 males in the 500 mg/kg/day group that were not paired for breeding was similar to that in the control group during Study Days 13 to 27. Mean body weights in the 500 mg/kg/day group F0 males remained lower than the control group during the recovery period (Study Days 28 to 41) despite mean body weight gains that were similar to or higher than the control group. Test substance-related, slightly lower mean body weight gains were noted for F0 males in the 100 and 200 mg/kg/day groups compared to the control group when the overall dosing period (Study Days 0 to 27) was evaluated due to lower mean body weight gains in these groups during Study Days 13 to 21. However, the magnitude of the differences were not of sufficient magnitude to affect absolute mean body weights and there were no corresponding decreases in mean food consumption in these groups; therefore, the decrements in body weight observed for the 100 and 200 mg/kg/day group F0 males were not considered adverse.

Test substance-related lower mean food consumption was noted for F0 females in the 200 and 500 mg/kg/day groups during the pre-mating period (Study Days 0 to 13). Mean body weights and body weight gains in these groups were similar to the control group during the pre-mating period (Study Days 0 to 13) and when the entire treatment period (Study Days 0 to 48) for F0 females in the 500 mg/kg/day group that were not paired for mating. Therefore, the lower mean food consumption noted in the 200 and 500 mg/kg/day groups during the pre-mating period was not considered adverse. A lower mean body weight gain was noted for F0 females in the 500 mg/kg/day group compared to the control group when the entire gestation period (Days 0 to 20) was evaluated due to lower mean body weight gains during Gestation Days 14 to 20. As a result, mean body weights for F0 females in this group were 15.8% lower than the control group on Gestation Day 20. Mean food consumption in the 500 mg/kg/day group F0 females was similar to the control group during gestation. The changes in mean gestational body weights were considered secondary to the lower live litter sizes and reduced mean pup body weights noted at 500 mg/kg/day. A mean body weight loss and corresponding decreased food consumption were noted for F0 females inthe 500 mg/kg/day group during Lactation Days 10 to 13 resulting in a lower mean body weight gain for this group when the overall lactation period (Days 1 to 13) was evaluated and mean body weights that were 8.7% lower than the control group on Lactation Day 13. Mean body weight gains, body weights, and food consumption in the 500 mg/kg/day group F0 females were similar to the control group during the recovery period (Study Days 49 to 62). In the 200 mg/kg/day group F0 females, mean body weights, body weight gains, and food consumption during gestation and lactation were similar to that in the control group. Mean body weights, body weight gains, and food consumption in the 35 mg/kg/day group F0 males and 35 and 100 mg/kg/day group F0 females and were unaffected by test substance administration throughout the study. No test substance-related effects were noted during the FOB or motor activity evaluations at any dosage level in the F0 generation.

F0 gestation length, oestrous cycle length, and reproductive performance (mating, fertility, and copulation/conception indices) in all test substance-treated groups and the process of parturition in the 35, 100, and 200 mg/kg/day groups were unaffected by test substance administration. A higher number of unaccounted-for sites (attributed to a single female) and a test substance-related lower number of implantation sites were noted in the 500 mg/kg/day group compared to the control group. As a result, a lower number of F1 pups born and live litter size on PND 0, with a corresponding greater number of pups that were found dead, were observed in this group. In addition, lower postnatal survival was noted for this group from birth to PND 4 due to 1 female with a total litter loss and 6 females in this group that had entirely resorbed litters. There were no test substance-related effects on unaccounted-for sites, implantation sites, or F1 survival and clinical condition in the 35, 100, and 200 mg/kg/day groups.

Test substance-related lower mean F1 pup body weight gains were noted in the 500 mg/kg/day group compared to the control group generally throughout the postnatal period resulting in mean F1 male and female body weights that were up to 13.1% and 14.7% lower, respectively. Mean F1 pup body weights and body weight gains in the 35, 100, and 200 mg/kg/day groups were similar to the control group. There were no test substance-related effects noted on F1 anogenital distance orareolae/nipple anlagen (males only) in the 35, 100, 200, and 500 mg/kg/day groups. No remarkable necropsy findings were noted for F1 pups that were euthanised on PND 13. There were no test substance-related effects on thyroid/parathyroid weights noted for F1 pups on PND 13. Test substance-related findings at the primary necropsy consisted of lower thymus weights which correlated with decreased thymic lymphocytes; decreased sternal bone marrow cellularity which correlated with lower reticulocyte parameters in the 500 mg/kg/day group F0 females; lower seminal vesicle weights in the 100 and 500 mg/kg/day group F0 males with no microscopic correlate; higher kidney weights in the 200 and 500 mg/kg/day group males with no microscopic correlate; and higher liver weights in the 500 mg/kg/day group F0 males with no microscopic correlate. Thymus, bone marrow, reticulocyte, and seminal vesicle changes were consistent with a test substance-related stress-response. Higher liver weights are a sensitive indication of hepatocellular microsomal enzyme induction, while microscopic evidence of hepatocellular hypertrophy is generally apparent only in livers with greater than 20% higher weights. Serum thyroxine (T4) values were lower in the 500 mg/kg/day group F0 males and F1 pups, with no thyroid weight changes or microscopic correlates in the F0 males; lower T4 values were attributed to enhanced metabolism of T4 secondary to test substance-related microsomal enzyme induction.

Under the conditions of this screening study, dystocia was noted for F0 females at 500 mg/kg/day. Therefore, a dosage level of 200 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for female reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. There were no test substance-related effects on reproductive parameters for F0 males at any dosage level. Therefore, a dosage level of 500 mg/kg/day, the highest dosage level tested, was considered to be the NOAEL for male reproductive toxicity. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 200 mg/kg/day based on adverse lower body weights, body weight gains, and reduced food consumption in the 500 mg/kg/day group males and females (during lactation). During the recovery period, the effects on body weight and food consumption were no longer observed in the 500 mg/kg/day group males and females with the exception of lower body weights whichcontinued to be observed in males. The NOAEL for neonatal toxicity was 200 mg/kg/day based on the lower number of F1 pups born, live litter size, lower postnatal survival and lower mean F1 pup body weights and body weight gains in the 500 mg/kg/day group.

Justification for classification or non-classification

Findings from reproductive/developmental toxicity screening are not currently considered to be relevant to humans and classification is not required under the terms of Regulation (EC) No 1272/2008 and subsequent amendments