Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Endpoint summary

Currently viewing:

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Extended 1-Generation Reproductive Toxicity with both developmental neuro- and immunotoxicity; Rat oral; OECD Guideline 443; Reliability = 1

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
yes
Remarks:
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusion.
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:
- Premating exposure duration for parental (P0) animals: F0 males were dosed orally by gavage for a minimum of 70 consecutive days prior to mating and continuing through the day prior to euthanasia. F0 females were dosed orally by gavage for a minimum of 70 consecutive days prior to mating
- Basis for dose level selection: The dose levels were selected based on the REACH registration dossier for reaction mass of divinylbenzene and ethylstyrene (EC number: 910-757-7) and 3 previous studies conducted at Charles River.
- Inclusion/exclusion of extension of Cohort 1B: Included
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: Included
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: Included
- Route of administration: The oral route of exposure was selected because this a possible route of human exposure
- Other considerations: The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. The testing facility has reproductive historical data for the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of reproductive toxicants. The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 443, Extended One-Generation Reproductive Toxicity Study, 28 Jul 2011, which recommends including a sufficient number of mating pairs to yield at least 20 pregnant females per dose group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality in each generation of the study, 25 to 30 F0 animals/sex/group was an appropriate number of animals to achieve the desired number of animals in each generation
Specific details on test material used for the study:
Purity: 98.3%
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. The testing laboratory has reproductive historical data for the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of reproductive toxicants. The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 443, Extended One-Generation Reproductive Toxicity Study, 28 Jul 2011, which recommends including a sufficient number of mating pairs to yield at least 20 pregnant females per dose group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality in each generation of the study, 25 to 30 F0 animals/sex/group was an appropriate number of animals to achieve the desired number of animals in each generation.
Sex:
male/female
Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: males were between 155 and 248 g and females were between 114 and 164 g
- Housing: In groups of 2–3 animals per cage during the premating period. Single/Individual following positive signs of mating. All offspring selected to constitute the F1 generation were group housed (2–3 animals of the same sex and same dosing group together) beginning at weaning and remained in these cages until euthanasia. All offspring selected to constitute Cohort 2B were single-housed overnight, prior to euthanasia on PND 22. Caging was solid-bottom cages containing heat-treated aspen bedding material
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
- Water (e.g. ad libitum): Municipal tap water, treated by reverse osmosis and ultraviolet irradiation, ad libitum via an automatic watering system. Glass water bottles were provided, if required.
- Acclimation period: After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing. The testes were palpated at least once for all males during acclimation.
- Enrichment: Animals were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68°F to 77°F (20°C to 25°C)
- Humidity (%): 30 to 70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark
Route of administration:
oral: gavage
Vehicle:
other: 0.5% Methylcellulose (400 cps) and 0.1% Cremophor EL in deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dose formulations were divided into aliquots where required to allow them to be dispensed on each dosing occasion. The test substance dose formulations were prepared approximately weekly. Storage conditions were set to maintain a target temperature of 5°C. Any residual volumes from each dosing occasion were discarded. Dosing formulations were prepared at appropriate concentrations to meet dose level requirements.

VEHICLE: Vehicle formulations were divided into aliquots where required to allow them to be dispensed on each dosing occasion. The vehicle dose formulations was prepared approximately weekly. Vehicle components were stored at a target temperature of 18-24°C. Prepared vehicle was stored at a target temperature of 5°C. Any residual volumes from each dosing occasion were discarded. Vehicle dosing volume was 10 mL/kg.
Details on mating procedure:
Following a minimum of 70 days of treatment, 1 female was cohabited with 1 male rat of the same treatment group, avoiding sibling mating (Charles River Laboratories supplied non-litter mates), in a solid-bottom cage for mating (home cage of the male). A maximum of 14 days was allowed for mating. If no evidence of mating was obtained after 14 days, the animals were separated without further opportunity for mating, and the female was placed in a solid-bottom cage containing bedding material. Detection of mating was confirmed by evidence of a copulatory plug in the vagina or by evidence of sperm in a vaginal lavage. After confirmation of mating, the female was returned to an individual solid-bottom cage, and the day was designated as Day 0 of gestation. F0 animals cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity: 1 mL was collected from the top, middle, and bottom of the first preparation at dose levels of 50 and 500 mg/kg. Four samples were collected from each stratum Two of the samples from each stratum were analyzed for homogeneity and the other 2 were stored as backup. The average result from homogeneity analysis of these 2 doses served as concentration verification for these groups from the first preparation.

Concentration: 1 mL was collected from the middle of the 1st, 4th, 8th, and last preparation at all dose levels. Four samples were collected from each stratum Two of the samples from each dose level were analyzed for concentration and the other 2 were stored as backup.

All samples to be analyzed were transferred to the Analytical Chemistry Department at the testing facility for same day analysis, where possible, or stored for analysis within known formulation stability period. Analyses were performed by high performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV) using a validated analytical procedure. Storage conditions were set to maintain a target temperature of 5°C. Acceptance criteria included mean sample concentration within 100% ± 15% of theoretical concentration, individual sample concentration of ±20%, and relative standard deviations (RSD) of concentrations ≤10% for each group.

The analyzed dosing formulations contained 87.9% to 103.6% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that
was administered to the control group (Group 1).
Duration of treatment / exposure:
F0 animals were dosed via oral gavage daily for 70 consecutive days prior to mating and continuing through the day prior to euthanasia. The F1 generation was potentially exposed in utero and through nursing during lactation. Following weaning, the offspring selected to become the F1 generation were dosed beginning at weaning (PND 21) and continuing until euthanasia (as appropriate for the relevant cohort).
Frequency of treatment:
Daily
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
25 for the 0, 50, and 200 mg/kg/day dose groups
30 for the 500 mg/kg/day dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral route of exposure was selected because this a possible route of human exposure. The dose levels were selected based on the REACH registration dossier for reaction mass of divinylbenzene and ethylstyrene (EC number: 910-757-7) and 3 previous studies conducted at Charles River. On a dose range-finding prenatal developmental toxicity study, time-mated female rats received the test substance via oral gavage at dose levels of 500, 600, 700, and 800 mg/kg/day. Test substance-related clinical signs were observed in a dose-dependent manner at approximately 1-hour post-dose at ≥ 600 mg/kg/day included slight to moderate dehydration, hunched posture, slight to moderate incoordination, and/or decreased activity. Mean maternal body weights were 8.3%, 11.3%, 7.2%, and 17.3% lower than controls in the 500, 600, 700, and 800 mg/kg/day groups, respectively. Higher mean litter proportions of post-implantation loss and lower mean fetal weights were noted at ≥ 500 mg/kg/day. Based on these results, dose levels of 100, 350, and 650 mg/kg/day were selected for the subsequent prenatal developmental toxicity study. On the prenatal developmental toxicity study (OECD 414), 1 female in the 650 mg/kg/day group was euthanized in extremis on Gestation Day 12 following body weight losses and severely reduced food consumption and clinical observations of hunched posture, erect fur, decreased activity, and thin body. Although all other females in this group survived to scheduled necropsy, 17 of 24 gravid females in the group showed similar body weight losses during the first week of the treatment period; therefore, a relationship of the observed moribundity to test substance administration could not be ruled out. At termination on Gestation Day 21, mean body weight in the 650 mg/kg/day group was 8.8% lower than the control group. Test substance-related lower mean fetal weights were observed in the 650 mg/kg/day group (6.476% to 6.740%; male/female/combined). On a subsequent reproduction/developmental toxicity screening study (modified OECD 421), parental (F0) male and female rats received the test substance via oral gavage at dose levels of 500, 600, and 700 mg/kg/day. F1 offspring were directly dosed via oral gavage beginning on PND 21 and continuing until PND 42. In the F0 generation, lower mean body weight gains or mean body weight losses, with correspondingly lower mean food consumption, were noted for both males and females in all test substance groups. Consequently, mean male body weights in the 600 and 700 mg/kg/day groups were 5.8% and 6.5% lower, respectively, then controls on Study Day 27. Mean female body weights in the same groups were 4.8% to 5.5% lower than controls on Study Day 13, 11.0% to 11.5% lower than controls on Gestation Day 20, and comparable to controls on Lactation Day 21. In the F1 generation, mean pup birth weights (PND 1) were 15.6% to 28.6% (males) and 16.2% to 27.0% (females) lower than the control group across the treated groups. Mean postnatal survival (% per litter) from birth to PND 4 was 76.9%, 70.7%, and 64.8% lower than control in the 500, 600, and 700 mg/kg/day groups, respectively, compared to 99.4% in the control group; differences were statistically significant for all test substance treated groups. At the time of weaning, mean pup body weights were 24.0% to 25.4% lower than controls in the 500 mg/kg/day group, and 12.8% to 20.1% lower than controls in the 600 and 700 mg/kg/day groups at the time of weaning. During the period of direct dosing for the F1 offspring (PND 21–42), mean body weight gains in the test substance treated groups remained sporadically lower than controls, and mean absolute body weights were 18.5%, 9.7%, and 9.7% lower, respectively, than controls, for the 500, 600, and 700 mg/kg/day group males, and 14.4% lower than controls for the 500 mg/kg/day females at termination (PND 42); differences were statistically significant for the males in the 500 mg/kg/day group only. In summary, administration of the test substance at a dose of 650 mg/kg/day resulted in moribundity resulting in euthanasia. At doses ≥ 600 mg/kg/day, the test substance administration resulted in adverse clinical observations (hunched posture, slight to moderate incoordination, and/or decreased activity, and slight to moderate dehydration) in pregnant rats. At doses ≥ 500 mg/kg/day, test substance administration resulted in higher mean litter proportions of post-implantation loss, lower mean fetal weights, lower mean pup birth weights (PND 1), and lower mean postnatal survival. Lastly, at doses ≥ 500 mg/kg/day, test substance administration resulted in lower mean pup body weight gains and lower mean pup weights during the preweaning and postweaning periods. Based on these data, 500 mg/kg/day was selected as the high dose for the current study. Lower doses of 50 and 200 mg/kg/day were selected identify potential dose-response relationships, and to establish no-observed-adverse-effect levels (NOAEL) for systemic, reproductive, and developmental toxicity.
- Rationale for animal assignment (if not random): F0 animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. To reduce variability among the F1 litters, 10 pups/litter, 5 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 10 pups. For the F1 generation, 3 F1 pups/sex/litter from all available litters (≥ 20 litters/group) were randomly selected prior to weaning and were assigned to the following cohorts. Cohorts 1A and 1B were assigned to reproductive/developmental toxicity testing. Animals assigned to Cohort 1B were maintained on study for possible breeding when the animals were between 90 and 120 days of age to generate an F2 generation; however, additional breeding was not required on this study. Cohorts 2A and 2B were assigned to developmental neurotoxicity testing. Cohorts 3 and 3A were assigned to immunotoxicity testing. Assignment of same-sex littermates to a particular cohort was avoided whenever possible. In addition, if there were an insufficient number of pups to fill a designated cohort, the following prioritization plan was used (highest to lowest priority): Cohort 1A, Cohort 1B, Cohort 2A, Cohort 2B, Cohort 3, and Cohort 3A.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All F0 animals were checked for mortality at least twice daily (morning and afternoon) beginning upon arrival through termination. Cage side observations were made once daily, conducted prior to dosing on days of scheduled dosing, approximately 1 hour post-dose. Cage side observations were not conducted prior to dosing on days that the detailed clinical observations were performed. All F0 females were observed twice daily for dystocia (prolonged or difficult labor) and other difficulties at parturition and any findings were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All F0 males were observed weekly, beginning with the initiation of dosing until euthanasia. Animals were also observed on the day of euthanasia. All F0 females were observed weekly beginning with the initiation of dosing until evidence of copulation was observed, or until euthanasia for females with no evidence of mating. Observations were also made on Gestation days 0, 4, 7, 10, 14, 17, and 20 and Lactation days 1, 4, 7, 10, 14, 17, and 21. Females that delivered and those that failed to deliver, continued to be observed weekly beginning on Lactation Day 21 and Post-mating Day 25, respectively, and continuing until euthanasia. Animals were also observed on the day of euthanasia.

BODY WEIGHT: Yes
- Time schedule for examinations: All F0 males were weighed weekly beginning with the initiation of dosing through euthanasia. Body weights were also recorded on the day of euthanasia. All F0 females were weighed weekly beginning with the initiation of dosing until evidence of copulation was observed, or until euthanasia for females with no evidence of mating (or those that failed to deliver). Females were also weighed on Gestation Days 0, 4, 7, 10, 14, 17 and 20 and Lactation Days 1, 4, 7, 10, 14, 17, and 21. Females that delivered, and those that failed to deliver, continued to be weighed weekly beginning on Lactation Day 21 and Post-mating Day 25, respectively, and continuing until euthanasia. Body weights were also recorded on the day of euthanasia.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Food consumption for all F0 males was collected weekly, on the days of body weight collection, beginning with the initiation of dosing through euthanasia. It was measured on a per cage basis, except during periods of individual housing, and normalized to the number of animals/cage and was reported in g/animal/day.Food efficiency was calculated and reported. Food consumption for all F0 females was collected weekly, on the days of body weight collection, beginning with the initiation of dosing until the initiation of breeding, on Gestation Days 0, 4, 7, 10, 14, 17, and 20, and on Lactation Days 1, 4, 7, 10, 14, 17, and 21. For females without evidence of mating or those that failed to deliver, food consumption was not recorded following the end of the breeding period or Postmating Day 25, respectively. Food consumption was also not recorded for females with total litter loss, following loss of the litter. Similarly, food consumption was not recorded following weaning of offspring, for females that deliver (between weaning and euthanasia). Food efficiency was calculated and reported.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
Daily vaginal lavages beginning 14 days prior to the initiation of the mating period and continuing during cohabitation until evidence of copulation was observed. Lavaging continued for those females with no evidence of mating until termination of the mating period. A vaginal lavage was also performed on the day of necropsy.

The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P], beginning 14 days prior to initiation of the mating period and continuing until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle. At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data. The percent cycling and percent cycling regularly were calculated.
Sperm parameters (parental animals):
Immediately upon euthanasia, the reproductive tract of each male was exposed via a ventral mid-line incision. The right cauda epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis, and it was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a minimum 10-minute incubation period, a sample of sperm was loaded onto a slide with a 100-μm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all pipettes, slides, and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported. The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and cauda epididymis from all males were weighed and stored frozen. The left cauda epididymis was homogenized and analyzed for determination of homogenization resistant spermatid count. An aliquot of each sample was added to a solution containing a DNA specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-μm chamber depth. Illumination from a xenon lamp within the analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes; To reduce variability among the litters, 10 pups from each litter, of equal sex distribution (if possible), were randomly selected on PND 4. Culled pups were used for specimen collections and subjected to a gross necropsy. For litters consisting of fewer than 10 pups, adjustments for litter size were not performed.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:

- Mortality: All F1 offspring were observed for mortality at least twice daily (morning and afternoon) for general appearance and behavior and survival from birth to weaning.

- Cage-Side Observations: All F1 offspring were observed once daily beginning on PND21; conducted prior to dosing on days of scheduled dosing, approximately 1 hour post-dose. Cage side observations were not conducted prior to dosing on days that the detailed clinical observations were performed.

- Detailed Clinical Observations: All F1 offspring were observed on PND 1, 4, 7, 14, and 21, and once weekly thereafter until euthanasia. Animals were also observed on the day of euthanasia.

- Pup Sexes: Pup sexes were examined in all F1 offspring on PND 0, 4, 14, and 21.

- Individual Body Weights: All F1 offspring were weighed on PND 1, 4 (before culling), 7, 14, and 21, and weekly thereafter until euthanasia. Body weight were also recorded on the day of euthanasia

- Food Consumption: Food consumption was recorded for all F1 offspring weekly, beginning on PND 21 (on the days of body weight collections) until euthanasia. Food consumption was not recorded for offspring assigned to Cohort 2b (scheduled for euthanasia on PND 22). Food consumption was measured on a per cage basis, except during periods of individual housing and was normalized to the number of animals/cage and reported in g/animal/day. Food efficiency was calculated and reported.

- Preweaning Developmental Landmarks: Anogenital Distance: All Fl offspring were observed on PND1. The absolute and relative values (to the cube root of body weight) were reported

- Preweaning Developmental Landmarks - Areolae/Nipple Anlagen Retention: All F1 males were observed on PND 13 and the number of nipples was recorded.

- Selection of F1 generation: Prior to PND 21, 5 F1 pups per sex per litter from all available litters were selected to constitute the F1 generation and for assignment to specific cohorts. Pups were selected randomly; only pups not expected to survive due to notable physical limitation and/or obvious runts (animals with body weight more than 2 standard deviations lower than the respective mean litter weight) were not available for selection.

- Postweaning Developmental Landmarks - Vaginal Patency: At least 3 female F1 pups/per litter were observed beginning on PND 25. Examination of the females was continued daily until vaginal patency was present. Any abnormalities of genital organs (e.g., persistent vaginal threads) were noted. The body weight of each female was recorded on the day of attainment of vaginal patency. Animals assigned to all postweaning cohorts (1, 2 and 3) were assessed for post weaning developmental landmarks.

- Postweaning Developmental Landmarks - Balanopreputial Separation: At least 3 male F1 pups per litter were observed beginning on PND 35. Examination of the males was continued daily until balanopreputial separation was present. Any abnormalities of genital organs (e.g., persistent preputial threads) were noted. The body weight of each male was recorded on the day of attainment of balanopreputial separation. Animals assigned to all post weaning cohort (1, 2, and 3) were assessed for postweaning developmental landmarks.

- Estrous cyclicity (Cohorts 1A and 1B): Beginning on the day vaginal opening was observed, vaginal lavages were performed daily for all F1 females assigned to Cohort 1A and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female until the first sign of estrus (cornified cells) was observed. The age of first vaginal estrus after vaginal opening was recorded. Vaginal lavages were also performed daily for all F1 females assigned to Cohort 1A and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female for 2 weeks during PND 75–91 (the day of necropsy); the stage of the estrous cycle was also evaluated on the day of necropsy for the Cohort 1B females. For Cohort 1A, the average cycle length was calculated and reported for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P]). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. At the end of the study, the overall pattern of each Cohort 1A female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
- Auditory Startle Response (Cohort 2A): Up to 10 F1 pups/sex/group (Cohort 2A) were observed for auditory startle response on PND 24 (±1 day). Animals were tested at approximately the same time on each test day. Testing of treatment groups was balanced across the day of testing and executed by appropriately trained technicians. Auditory startle response testing was performed in a room equipped with a white noise generator set to operate at 70 ± 10 dB. The test sessions consisted of a 5-minute acclimation period with a 70 ± 2-dB broadband background white noise. The startle stimulus for each trial was a 115 ± 5-dB mixed frequency noise burst stimulus (approximately 20 ms in duration). Responses were recorded during the first 100 ms following onset of the startle stimulus for each trial. The test session consisted of 50 trials with an 8-second intertrial interval. Startle response data were analyzed in 5 blocks of 10 trials each. Startle response measures obtained are peak response amplitude (PEAK) and
latency to peak response (Tpeak) in milliseconds.
- FOB Assessments (Cohort 2A): Up to 10 F1 pups/sex/group (Cohort 2A) were observed for FOB assessment on PND 65 (prior to the locomotor activity assessment). Testing was performed by the same trained technicians, wherever possible, who did not know the animal’s group assignment and was performed at approximately the same time each day. The FOB was counterbalanced across testing time, sex, and group. The FOB was performed in a sound-attenuated room equipped with a white noise generator. All animals were observed for the following parameters. Home cage observations included biting, convulsions/tremors, feces consistency, palpebral (eyelid) closure, and posture. Handling observations included ease of handling animal in hand, ease of removal from cage, eye prominence, fur appearance, lacrimation/chromodacryorrhea, mucous membranes/eye/skin color, muscle tone, palpebral closure, piloerection, red/crusty deposits, respiratory rate/character, and salivation. Open field observations (evaluated over a 2-minute observation period) included arousal, backing, bizarre/stereotypic behavior, convulsions/tremors, gait, gait score, grooming, mobility, rearing, time to first step (seconds), urination/defecation. Sensory observations included air righting reflex, approach response, eyeblink response, forelimb extension, hindlimb extension, olfactory orientation, pupil response, startle response, tail pinch response, touch response. Neuromuscular observations included grip strength-hind and forelimb, hindlimb extensor strength, hindlimb foot splay, and rotarod performance. Physiological observations included body temperature, body weight, and catalepsy.
- Motor Activity (Cohort 2A): Up to 10 F1 pups/sex/group (Cohort 2A) were observed for motor activity on PND 65. Locomotor activity sessions were performed in a sound-attenuated room equipped with a white noise generation system set to operate at 70 ± 10 dB. The same animals were tested at each interval using a series of infrared photobeams to quantify each animal’s motor activity. The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator, and black enclosures were used to decrease the potential for distraction. Data were collected in 5-minute epochs over a period of 60 minutes, and the data were reported in 10-minute subintervals. Total motor activity was defined as a combination of fine motor skills (i.e., grooming; interruption of 1 photobeam) and ambulatory motor activity (e.g., interruption of 2 or more consecutive photobeams).

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: All animals in Cohort 3 (10 rats/sex/group, representing as many litters as possible) and Cohort 3A (10 rats in the control group, representing as many litters as possible) were administered a single intravenous immunization of sheep red blood cells (sRBC) via a lateral tail vein on PND 54 to elicit a primary antibody response. Each sRBC injection was composed of 0.5 mL of Earle’s Balanced Salt Solution with HEPES buffer solution and contained approximately 2E8 sRBC. Positive control rats in Cohort 3A also received 10 mL/kg/day (25 mg/kg/day) of cyclophosphamide (CPS) by intraperitoneal injection for 5 consecutive days (PND 54–58) prior to and including the day prior to the scheduled necropsy (PND 59). CPS is a known immune suppressant and was prepared in phosphate-buffered saline at a concentration of 2.5 mg/mL, apportioned into at least 5 separate (daily) aliquots and stored at ≤ -10ºC until needed. On the day of injection, the CPS was thawed, stored on ice, and mixed prior to injection. On PND 59 (approximately 120 hours following immunization with sRBC), all animals were euthanized by carbon dioxide inhalation. Approximately 1 mL to 1.5 mL of blood was collected via the inferior vena cava into tubes without anticoagulant. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70°C. Samples were transferred to the Immunotoxicology Department; t-cell dependent antibody response analysis (TDAR) was conducted using a sRBC IgM ELISA kit. Following blood collection, a gross necropsy was conducted on each animal. The thymus and spleen were weighed and retained in 10% neutral buffered formalin for possible future histopathology.
Postmortem examinations (parental animals):
THYROID HORMONE ANALYSIS: Blood samples for thyroid hormone analyses were collected (prior to 1200 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels) from adult F0 animals (10/sex/group) from all groups on the day of scheduled necropsy. At least 1.0 mL/sample was collected via venipuncture from the jugular vein using the hand-held restraint method. The site of collection (including right or left) was documented. The same animals had blood collected for clinical pathology assessments. Two aliquots were collected: one aliquot approximately 150 µL for total T4 and a second aliquot approximately 100 µL for TSH. Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70°C. Blood samples were analyzed for Thyroxine (Total T4) and Thyroid Stimulating Hormone (TSH). Samples to be analyzed for T4 were transferred to the Bioanalytical Chemistry Department; analyses were conducted using validated ultra-high performance liquid chromatography with dual mass spectroscopy (UHPLC/MS/MS) assays. Samples to be analyzed for TSH concentrations were transferred to the Immunotoxicology Department; analyses were conducted using a validated Luminex Bead Based (TSH) assay.

CLINICAL PATHOLOGY/HEMATOLOGY/URINALYSIS/COAGULATION: Animals were fasted overnight prior to blood collection. Urine was collected overnight using metabolism cages. Blood samples for hematology and/or clinical chemistry were collected from the jugular vein. Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation. K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was used for samples collected for clotting determinations. Samples for clinical chemistry were collected without anticoagulants. In addition, blood smears were prepared, stained with Wright-Giemsa stain, cover-slipped, and retained for possible future evaluation. Samples were collected from F0 adult males and females (10/sex/group; all groups) on the day of scheduled necropsy. All animals (including those not scheduled for clinical pathology assessments) were fasted overnight prior to blood collection. Individual body weights were recorded prior to and after fasting of animals. Hematology samples (0.5 mL) and clinical chemistry samples (1.5 mL) were collected via venipuncture from the jugular vein using the hand-held restraint method (if attempts were unsuccessful, blood was collected, as possible, from the vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation). Coagulation samples (1.8 mL) were collected via venipuncture from the vena cava (following euthanasia). Urine was collected at least 8 hours (no more than 24 hours) using metabolism cages.

- Urinalysis: Urine samples were processed and analyzed for specific gravity (SG), pH, urobilinogen (URO), Total volume (TVOL), color (COL), clarity (CLA), Protein (PRO), glucose (GLU), ketones (KET), microscopy of sediment, bilirubin (BIL), occult blood (BLD), and leukocytes (LEU).

- Hematology: Blood samples were analyzed for total leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (Platelet), reticulocyte count, Differential leukocyte count - neutrophil (NEU), lymphocyte (LYMPH), monocyte (MONO), eosinophil (EOS), basophil (BASO), large unstained cell (LUC), red cell distribution width (RDW), hemoglobin distribution width (HDW), platelet estimate, red cell morphology (RBC Morphology)

- Coagulation: Blood samples were processed for plasma, and the plasma was analyzed for activated partial thromboplastin time (APTT), fibrinogen, and prothrombin time (PT)

- Clinical Chemistry: Blood samples were processed for serum, and the serum was analyzed for albumin, total protein, globulin [by calculation], albumin/globulin ratio (A/G Ratio) [by calculation], total bilirubin (Total BILI) (when total bilirubin was >0.5 mg/dL or >8.55 µmol/L, direct bilirubin was also measured and indirect bilirubin was calculated), urea nitrogen, creatinine, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT), glucose, total cholesterol (Cholesterol), calcium, chloride, phosphorus, potassium, sodium, sorbitol dehydrogenase (SDH), triglycerides (Triglyceride), bile acids, and appearance (including degree of hemolysis, lipemia, and icterus).

SACRIFICE: A necropsy was conducted for animals that died on study, and specified tissues were saved. For one female that was found dead during lactation, the number of corpora lutes (through Lactation Day 4) and former implantation sites were recorded. All pups were euthanized by an intraperitoneal injection of sodium pentobarbital in the scapular region and necropsied. All surviving animals, including females that failed to deliver or with total litter loss, were euthanized by carbon dioxide inhalation following the selection of the F1 generation.

GROSS NECROPSY: All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. The numbers of implantations sites and former implantation sites were recorded for females that delivered. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.

ORGAN WEIGHTS: Organs weighed at necropsy included adrenal glands, brain, epididymides (total and cauda; weighed separately), heart, kidneys, liver, ovaries, pituitary gland, prostate gland, seminal vesicles with coagulating glands (with accessory fluids), spleen, tested (weighed separately), thymus gland, thyroids with parathyroids (weighed after fixation), and uterus with oviducts and cervix. Unless otherwise noted above, paired organs were weighed together. Organ to body weight ration (using the terminal body weight) and organ to brain weight ratios were calculated. Organ weights were not recorded for animals found dead.

HISTOPATHOLOGY: Representative samples of the following tissues were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated: adrenal glands (2), aorta, bone with marrow (sternebrae), brain, coagulating glands (2), eyes with optic nerve (2; fixed in Davidson's solution), gastrointestinal tract, esophagus, stomach, duodenum, Peyer’s Patches, jejunum, ileum, cecum, colon, rectum, heart, kidneys (2), lacrimal/Harderian glands, liver (section of 2 lobes), lungs (including bronchi, fixed by inflation with fixative), lymph node (axillary [2], iliac [2], mandibular [2; only 1 examined], and mesenteric), ovaries (fixed in 10% neutral buffered formalin for approximately 48 hours and then transferred to 70% ethanol) and oviducts (2), pancreas, peripheral nerve (sciatic; only 1 examined), pituitary, prostate, mandibular salivary gland (2; only 1 examined), seminal vesicles (2), skeletal muscle (quadriceps), skin with mammary gland (for females, a corresponding section of skin was taken from same anatomic area for males), spinal cord (cervical), spleen, testes with epididymides (2) and vas deferens (1) (NOTE: right testes and right and remainder of left epididymis were fixed in modified Davidson's solution; both testes and epididymides from found dead animals were fixed in modified Davidson's solution; if the left testis or epididymis was noted with a gross lesion, the left testis and epididymis were fixed in modified Davidson's solution and the right testis and epididymis were used for sperm evaluation), thymus, thyroids with (with parathyroids, if present [2]), trachea, urinary bladder, uterus with cervix and vagina, all gross lesions (all groups). Tissue trimming was performed at the Testing Facility. Ovaries were processed at PAI Durham. Tissues identified above from all animals in the control and high-dose groups and from all animals found dead and euthanized in extremis, as well as gross lesions from all animals in all groups, and reproductive organs of all animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected, were embedded in paraffin, sectioned, mounted on glass slides and stained with hematoxylin and eosin. Processing of the testes, epididymides, and ovaries were performed as noted below. Sections of 2–4 microns of the testis (transverse) and epididymis (longitudinal) were stained with PAS and hematoxylin staining in addition to the routine hematoxylin and eosin (H&E) staining. Testes and epididymides from males that were found dead or euthanized in extremis were stained with H&E only. The following regions of the epididymis were embedded in paraffin: caput, corpus, and cauda; the vas deferens was examined when possible. A single section was taken from all F0 females for a qualitative bilateral evaluation of each ovary. Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified for microscopic examination were evaluated from all animals in the control and high-dose groups and from all animals found dead and euthanized in extremis. Gross lesions were examined from all animals in all groups. In addition, reproductive organs of all animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected, were subjected to a histopathologic evaluation. Reproductive organs consisted of ovaries, oviducts, uterus, cervix, and vagina of females, and testes, epididymides, prostate gland, seminal vesicles, and coagulating glands of males. Uterine and ovarian histopathology were considered in light of the terminal estrous stage. The vas deferens and rete testis were evaluated, if possible. Histopathological examination of the testis included a qualitative assessment of the stages of spermatogenesis. For males that survived to the scheduled necropsy, microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules. The progression of these cellular associations defines the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).
Postmortem examinations (offspring):
F1 GENERATION (LITTERS):

THYROID HORMONE ANALYSIS: Blood samples for thyroid hormone analyses were collected (prior to 1200 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels) from culled (PND 4) and non-selected (PND 21) offspring, and from F1 (Cohort 1A, PND 91) animals. Samples collected at termination for PND 4 F1 culled pups (10 litters/group; all groups) (10/sex/group) were pooled by litter, regardless of sex. The first 10 litters with sufficient numbers of culled pup in each group were selected. Additional litters may have been used if there was an insufficient number of pups in the first 10 litters to obtain the desired blood volume. Samples collected at termination for PND 21 male and female non-selected pups (10/sex/group; all groups) may have had samples pooled by litter (if necessary) and separated by sex. Additional non-selected male or female pups from the same litter may have been used to obtain the desired blood volume. At least 1.0 mL/sample was collected via cardiac puncture under isoflurane anesthesia. Following blood collection, pups were euthanized by exsanguination (bilateral thoracotomy, if necessary) and examined macroscopically. Samples were collected from the F1 (Cohort 1A) males and females on PND91 (day of scheduled necropsy) (10/sex/group) from all groups. At least 1.0 mL/sample was collected via venipuncture from the jugular vein using the hand-held restraint method. The site of collection (including right or left) was documented. The same animals had blood collected for clinical pathology assessments. Two aliquots were collected: one aliquot approximately 150 µL for total T4 and a second aliquot approximately 100 µL for TSH. Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70°C. Blood samples were analyzed for Thyroxine (Total T4) and Thyroid Stimulating Hormone (TSH). Samples to be analyzed for T4 were transferred to the Bioanalytical Chemistry Department; analyses were conducted using validated ultra-high performance liquid chromatography with dual mass spectroscopy (UHPLC/MS/MS) assays. Samples to be analyzed for TSH concentrations were transferred to the Immunotoxicology Department; analyses were conducted using a validated Luminex Bead Based (TSH) assay.

CLINICAL PATHOLOGY/HEMATOLOGY/URINALYSIS/COAGULATION: Animals were fasted overnight prior to blood collection. Urine was collected overnight using metabolism cages. Blood samples for hematology and/or clinical chemistry were collected from the jugular vein. Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation. K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was used for samples collected for clotting determinations. Samples for clinical chemistry were collected without anticoagulants. In addition, blood smears were prepared, stained with Wright-Giemsa stain, cover-slipped, and retained for possible future evaluation. Samples were collected from F1 adult males and females (10/sex/group; all groups) on the day of scheduled necropsy (PND 91). All animals (including those not scheduled for clinical pathology assessments) were fasted overnight prior to blood collection. Individual body weights were recorded prior to and after fasting of animals. Hematology samples (0.5 mL) and clinical chemistry samples (1.5 mL) were collected via venipuncture from the jugular vein using the hand-held restraint method (if attempts were unsuccessful, blood was collected, as possible, from the vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation). Coagulation samples (1.8 mL) were collected via venipuncture from the vena cava (following euthanasia). Urine was collected at least 8 hours (no more than 24 hours) using metabolism cages.

- Urinalysis: Urine samples were processed and analyzed for specific gravity (SG), pH, urobilinogen (URO), Total volume (TVOL), color (COL), clarity (CLA), Protein (PRO), glucose (GLU), ketones (KET), microscopy of sediment, bilirubin (BIL), occult blood (BLD), and leukocytes (LEU).

- Hematology: Blood samples were analyzed for total leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (Platelet), reticulocyte count, Differential leukocyte count - neutrophil (NEU), lymphocyte (LYMPH), monocyte (MONO), eosinophil (EOS), basophil (BASO), large unstained cell (LUC), red cell distribution width (RDW), hemoglobin distribution width (HDW), platelet estimate, red cell morphology (RBC Morphology)

- Coagulation: Blood samples were processed for plasma, and the plasma was analyzed for activated partial thromboplastin time (APTT), fibrinogen, and prothrombin time (PT)

- Clinical Chemistry: Blood samples were processed for serum, and the serum was analyzed for albumin, total protein, globulin [by calculation], albumin/globulin ratio (A/G Ratio) [by calculation], total bilirubin (Total BILI) (when total bilirubin was >0.5 mg/dL or >8.55 µmol/L, direct bilirubin was also measured and indirect bilirubin was calculated), urea nitrogen, creatinine, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), glucose, total cholesterol (Cholesterol), calcium, chloride, phosphorus, potassium, sodium, sorbitol dehydrogenase (SDH), triglycerides (Triglyceride), bile acids, and appearance (including degree of hemolysis, lipemia, and icterus).

SACRIFICE: A necropsy was conducted for animals that died on study, and specified tissues were saved. Pups euthanized due to the death of dam were euthanized as per Testing Facility SOPs. These animals underwent necropsy, and specified tissues were retained. Intact offspring that were found dead or euthanized for humane reasons during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. Findings were recorded as developmental variations or malformations, as appropriate. A gross necropsy was performed on any pup found dead or euthanized for humane reasons after PND 4. On PND 4, culled pups were euthanized by exsanguination (those pups used for blood/thyroid collection) or an intraperitoneal injection of sodium pentobarbital. On PND 21, non-selected pups were euthanized by carbon dioxide inhalation. PND 21 pups selected for blood collection were anesthetized using isoflurane and euthanized by exsanguination following blood collection.

GROSS NECROPSY: On PND 4, 1 culled pup/sex/litter was subjected to a complete necropsy examination. Pups were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. All remaining culled pups were discarded without examination. On PND 21, non-selected pups were subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system.

ORGAN WEIGHTS: Brain, liver, spleen, thymus, and thyroids (after fixation) were weighed at necropsy from 10 non-selected F1 pup/sex/group (same animals used for blood collection) on PND 21. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

HISTOPATHOLOGY: Representative specimens with malformations and gross lesions from offspring found dead or euthanized for humane reasons were preserved in 10% neutral buffered formalin. Representative samples of trachea (with thyroid gland) and all gross lesions were collected from 1 culled pup/sex/litter on PND 4 and preserved in 10% neutral buffered formalin. Representative samples of brain, liver, ovaries (2), skin with mammary gland (for females; corresponding section of skin was taken from the same anatomical area for males), spleen, testes (2; fixed in modified Davidson's solution), thymus, thyroids, and all gross lesions were collected from 10 non-selected F1 pup/sex/litter (same animals used for blood collection) on PND 21 and preserved in 10% neutral buffered formalin.

F1 GENERATION (COHORT 1):

SACRIFICE: A necropsy was conducted for animals that died on study, and specified tissues were saved. All surviving animals were euthanized by carbon dioxide inhalation.

SPERM EVALUATIONS (Cohort 1A): Immediately upon euthanasia, the reproductive tract of each male was exposed via a ventral mid-line incision. The right cauda epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis, and it was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a minimum 10-minute incubation period, a sample of sperm was loaded onto a slide with a 100-μm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all pipettes, slides, and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported. The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and cauda epididymis from all males were weighed and stored frozen. The left cauda epididymis was homogenized and analyzed for determination of homogenization resistant spermatid count. An aliquot of each sample was added to a solution containing a DNA specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-μm chamber depth. Illumination from a xenon lamp within the analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample.

GROSS NECROPSY: All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system.

ORGAN WEIGHTS: Organs weighed at necropsy included adrenal glands, brain, epididymides (total and cauda; weighed separately; also weighed for Cohort 1B where they were weighed whole - total weight only), heart, kidneys, liver, lymph nodes - iliac; lymph nodes - mandibular; ovaries (also weighed for Cohort 1B), pituitary gland (also weighed for Cohort 1B), prostate gland (also weighed for Cohort 1B), seminal vesicles with coagulating gland and fluid (also weighed for Cohort 1B), spleen, testes (weighed separately; also weighed for Cohort 1B), thymus, thyroids with parathyroids (weighed after fixation), and uterus with oviducts and cervix (also weighed for Cohort 1B). Unless otherwise noted above, paired organs were weighed together. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis.

HISTOPATHOLOGY: Gross lesions were preserved in 10% neutral buffered formalin for all Cohort 1 surplus animals. Representative samples of the following tissues were collected from all animals in Cohorts 1A and 1B and preserved in 10% neutral buffered formalin, unless otherwise indicated: adrenal glands (2), aorta, bone with marrow (sternebrae), brain, coagulating glands (2), eyes with optic nerve (2; fixed in Davidson's solution), gastrointestinal tract, esophagus, stomach, duodenum, Peyer’s Patches, jejunum, ileum, cecum, colon, rectum, heart, kidneys (2), lacrimal/Harderian glands, liver (section of 2 lobes), lungs (including bronchi, fixed by inflation with fixative), lymph node (axillary [2], iliac [2], mandibular [2; only 1 examined], and mesenteric), ovaries (fixed in 10% neutral buffered formalin for approximately 48 hours and then transferred to 70% ethanol) and oviducts (2), pancreas, peripheral nerve (sciatic; only 1 examined), pituitary, prostate, mandibular salivary gland (2; only 1 examined), seminal vesicles (2), skeletal muscle (quadriceps), skin with mammary gland (for females, a corresponding section of skin was taken from same anatomic area for males), spinal cord (cervical), spleen, testes with epididymides (2) and vas deferens (1) (NOTE: right testes and right and remainder of left epididymis were fixed in modified Davidson's solution; both testes and epididymides from found dead animals were fixed in modified Davidson's solution; if the left testis or epididymis was noted with a gross lesion, the left testis and epididymis were fixed in modified Davidson's solution and the right testis and epididymis were used for sperm evaluation), thymus, thyroids with (with parathyroids, if present [2]), trachea, urinary bladder, uterus with cervix and vagina, all gross lesions (all groups). Tissue trimming was performed at the Testing Facility. Ovaries were processed at PAI Durham. All other tissues were processed at the testing facility. Tissues identified above from F1 animals in Cohort 1A in the control and high-dose groups and from all animals found dead, as well as gross lesions from all animals in all groups were embedded in paraffin, sectioned, mounted on glass slides and stained with hematoxylin and eosin. Processing of the testes, epididymides, and ovaries were performed as noted below. Sections of 2–4 microns of the testis (transverse) and epididymis (longitudinal) were stained with PAS and hematoxylin staining in addition to the routine hematoxylin and eosin (H&E) staining. Testes and epididymides from males that were found dead were stained with H&E only. Five (5) sections were taken approximately 100 µm apart from the inner third of each ovary from all F1 Cohort 1A females at the scheduled termination. In addition, a single section was taken from all F1 Cohort 1A females for a qualitative bilateral evaluation of each ovary. For females found dead, a single section from each ovary was qualitatively evaluated. The coagulating glands, ovaries, pituitary gland, prostate gland, seminal vesicles, testes with epididymides, uterus with cervix and vagina, and gross lesions from Cohort 1B animals were processed to the block stage (in paraffin). Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified for microscopic examination were evaluated from all animals in the control and high-dose groups and from all animals found dead. Gross lesions were examined from all animals in all groups. In addition, reproductive organs of all animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected, were subjected to a histopathologic evaluation. Histopathological examination of the testis included a qualitative assessment of the stages of spermatogenesis. For males that survived to the scheduled necropsy, microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules. The progression of these cellular associations defines the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). When possible, sections of the rete testis were examined in the F1 Cohort 1A males. For all F1 Cohort 1A females in the control and high-dose groups at scheduled termination, a quantitative histopathologic evaluation of multiple sections of the ovaries was conducted. This examination included enumeration of the total number of primordial follicles. Uterine and ovarian histopathology were considered in light of the terminal estrous stage.

SPLENIC LYMPHOCYTE IMMUNOPHENOTYPING (COHORT 1A): The spleen from 10 F1 animals/sex/group in Cohort 1A was harvested, weighed, and cut in half. One-half was placed into chilled Hank’s Balanced Buffer Salt solution with 2% fetal bovine serum; the remaining half was placed in 10% neutral buffered formalin. Immunophenotyping results were enumerated as relative (% gated) and absolute tissue (tissue cells/10e6 cells) values for each leukocyte subset. The absolute lymphocyte count (cells/organ) of each subset was calculated by multiplying each leukocyte subset frequency (%) with the total leukocyte count. The following formula was used in generating the absolute number of different subsets of lymphocytes in the spleen:

Absolute number of cells (10E6/tissue) = ([whole organ wt * live cell conc * vol of cells]/partial organ weight) X (cell subset frequency/100) X (0.000001)

NEUROPATHOLOGY (Cohort 2): Due to the large number of animals to be perfused for neuropathological assessment, perfusions for Cohorts 2A and 2B were performed over several days. The perfusions were performed such that both sexes and all treatment groups were approximately equally represented across each day. The order of perfusions each day was also counterbalanced by sex and treatment group (i.e., 1 male from Group 1, 1 male from Group 2, 1 male from Group 3, 1 male from Group 4, 1 female from Group 1, etc.). Brains from all animals on PND 22 (Cohort 2B) and the central and peripheral nervous system tissues from all animals on PND 78 (Cohort 2A) were dissected. Any abnormal coloration or lesions of the external brain and spinal cord were recorded. The whole brains were removed (including olfactory bulbs), weighed, and the dimensions (length [excluding olfactory bulbs] and width) were recorded. Brains from all animals perfused in situ on PND 22 (Cohort 2B) were collected and preserved in 10% neutral buffered formalin. In addition, representative samples of the following tissues were collected from all animals perfused in situ on PND 78 (Cohort 2A), unless otherwise indicated: brain including olfactory bulbs, cerebral cortex (2 levels), hippocampus/dentate gyrus, basal ganglia, thalamus, hypothalamus, midbrain, cerebellum, pons, and medulla oblongata (full coronal section prepared); spinal cord including at cervical swellings C3-C7 & at lumbar swellings T13-L4, trigeminal ganglia/nerves (both tissues processed), lumbar dorsal root ganglia at T13-L4 (4-6 tissues collected; 2 evaluated microscopically), lumbar dorsal root fibers at T13-L4 (4-6 tissues collected; 2 evaluated microscopically), lumbar ventral root fibers at T13-L4 (4-6 tissues collected; 2 evaluated microscopically), cervical dorsal root ganglia at C3-C7 (4-6 tissues collected; 2 evaluated microscopically), cervical dorsal root fibers at C3-C7 (4-6 tissues collected; 2 evaluated microscopically), cervical ventral root fibers at C3-C7 (4-6 tissues collected; 2 evaluated microscopically), pituitary gland, cauda equina nerves (2 sections prepared; cross section embedded in hard plastic - Spurr's resin, Epon, methyl methacrylate, etc.; longitudinal section embedded in paraffin; right nerve processed for microscopic examination; tissue from left hind leg collected and preserved for possible future evaluation), sciatic nerves (2 sections prepared; cross section embedded in hard plastic - Spurr's resin, Epon, methyl methacrylate, etc.; longitudinal section embedded in paraffin; right nerve processed for microscopic examination; tissue from left hind leg collected and preserved for possible future evaluation), sural nerves (2 sections prepared; cross section embedded in hard plastic - Spurr's resin, Epon, methyl methacrylate, etc.; longitudinal section embedded in paraffin; right nerve processed for microscopic examination; tissue from left hind leg collected and preserved for possible future evaluation), tibial nerves (2 sections prepared; cross section embedded in hard plastic - Spurr's resin, Epon, methyl methacrylate, etc.; longitudinal section embedded in paraffin; right nerve processed for microscopic examination; tissue from left hind leg collected and preserved for possible future evaluation), peroneal nerves (2 sections prepared; cross section embedded in hard plastic - Spurr's resin, Epon, methyl methacrylate, etc.; longitudinal section embedded in paraffin; right nerve processed for microscopic examination; tissue from left hind leg collected and preserved for possible future evaluation), nasal tissue with olfactory epithelium, optic nerves (both tissues processed), Eyes (both tissues processed), skeletal muscle: gastrocnemius, and other sites if deemed necessary. Tissues were processed at PAI Durham. The brains from all animals perfused in situ on PND 22 (Cohort 2B) were embedded in paraffin. For all animals perfused in situ on PND 78 (Cohort 2A), the central nervous system tissues identified were embedded in paraffin, and the peripheral nervous system tissues were embedded in plastic. Tissues were sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Neuropathological evaluation was performed by a board-certified veterinary pathologist. For animals perfused in situ on PND 22 (Cohort 2B), sections from all major brain regions (including olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, midbrain, brainstem, and cerebellum) were evaluated from all animals in the control and high-exposure group. For all animals perfused in situ on PND 78 (Cohort 2A), tissues identified for microscopic examination were evaluated from all animals in the control and high-exposure group.

Cohort 2B was euthanized on PND 22. Necropsy, tissue collection, and organ weights were performed for 10 animals/sex/group. Histology and histopathology of the brain was performed on 10 animals/group for the control and high dose group only. Cohort 2A was euthanized on PND 78. Necropsy, tissue collection, and organ weights were performed for 10 animals/sex/group. Histology and histopathology of nervous system tissues, and morphometry was performed on 10 animals/group for the control and high dose group only (9 females in the control group).

MORPHOMETRIC ANALYSIS (COHORT 2A): For F1 Cohort 2A animals (PND 78), microscopic morphometric measurements were performed on highly homologous sections of brain from 3 of the 10 blocks and stained with Luxol fast blue/Cresyl violet. The 3 slides corresponding to these blocks were scanned using a Hamamatsu Nanozoomer whole slide scanner and imported into the Visiopharm software for measurements. Microscopically, 11 linear morphometric measurements, were taken in a blinded random fashion from control and high dose group animals. Morphometric measures taken bilaterally were averaged for each rat. These 11 linear measurements were as follows:
● Thickness of the frontal cortex bilaterally. This measurement was taken from the dorsal portion of the cerebral cortex within the coronal section passing through the region of the optic chiasm.
● Thickness of the parietal cortex bilaterally. This measurement was taken from the dorsolateral portion of the cerebral cortex within the coronal section taken through the optic chiasm.
● Diagonal width (maximum cross-sectional width) of the caudate-putamen bilaterally. This measurement was performed on the coronal section taken at the level of the optic chiasm.
● Thickness of the corpus callosum bilaterally just lateral to its midpoint at the level of Layer 2 of the overlying cingulate gyrus. This measurement was taken within the section passing through the optic chiasm.
● Thickness of all hippocampal layers combined, bilaterally, extending from the alveus to just lateral to the inferior blade of the dentate gyrus. This measurement was performed within the section taken at the level of the infundibulum.
● Maximum height of the cerebellum at the level of the deep cerebellar nuclei, extending from the roof of the fourth ventricle to the dorsal surface. Note that Lobules 1 or 10 of the cerebellum (which protrude into the fourth ventricle) may have been excluded from measurement if inconsistently present on section.

IMMUNOTOXICOLOGY (COHORT 3): All animals in Cohort 3 (10 rats/sex/group, representing as many litters as possible) and Cohort 3A (10 rats in the control group, representing as many litters as possible) were administered a single intravenous immunization of sheep red blood cells (sRBC) via a lateral tail vein on PND 54 to elicit a primary antibody response. Each sRBC injection was composed of 0.5 mL of Earle’s Balanced Salt Solution with HEPES buffer solution and contained approximately 2E8 sRBC. Positive control rats in Cohort 3A also received 10 mL/kg/day (25 mg/kg/day) of cyclophosphamide (CPS) by intraperitoneal injection for 5 consecutive days (PND 54–58) prior to and including the day prior to the scheduled necropsy (PND 59). CPS is a known immune suppressant and was prepared in phosphate-buffered saline at a concentration of 2.5 mg/mL, apportioned into at least 5 separate (daily) aliquots and stored at ≤ -10ºC until needed. On the day of injection, the CPS was thawed, stored on ice, and mixed prior to injection. On PND 59 (approximately 120 hours following immunization with sRBC), all animals were euthanized by carbon dioxide inhalation. Approximately 1 mL to 1.5 mL of blood was collected via the inferior vena cava into tubes without anticoagulant. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70°C. Samples were transferred to the Immunotoxicology Department; t-cell dependent antibody response analysis (TDAR) was conducted using a sRBC IgM ELISA kit. Following blood collection, a gross necropsy was conducted on each animal. The thymus and spleen were weighed and retained in 10% neutral buffered formalin for possible future histopathology.
Statistics:
See "Any other information on materials and methods"
Reproductive indices:
Male mating index (%), female mating index (%), male fertility index (%), female fertility index (%), male copulation index (%), female conception index (%)
Offspring viability indices:
Offspring indices included mean live litter size, postnatal survival between birth and PND 0 or PND 4 (pre-selection ) (% per litter), and postnatal survival for all other intervals (% per litter)
Clinical signs:
no effects observed
Description (incidence and severity):
No test substance-related clinical findings were noted during the generation at the daily examinations and, other than sporadic occurrences of salivation-related findings, no significant clinical observations at approximately 1-hour post-dose. Other findings noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no test substance-related mortalities in this study. One male in the 200 mg/kg/day group and one female in the 500 mg/kg/day group were found dead on Study Day 52 and Lactation Day 3, respectively, both with findings consistent with dosing error. The male died during dosing and had red fluid in the thoracic cavity, yellow fluid in the abdominal cavity, and dark red discoloration of the duodenum, jejunum, ileum, jejunum, and thymus gland, and a swollen liver at necropsy. The female was noted with labored respiration on the day of death; necropsy findings consisted of a perforated esophagus at the approximate level of thoracic vertebra No. 8 and red fluid in the thoracic cavity. All other F0 animals survived to the scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Weekly: No test substance-related effects on mean F0 body weights and body weight gains were noted in the 50, 200, and 500 mg/kg/day groups. The values in the test substance-treated groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males). Sporadic statistically significant differences from the control group occurred in males only, but the changes were transient and/or did not occur in a dose-related manner.

Gestation: Mean F0 maternal body weights and body weight gains were unaffected by test substance administration during gestation. Differences between the control, 50, 200, and 500 mg/kg/day groups were slight and not statistically significant.

Lactation: Mean F0 maternal body weights and body weight gains were unaffected by test substance administration during lactation. The only statistically significant differences from the control group were higher mean body weight gains noted in the 500 mg/kg/day group during Lactation Days 1–4 and when the overall lactation period (Days 1–21) was evaluated. These differences occurred in a direction (higher) that is not considered toxicologically relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Weekly: Mean F0 food consumption, evaluated as g/animal/day, and food efficiency in the 50, 200, and 500 mg/kg/day groups was unaffected by test substance administration. Occasional statistically significant differences from the control group occurred in all groups; however, these differences occurred in a direction (higher mean food consumption) that is not considered toxicologically relevant, and there were no effects on mean absolute body weights.

Gestation: Mean F0 maternal food consumption and food efficiency were unaffected by test substance administration during gestation. Although mean food consumption in the 500 mg/kg/day group females was sporadically statistically significant compared to the control group, these differences occurred in a direction (higher mean food consumption) that is not considered toxicologically relevant, and there were no effects on mean absolute body weights or food efficiency.

Lactation: Mean F0 maternal food consumption and food efficiency were unaffected by test substance administration during lactation. Sporadic statistically significant differences from the control group occurred in a direction (higher mean food consumption) that is not considered toxicologically relevant, and there were no effects on mean absolute body weights.
Food efficiency:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on hematology parameters. Any statistically significant differences from the control group were generally observed in a non-dose-responsive manner and/or did not show correlating patterns of changes in other parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on coagulation parameters. Differences noted were generally observed in a non-dose-responsive manner and/or did not show correlating patterns of changes in other parameters.

There were no test substance-related effects on clinical chemistry parameters. Differences noted were generally observed in a non-dose-responsive manner and/or did not show correlating patterns of changes in other parameters.
Endocrine findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on thyroid hormones. Any statistically significant
differences from the control group did not occur in a dose-related manner.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on urinalysis parameters. Although the mean pH values for the 200 and 500 mg/kg/day group males (7.3 and 6.9, respectively) were statistically significantly lower than the concurrent control group, the values were within the historical control group data range (6.1–7.5). However, the concurrent control group value (8.0) was higher than the maximum mean value in the historical control data. Therefore, the differences were attributed to the atypically high control group value, and not attributed to test substance administration.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test substance-related microscopic findings in any of the male or female dose
groups. All microscopic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than test substance administration. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
All microscopic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than test substance administration. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental
tissue alterations.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean lengths of estrous cycles in these groups were similar to the control group value, ranging from 4.2 to 4.4 days across all groups. In addition, 100% of the females across all groups were cycling, and between 43.3% to 60.0% were cycling regularly. None of these differences were statistically significant.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No test substance-related effects were observed on F0 sperm parameters (mean epididymal sperm numbers, motility, progressive motility, and morphology) in males at any dose level. Differences from the control group were slight and were not statistically significant.
Reproductive performance:
no effects observed
Description (incidence and severity):
No test substance-related effects on F0 reproductive performance were observed at any dose level. No statistically significant differences were noted between the control and test substance-treated groups. Two, 1, and 3 mating pairs in the control, 50, and 200 mg/kg/day groups, respectively, did not produce a litter. There were no microscopic findings in these animals that would account for the reduced fertility. Twenty, 24, 22, and 30 females in the control, 50, 200, and 500 mg/kg/day, respectively, were gravid. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value.
No test substance-related effects were noted on mean gestation lengths or the process of parturition at any dose level. The mean duration of gestation ranged from 21.8 to 22.0 days across all groups. Differences from the control group were slight and were not statistically significant.
Key result
Dose descriptor:
NOAEL
Remarks:
P0 reproductive and systemic toxicity
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: no adverse effects at highest dose tested
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Eighteen (2) pups (litters) in the 500 mg/kg/day group were noted with a cool body. The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was otherwise unaffected by test substance administration. Sixteen (10), 10(9), 5(5), and 31(9) pups (litters) in the control, 50, 200, and 500 mg/kg/day groups, respectively, were found dead or euthanized in extremis. Seven (5), 4(4), 2(1), and 20(9) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. Eight pups from 1 litter in the 500 mg/kg/day group were euthanized due to the death of the dam.

Post-weaning, no test substance-related clinical findings were noted during the generation at the daily examinations and approximately 1-hour post-dose. Findings noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The mean number of pups born, live litter size, or percentage of males per litter at birth in the 50, 200, and 500 mg/kg/day groups and postnatal survival in the 50 and 200 mg/kg/day groups were comparable to the control group; no statistically significant differences were noted. Lower postnatal survival was noted in the 500 mg/kg/day group during PND 0–1 (90.1%), resulting in lower mean postnatal survival from birth to PND 4 (89.3%); the differences from the control group were not statistically significant. These differences were attributed to 7 of 30 females in this group which <90% mean pup survival during this interval. In comparison, mean pup survival in the 50 and 200 mg/kg/day groups was 97.8% and 99.2% during this interval and only 2 of 24 females in the control group had pup survival <90% during PND 0–1. Because lower mean postnatal survival during birth-PND 4 was also noted at this dose level on a previous reproduction/developmental toxicity screening study, the lower pup survival at 500 mg/kg/day was considered test substance related and adverse.

One male and 1 female each in the control and 200 mg/kg/day groups were found dead within 2 weeks of weaning. The male and female in the 200 mg/kg/day group were found dead on PND 25 and 26, respectively; there were no clinical observations of necropsy findings for these animals. The male in the control group was found dead on PND 22; there were no clinical findings for this animal prior to death, and autolysis precluded necropsy evaluation. A single female in the control group was found dead on PND 34. A swollen head was noted for this female for several days prior the death. Findings noted at veterinary examination included active with normal posture; slightly uncoordinated gait; moderate firm cranial swelling; left hindlimb mildly splayed; no obvious fractures or swelling; and good range of motion. At necropsy, advanced degree of autolysis precluded complete examination. In the brain, dilated ventricles and dark red contents in the cranial cavity surrounding the brain and pituitary gland. All other F1 parental animals in the control, 50, 200, and 500 mg/kg/day groups survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean male and female birth weights (PND 1) in the 500 mg/kg/day group were marginally lower (5.6% and 5.8%, respectively) than the control group. Mean body gains in these pups were comparable to the control group throughout the preweaning period, such that mean body weights thereafter were comparable through PND 21. Mean male and female pup body weights and body weight changes in the 50 and 200 mg/kg/day groups were unaffected by test substance administration throughout the postnatal period. No statistically significant differences from the control group were noted.

Post-weaning, no test substance-related effects on mean F1 body weights and body weight gains were noted in the 50, 200, and 500 mg/kg/day groups. The values in the test substance-treated groups were generally similar to the control group values for the entire generation. Sporadic statistically significant differences from the control group were transient and/or did not occur in a dose-related manner.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Post-weaning, mean F1 food consumption, evaluated as g/animal/day, in the 50, 200, and 500 mg/kg/day groups was unaffected by test substance administration. Higher mean food consumption were noted for males in the 500 mg/kg/day group compared to the control group during PND 21–28, and 35–56, and was considered incidental as the animals showed correspondingly higher mean body weight gains during PND 35–42 and lower mean body weight gains during PND 42–49. Furthermore, higher food consumption is generally not considered toxicologically relevant. Occasional statistically significant differences were noted in mean F1 female food consumption but not in a dose-responsive manner and generally not in a direction considered toxicologically relevant.
Food efficiency:
no effects observed
Description (incidence and severity):
Post-weaning, mean F1 food efficiency in the 50, 200, and 500 mg/kg/day groups was unaffected by test substance administration. Lower mean food efficiency were noted for males in the 500 mg/kg/day group compared to the control group during PND 21–28, and 35–56, and was considered incidental as the animals showed correspondingly higher mean body weight gains during PND 35–42 and lower mean body weight gains during PND 42–49. Occasional statistically significant differences were noted in mean F1 female food consumption but not in a dose-responsive manner and generally not in a direction considered toxicologically relevant.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on hematology parameters. Differences noted were generally observed in a non-dose-responsive manner and/or did not show correlating patterns of changes in other parameters
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on coagulation parameters. No statistically significant differences were noted.

There were no test substance-related effects on clinical chemistry parameters. Differences noted were generally observed in a non-dose-responsive manner and/or did not show correlating patterns of changes in other parameters.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on urinalysis parameters.
Sexual maturation:
no effects observed
Description (incidence and severity):
Balanopreputial Separation: Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance administration. None of the differences from the control group were statistically significant.

Vaginal Patency: Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance administration. None of the differences from the control group were statistically significant.

Estrous Cycle Data (Cohort 1A): There were no test substance-related effects on the mean age at first estrus or the mean duration from vaginal opening to first estrus. In young adult females, there was also no test substance-related effect on mean estrous cycle length, which ranged between 3.9 to 4.3 days. Mean cycle status was generally comparable across all groups with 100% of the females across all groups showing evidence of estrous cyclicity, and a comparable percentage of females showing regular cyclicity across the control and high dose group.

Sperm Evaluations (Cohort 1A): No test substance-related effects were observed on F1 sperm parameters (mean epididymal sperm numbers, motility, progressive motility, and morphology) in males at any dose level. Differences from the control group were slight and were not statistically significant.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distances (absolute, relative to pup body weight, and relative to the cube root of pup body weight) in the 50, 200, and 500 mg/kg/day groups were similar to the control group values. A higher mean relative anogenital distance (to cube root of pup body weight) was noted for female pups in the 200 and 500 mg/kg/day groups; the difference from the control group was statistically significant only in the 500 mg/kg/day group and the mean value (0.93 for both groups, versus 0.88 in the control group) was within the range of the historical control data and hence considered incidental.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
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.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No test substance-related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed for F1 pups on PND 21 at any dose level when the test substance-treated groups were compared to the control group. Any statistically significant differences did not occur in a dose-related manner.

Potential test substance-related alteration of organ weights were noted in the following F1 Cohort 1A groups:

Mean final body weight of the 500 mg/kg/day F1 males was 9.8% lower than concurrent control values. Mean final body weight of the 500 mg/kg/day F1 females was only 1.5% lower than concurrent control values.

Brain weight typically remains relatively constant in the presence of lower body weight, which explains the slightly lower mean absolute brain weight but higher brain weight relative to final body weight in 500 mg/kg/day males.

Mean liver weight of 500 mg/kg/day F1 females was higher than concurrent control values when expressed as ABS, RBr, or RBr. There was no microscopic correlate of the altered liver weight. Mean absolute liver weight of 500 mg/kg/day females was only 11.7% higher than concurrent control values; therefore, would not be readily detected by microscopic examination. Mean liver weight of the 500 mg/kg/day F1 males was higher than concurrent control values when expressed RBo but not when expressed as ABS or RBr. There was no microscopic correlate of the altered liver weight. Interpretation of the liver weight alteration in the 500 mg/kg/day males was complicated by the body weight decrement; therefore, the higher liver weight in the 500 mg/kg/day F1 males was not conclusively an effect of test substance administration. Though the interpretation of liver weights in the 500 mg/kg/day males was complicated by body weight decrement, the constellation of liver weight changes in F1 males and females suggested a minor increase in liver weight associated with test substance administration. The higher liver weight was suspected to be associated with a non-adverse metabolic adaptation such enzyme induction.

Mean kidney weight of the 500 mg/kg/day F1 males was not statistically significantly lower than concurrent control values when expressed as ABS or RBr but statistically significantly higher than concurrent control values when expressed RBo. There was no microscopic correlate of the altered kidney weight. The inconsistent dose-related trend suggests the kidney weight alterations were not associated with test substance administration.

Mean mandibular lymph node weight of the 500 mg/kg/day F1 females was higher than concurrent control values when expressed as ABS, RBr, or RBo. Mean mandibular lymph node weight of the 500 mg/kg/day females was 47.6% higher than concurrent control values while mean mandibular lymph node weight of 50 and 200 mg/kg/day females was 7.7% or 5.8% (respectively) lower than concurrent control values. Mandibular lymph nodes were weighed in only 10 rats/group, and standard deviations were substantial. It was noted that 3 rats from the 500 mg/kg/day group had the highest mandibular lymph node weights of any females in this cohort. This combination of factors suggested the alteration in mandibular lymph node weight was not associated with test substance administration.

Mean spleen weight of 500 mg/kg/day F1 males was lower than concurrent control values when expressed as ABS or RBr but not when expressed RBo. There was no microscopic correlate of the altered spleen weight. This variation pattern in the organ weight analysis suggests an alteration associated with body weight changes. Mean spleen weight of 500 mg/kg/day F1 females was slightly lower than concurrent control values when expressed as ABS, RBr, or RBo, but the differences were not statistically significant. There was no microscopic correlate of the altered spleen weight. Mean thymus weight of 500 mg/kg/day F1 males was lower than concurrent control values when expressed as ABS, RBr, or RBo. There was no microscopic correlate of the altered thymus weight. Mean thymus weight of the 500 mg/kg/day females was slightly lower than concurrent control values when expressed as ABS, RBr or RBo, but the differences were not statistically significant. There was no microscopic correlate of the altered thymus weight. Altered thymus and spleen weight can be a manifestation of a stress response but there was no direct microscopic evidence of organ damage that would result in a stress response. The clinical pathology data did not reveal a consistent pattern of changes that would support an interpretation of a stress response. The stress response system of rodents is known to be exquisitely sensitive, as evidenced by the hematologic and organ weight changes that accompany routine movement of the housing cages of laboratory mice. The overall presentation of organ weight, microscopic, and clinical laboratory data suggest a non-adverse test substance-associated decrement in thymus and spleen weights of 500 mg/kg/day males that was of uncertain pathogenesis but possibly related to the lower final body weights.

In Cohort 1B, mean final body weight of the 500 mg/kg/day F1 males was lower than concurrent control values but the difference was not statistically significant. Mean thyroid/parathyroid gland weights of the 500 mg/kg/day F1 males was statistically significantly lower than concurrent control values when the organ weights were expressed as ABS but not when expressed RBo. The potential effect on thyroid/parathyroid gland weight was not apparent in the females, which had less decrement in final body weight than the males. There was no microscopic correlate of the apparent thyroid/parathyroid gland weight. It was concluded the apparent alteration in thyroid/parathyroid gland weight in the 500 mg/kg/day males was associated with the body weight alteration.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Unscheduled Deaths: No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead or euthanized in extremis. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

PND 4 Culled Pups: There were no internal findings at the necropsy of culled pups euthanized on PND 4.

PND 21 Non-selected Pups and Pups Euthanized Due to Death of Dam: No internal findings that could be attributed to parental administration with the test substance were noted at the necropsy of non-selected pups euthanized on PND 21.

PND 21 Pups Selected for Organ Weights: No internal findings that could be attributed to parental administration with the test substance were noted at the necropsy of pups selected for organ weights euthanized on PND 21.

F1 Generation Following Weaning: At the scheduled F1 male and female necropsies of Cohorts 1A and 1B, no test substance-related internal findings were observed at any dose level. Macroscopic findings observed in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related. A small number of pups in the Cohort 1 surplus pups in the test article-treated groups were noted with dark red discoloration of the lungs on PND 52. However, this finding was not noted in the control group at other ages examined, and was considered incidental.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no test substance-related microscopic findings in any of the male or female dose groups. All microscopic findings observed were considered incidental, of the nature commonly observed in rats of this strain and age, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to test substance administration.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis (PND 4 Culled Pups): There were no test substance-related effects on thyroid hormones in PND 4 culled pups.

Thyroid Hormone Analysis (PND 21 Selected Pups): There were no test substance-related effects on thyroid hormones in in PND 21 pups selected for hormone analysis.

Thyroid Hormone Analysis (Cohort 1A): On PND 91, mean T4 levels were 25.9% to 38.3% lower than the control group in the 50, 200, and 500 mg/kg/day group males and 24.2% to 29.6% lower than the control groups in the 200 and 500 mg/kg/day group females. Mean TSH levels were 25.5% to 94.0% higher than the control group in males and 51.3% to 86.9% higher than the control group in the 50, 200, and 500 mg/kg/day group females. Although the differences in T4 in both males and females appear dose responsive, correspondingly dose responsive differences in TSH were evident only in the males (females were non-dose responsive). Furthermore, there were no corresponding changes in thyroid gland weights, thyroid histopathology or any evidence of thyroid-disruption related alterations in neurobehavior, brain weights or measurements, brain morphometry or histopathology. Taken together these data indicate that the differences in mean T4 levels noted above were incidental and not biologically significant to influence downstream changes in organ system development or histopathology.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Auditory Startle Response: The auditory startle response habituation paradigm was conducted as a longitudinal assessment with selected F1 animals evaluated on PND 24. Administration of 50, 200, and 500 mg/kg/day had no significant effect on auditory startle responsiveness. The PEAK and Tpeak values for all trials combined were generally similar in the F1 males and females among the test substance-treated and the control groups. The only statistically significant differences from the control group were higher PEAK values in the 50 and 200 mg/kg/day group females when all trials were combined; however, there was no dose-response relationship and the PEAK values in the 500 mg/kg/day group, the highest dose level tested, were comparable to the concurrent control group. No effects were noted in the pattern of the habituation response over the entire 50-block test session in adult animals.

Functional Observational Battery: Home cage parameters, handling parameters, open field parameters, sensory parameters, neuromuscular parameters, and physiological parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated males and females when compared to the control group on PND 65.

Locomotor Activity: Locomotor activity patterns (total activity as well as ambulatory activity counts) in F1 males and females were unaffected by maternal test substance at all dose levels on PND 65. Values obtained from the 6 epochs evaluated (0–10, 11–20, 21–30, 31–40, 41–50, and 51–60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the historical control data. Differences from the control group were slight, not statistically significant, within the historical control data ranges, and/or did not occur in a dose-related manner. No remarkable shifts in the pattern of habituation occurred in any of the F1 Cohort 2A test substance-treated groups.

Neuropathology (Cohorts 2A and 2B):
- Macroscopic Examination: At the scheduled F1 Cohort 2A and 2B male and female necropsies, no macroscopic findings were observed at any dose level.
- Brain Weights and Measurements: On PND 22, mean final body weights, brain weights, and brain lengths and widths were comparable across all groups for F1 males and females selected for Cohort 2A. A single statistically significant value was noted in a non-dose responsive manner and was considered incidental and attributed to the higher variability noted for this group. On PND 78, mean final body weights for F1 Cohort 2A females in the 500 mg/kg/day group were 9.1% lower than the control group at termination; the difference was not statistically significant, and mean final body weights for F1 Cohort 2A females in the 50 and 200 mg/kg/day groups, and for F1 Cohort 2A males across all test substance-treated groups were comparable to the control group. At termination, brain weights, brain lengths, and widths for both males and females were comparable to the control group.
- Microscopic Examination: The neurohistopathological evaluation revealed no test substance-related findings in any of the male or female dose groups. All microscopic findings were considered incidental, of the nature commonly observed in rats of this strain and age, and/or were of similar incidence and severity in control and test substance-treated animals and, therefore, were considered unrelated to test substance exposure.
- Morphometric Analysis: There was a statistically significant increase in S6 (cerebellum thickness) among the 500 mg/kg/day group F1 Cohort 2A males compared to the control group. There were no other statistically significant differences compared to the control group.
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):

Test substance administration to male and female rats elicited no changes in splenic cell subsets
on PND 91. Discernable decreases noted in the absolute values of NK-T cells for males (26.3%) and females (30.2%) at 500 mg/kg/day compared to the control group were not considered test substance-related due to lack of correlation with the % gated values. Other differences in splenic immunophenotyping parameters were not considered test substance related based on their small magnitude, inconsistent direction, absence of a dose response, and general overlap of individual values with the range of control values.

Subsequent to immunization with sRBC, the CPS (a known immunosuppressant)-treated positive control group males and females (F1 Cohort 3A) had statistically significant, markedly lower mean sRBC IgM concentrations (-99.1% and -96.8%, respectively) when compared to the concurrent control group. Immunosuppressive changes noted in the positive control group were indicative of the pharmacodynamic effect of cyclophosphamide and demonstrated the acceptability of the sRBC IgM ELISA assay to detect a decrease in the T cell-mediated antibody response to an antigen. Increases in the sRBC-IgM concentration noted in females at 50 and 500 mg/kg/day were not considered test substance related due to lack of dose-response and presence of several high responders in test substance-dosed animals. High-responder females in the 50 mg/kg/day group and in the 500 mg/kg/day group) exhibited high sRBC-IgM concentrations that were out of range of historical data control ranging from 73.1 to 44905.4 µ/mL In addition, high responder males in the 50 mg/kg/day group and in the 500 mg/kg/day group) exhibited high sRBC-IgM concentrations that were out of range of historical data control ranging from 67.0 to 37360.9 µ/mL. Taken together, based on comparison to the CPS positive control group, which demonstrated an overt suppression of immune response as indicated by almost a complete ablation of the sRBC IgM responses and in conjunction with lack of test substance-related effects on splenic cell subsets, it was concluded that the test substance did not exert inhibitory effects on the humoral immune system of the dosed animals.
Key result
Dose descriptor:
NOAEL
Remarks:
F1 neurotoxicity and immunotoxicity
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
developmental neurotoxicity
developmental immunotoxicity
Key result
Dose descriptor:
NOAEL
Remarks:
F1 neonatal and developmental toxicity
Generation:
F1
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Key result
Dose descriptor:
NOAEL
Remarks:
F1 systemic toxicity
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Remarks on result:
other: no systemic toxicity at 500 mg/kg (highest dose tested)
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Conclusions:
F0 NOAEL (reproductive toxicity): 500 mg/kg (highest dose tested)
F0 NOAEL (systemic toxicity): 500 mg/kg (highest dose tested)
F1 NOAEL (systemic toxicity): 500 mg/kg (highest dose tested)
F1 NOAEL (neonatal/developmental toxicity): 200 mg/kg (lower postnatal pup survival between birth and PND4 at 500 mg/kg)
F1 NOAEL (neurotoxicity/immunotoxicity): 500 mg/kg (highest dose tested)
Executive summary:

The objective of this study was to evaluate the potential adverse effects of the test substance, when given by oral (gavage) administration to rats, on reproduction in an extended one-generation study. This included evaluation of life stages not covered by other types of toxicity studies and test for effects that may occur as a result of pre- and postnatal chemical exposure. Male and female rats, 25/sex/dose were exposed to 0, 50, or 200 mg/kg, and 30/sex/dose were exposed to 500 mg/kg. F0 animals were dosed via oral gavage daily for 70 consecutive days prior to mating and continuing through the day prior to euthanasia. The F1 generation was potentially exposed to the test substance in utero and through nursing during lactation. Following weaning, the offspring selected to become the F1 generation were dosed beginning at weaning (PND 21) and continuing until euthanasia (as appropriate for the relevant cohort). The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, pre- and postweaning developmental landmarks, neurobehavior, thyroid hormones, clinical pathology, macroscopic findings, sperm parameters, immunophenotyping, TDAR analysis, organ weights and measurements, microscopic examinations, and neuropathologic and brain morphometric examinations. F1 animals were further subdivided into cohorts following weaning and specifically evaluated for the following: Cohort 1 (A and B) for reproductive/developmental toxicity, Cohort 2 (A and B) for developmental neurotoxicity, and Cohort 3 (and 3A) for immunotoxicity testing. Animals assigned to Cohort 1A were evaluated on PND 91 (including estrous cycles and sperm evaluations); Cohort 1B animals were maintained on study for the assessment of reproductive performance and to generate F2 offspring, which was not warranted, and all animals were evaluated on PND 120. Animals assigned to Cohort 2B were evaluated on PND 22 (brain morphometry); Cohort 2A animals were maintained on study until PND 78 for neurobehavioral testing and for evaluation of adult neuropathology. Animals assigned to Cohort 3A served as a positive control group for the sRBC challenge beginning on PND 54, and for comparison versus Cohort 3, which was evaluated on PND 59 (TDAR analysis).


 


F0 Generation: There were no test substance-related mortalities. One male in the 200 mg/kg/day group was found dead during dosing on Study Day 52; necropsy findings for this male included red fluid in the thoracic cavity; yellow fluid in the abdominal cavity; dark red discoloration of the small intestine and thymus gland; and a swollen liver. One female in the 500 mg/kg/day group was found dead following a dosing error on Lactation Day 3 following an observation of labored respiration; a perforated esophagus and red fluid in the thoracic cavity were noted at necropsy. Although the cause of death for the male was undetermined, the macroscopic findings were consistent with a possible dosing error. All other F0 males and females survived to the scheduled necropsy. No test substance-related observations were noted at the detailed or cage-side observations at any dose level. There were no test substance-related effects on mean absolute body weights, body weight gains, food consumption, or food efficiency for F0 males during the pre- and postmating periods or for F0 females during the pre-mating, gestation, and lactation periods at any dose level. There were no test substance related effects on F0 estrous cyclicity, precoital intervals, reproductive performance (male and female mating and fertility, male copulation, and female conception indices), gestation lengths, and the process of parturition. Mean numbers of implantation sites and unaccounted-for sites were comparable across groups. No test substance-related effects on F0 sperm parameters (mean epididymal sperm numbers, motility, progressive motility, and morphology) were noted at any dose level. No test substance-related effects were noted on thyroid hormone concentrations for F0 males and females in the 50, 200, and 500 mg/kg/day groups. There were no test substance-related effects on hematology, coagulation, clinical chemistry, or urinalysis for F0 males and females at any dose level. There were no test substance-related macroscopic or microscopic findings noted at the scheduled necropsy for the F0 generation. Higher mean F0 liver weights (absolute and relative to final body and brain weight) in the 200 mg/kg/day males and 500 mg/kg/day males and females were noted compared to the control group. The higher liver weight was suspected to be associated with non-adverse metabolic enzyme induction. Higher mean kidney weight (absolute and relative to final body and brain weight) was noted in the 200 and 500 mg/kg/day F0 males compared to the control group, which was of undefined specific pathogenesis but suspected to be associated with the higher liver weight.


 


F1 Generation: The mean number of pups born, live litter size, or percentage of males per litter at birth in the 50, 200, and 500 mg/kg/day groups and postnatal survival in the 50 and 200 mg/kg/day groups were comparable to the control group. Lower postnatal survival was noted in the 500 mg/kg/day group during PND 0–1, resulting in lower mean postnatal survival from birth to PND 4, which was considered to be test substance related and adverse. A slight increase in the incidence of cool body was noted in 2 litters in the 500 mg/kg/day group. No other noteworthy observations were noted in F1 pups. Mean male and female birth weights (PND 1) in the 500 mg/kg/day group were marginally lower (5.6% and 5.8%, respectively) than the control group. Mean body gains in these pups were comparable to the control group throughout the preweaning period, such that mean body weights thereafter were comparable through PND 21. There were no test substance-related effects on mean F1 pup weights in the 50 and 200 mg/kg/day groups. There were no test substance related clinical observations or effects on absolute and relative anogenital distances for F1 males and females pups at any dose level. No retained nipples/areolae were noted for F1 male pups at any dose level on PND 13. There were no test substance–related effects on mean thyroid hormone concentrations, macroscopic findings, and/or effects on organ weights noted for F1 pups that died, or for PND 4 (culled) or PND 21 (non-selected or selected for hormone analysis) pups that could be attributed to F0 parental test substance administration. Following weaning and initiation of direct test substance administration on PND 21, there were no test substance-related effects on survival in the F1 generation. One male and 1 female each in the control and 200 mg/kg/day groups were found dead within 2 weeks of weaning (PND 25-34). No clinical observations or necropsy findings were noted for the animals in the 200 mg/kg/day group that were found dead. One female in the control group that was found dead had a swollen head for several days prior to death and veterinary observations that included slightly uncoordinated gait, mildly splayed hindlimb, and moderate firm cranial swelling. At necropsy, dilated ventricles of the brain and dark red contents in the cranial cavity were noted. All other F1 males and females survived to the scheduled necropsy following weaning. No test substance-related clinical observations were noted at the detailed or cage-side observations throughout the generation at any dose level. There were no test substance-related effects on mean absolute body weights, body weight gains, food consumption, and food efficiency for F1 males and females in the 50, 200, and 500 mg/kg/day group F1 males and females. The mean ages of attainment for balanopreputial separation and vaginal patency and the mean body weights at the age of attainment for F1 males and females, respectively, in the 50, 200, and 500 mg/kg/day groups were unaffected by test substance exposure.


In F1 Cohort 1A females, there were no test substance-related effects on the mean age at first estrus or the mean duration from vaginal opening to first estrus, mean estrous cycle length, and the percentage of females cycling regularly. There were no test substance-related effects on sperm parameters for F1 Cohort 1A males. Generally dose-responsive lower mean T4 levels were noted in Cohort 1A in the 50, 200, and 500 mg/kg/day group males and the 200 and 500 mg/kg/day group females compared to the control group. Higher mean TSH levels were noted in males and females in all groups compared to the control group; however, a dose-response relationship was noted only in males. Given the lack of effects on thyroid gland weights and histopathology or evidence of thyroid disruption-related alterations on the brain or neurobehavior, the differences in mean T4 levels noted above were considered incidental and not biologically significant to influence downstream changes in organ system development or histopathology. There were no test substance-related effects on hematology, coagulation, clinical chemistry, and urinalysis parameters for F1 Cohort 1A males and females on PND 91. There were no test substance-related effects on auditory startle response, FOB, or locomotor activity noted for F1 Cohort 2A males and females at any dose level. There were no test substance-related macroscopic or microscopic findings noted at the scheduled necropsies for animals assigned to Cohorts 1A and 1B. No test substance related effects on ovarian follicle count were noted for F1 Cohort 1A females at any dose level. On PND 22 and 78, there were no test substance-related effects on mean brain weights and measurements or macroscopic findings at any dose level for Cohort 2A and 2B animals; brain morphometry was also unaffected by test substance administration for F1 Cohort 2A animals. Mean final body weights in the 500 mg/kg/day group F1 Cohort 1A males selected for organ weights were 9.8% lower than the control group. However, mean final body weights for the entire group on PND 91 were comparable to the control group, and the difference was attributed to the specific subset of animals selected for organ weights, and unrelated to test substance administration. Mean liver weights (absolute and relative to final body and brain weight) in the 500 mg/kg/day group females and mean liver weight relative to final body weight in the 500 mg/kg/day group males were higher than the control group. Although the interpretation of liver weights in the 500 mg/kg/day males was complicated by body weight decrement, the constellation of liver weight changes in F1 males and females suggested a minor increase in liver weight associated with test substance administration. The higher liver weight was suspected to be associated with a non-adverse metabolic adaptation such enzyme induction. In addition, the overall presentation of F1 Cohort 1A organ weight, microscopic, and clinical laboratory data suggest a non-adverse test substance-associated decrement in thymus and spleen weights of 500 mg/kg/day males that was of uncertain specific genesis but possibly related to the lower final body weights for F1 Cohort 1A males selected for organ weights. Test substance administration elicited no changes in splenic immunophenotyping parameters at any dose level in the F1 Cohort 1A animals evaluated on PND 91. For animals assigned to F1 Cohort 3, there were no test substance-related effects on the humoral immune response in a T cell-mediated antibody response assay. Taken together, it was concluded that the test substance did not alter splenic cell subsets and did not exert an inhibitory effect on the humoral immune system.


 


Based on lack of any adverse effects on the parameters evaluated, a dose level of 500 mg/kg/day was considered to be the NOAEL (no observed adverse effect level) for F0 reproductive toxicity and F0 and F1 systemic toxicity. Based on the lower postnatal pup survival between birth and PND 4 at a dose level of 500 mg/kg/day, 200 mg/kg/day was considered to be the NOAEL for F1 neonatal and developmental toxicity. Based on the lack of any adverse effects on F1 thyroid hormones, neurobehavior, neuropathology or brain morphometry or immunotoxicity parameters, a dose levels of 500 mg/kg/day, the highest dose level tested was considered to be the NOAEL for F1 neurotoxicity, and F1 immunotoxicity of the test substance when administered orally to Crl:CD(SD) rats.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Species:
rat
Quality of whole database:
GLP Guideline study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a GLP study conducted according to OECD Guideline 443, male and female rats, 25/sex/dose, were exposed to 0, 50, or 200 mg/kg, and 30/sex/dose were exposed to 500 mg/kg. F0 animals were dosed via oral gavage daily for 70 consecutive days prior to mating and continuing through the day prior to euthanasia. The F1 generation was potentially exposed to the test substance in utero and through nursing during lactation. Following weaning, the offspring selected to become the F1 generation were dosed beginning at weaning (PND 21) and continuing until euthanasia (as appropriate for the relevant cohort). The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, pre- and postweaning developmental landmarks, neurobehavior, thyroid hormones, clinical pathology, macroscopic findings, sperm parameters, immunophenotyping, TDAR analysis, organ weights and measurements, microscopic examinations, and neuropathologic and brain morphometric examinations. F1 animals were further subdivided into cohorts following weaning and specifically evaluated for the following: Cohort 1 (A and B) for reproductive/developmental toxicity, Cohort 2 (A and B) for developmental neurotoxicity, and Cohort 3 (and 3A) for immunotoxicity testing. Animals assigned to Cohort 1A were evaluated on PND 91 (including estrous cycles and sperm evaluations); Cohort 1B animals were maintained on study for the assessment of reproductive performance and to generate F2 offspring, which was not warranted, and all animals were evaluated on PND 120. Animals assigned to Cohort 2B were evaluated on PND 22 (brain morphometry); Cohort 2A animals were maintained on study until PND 78 for neurobehavioral testing and for evaluation of adult neuropathology. Animals assigned to Cohort 3A served as a positive control group for the sRBC challenge beginning on PND 54, and for comparison versus Cohort 3, which was evaluated on PND 59 (TDAR analysis). 


Based on lack of any adverse effects on the parameters evaluated, a dose level of 500 mg/kg/day was considered to be the NOAEL (no observed adverse effect level) for F0 reproductive toxicity and F0 and F1 systemic toxicity. Based on the lower postnatal pup survival between birth and PND 4 at a dose level of 500 mg/kg/day, 200 mg/kg/day was considered to be the NOAEL for F1 neonatal and developmental toxicity. Based on the lack of any adverse effects on F1 thyroid hormones, neurobehavior, neuropathology or brain morphometry or immunotoxicity parameters, a dose levels of 500 mg/kg/day, the highest dose level tested was considered to be the NOAEL for F1 neurotoxicity, and F1 immunotoxicity of the test substance when administered orally to Crl:CD(SD) rats.

Effects on developmental toxicity

Description of key information

Developmental Toxicity; Rat oral; OECD Guideline 414; Reliability = 1


Developmental Toxicity; Rabbit oral; OECD Guideline 414; Reliability = 1

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Principles of method if other than guideline:
The objective of the study was to determine dose levels of test substance to be evaluated in a definitive prenatal developmental toxicity study in rabbits when administered via oral gavage.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
Purity: 98.3%
Species:
rabbit
Strain:
New Zealand White
Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose (400 cps) and 0.1% Cremophor EL in deionized water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and concentration analyses were performed.
Details on mating procedure:
Animals were received time-mated.
Duration of treatment / exposure:
Days 7-28 of gestation
Frequency of treatment:
daily
Duration of test:
Animals were sacrificed on Day 29G
Dose / conc.:
800 mg/kg bw/day
Dose / conc.:
700 mg/kg bw/day
Dose / conc.:
600 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
6 females
Control animals:
yes, concurrent vehicle
Maternal examinations:
Parameters examined included mortality, clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, and macroscopic examinations.
Fetal examinations:
Parameters examined included intrauterine growth and survival and external fetal morphology.
Statistics:
Numerical data and clinical and necropsy observation data were summarized by sex and occasion or by litter. Statistical analyses were performed on body weight, body weight gains, food consumption, ovarian and uterine content, gravid uterine weight and adjusted maternal body weights, litter means, and litter % of fetuses with gross/external abnormalities.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical observations noted for females that were moribund or found dead included decreased fecal output, thin body condition, labored respiration, splayed hindlimbs, decreased activity, lying on side, and/or body cold to touch. In surviving animals, decreased fecal output was noted for 1 female each in the 500, 600, and 700 mg/kg/day groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
Test substance-related moribundity/mortality was observed at ≥ 600 mg/kg/day as evidenced by 1, 1, and 3 females, respectively, in the 600, 700, and 800 mg/kg/day groups that were found dead, aborted, or euthanized in extremis during Gestation Days 11–24, all with similar manifestation of toxicity. Based on the moribundity/abortion/mortality and mean body weight losses with reduced food consumption for the majority of the surviving females at 800 mg/kg/day, this level was considered to have exceeded the maximum tolerated dose in rabbits and the remaining animals in this group were euthanized due to early group termination on Gestation Day 22, precluding further evaluation at this dose level. All other animals survived to the scheduled necropsy on Gestation Day 29.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Moribund/found dead females exhibited body weight losses leading up to death/abortion/euthanasia.

Lower mean body weight gains and/or body weight losses were noted in the 700 mg/kg/day group during Gestation Days 7–20. As a result, mean absolute body weights were 9.9% lower than the control group on Gestation Day 20. Mean body weight gains in this group were higher during Gestation Days 20–29, resulting in a slightly lower (5.3%) mean absolute body weight on Gestation Day 29. Slightly lower mean body weight gains were also noted in the 500 and 600 mg/kg/day groups, compared to the control group, generally throughout the treatment period (Gestation Days 7–29); mean absolute body weights in these groups were generally comparable to the control group throughout the study. Mean gravid uterine weights, adjusted body weights, and adjusted body weight gains were lower compared to the control group at 500, 600, and 700 mg/kg/day but did not occur in a clear dose-responsive manner.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Moribund/found dead females exhibited reduced food consumption leading up to death/abortion/euthanasia.

Lower food consumption was noted in the 700 mg/kg/day group during Gestation Days 7–20. During Gestation Days 20–29 mean food consumption remained lower than the control group. Slightly lower mean food consumption was also noted in the 500 and 600 mg/kg/day groups, compared to the control group, generally throughout the treatment period (Gestation Days 7–29).
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the scheduled necropsy on Gestation Day 29, the only remarkable macroscopic finding was
dark, red foci in the lungs of a single female in the 700 mg/kg/day group.
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
One female aborted at 800 mg/kg.
Pre- and post-implantation loss:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
Based on moribundity, mortality, abortion, clinical observations, and maternal body weight losses with corresponding severely reduced food consumption at 800 mg/kg/day and mortality, maternal body weight losses with reduced food consumption at 700 mg/kg/day, dose levels of 100, 350, and 650 mg/kg/day were selected for a definitive prenatal developmental toxicity study in rabbits.
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean fetal body weights in the 500, 600, and 700 mg/kg/day groups were lower (10.31% to 13.25%) than the control group. However, the differences in mean fetal weights were not dose-responsive and all groups, including controls, were below the minimum mean value in the historical control data for definitive studies. As such, mean fetal body weights were deemed inconclusive in terms of assisting with dose level selection for the definitive study.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Remarks on result:
not determinable
Remarks:
Mean fetal weights changes were deemed inconclusive in assisting with dose level selection for the definitive study. Intraueterine survival was unaffected by test substance administration at 500, 600, or 700 mg/kg/day, and there were no external fetal abnormalities observed.
Conclusions:
Based on moribundity, mortality, abortion, clinical observations, and maternal body weight losses with corresponding severely reduced food consumption at 800 mg/kg/day and mortality, maternal body weight losses with reduced food consumption at 700 mg/kg/day, dose levels of 100, 350, and 650 mg/kg/day were selected for a definitive prenatal developmental toxicity study of the test substance when administered orally by gavage to time-mated New Zealand White rabbits.
Executive summary:

The objective of this study was to determine dose levels of the test substance to be evaluated in a definitive prenatal developmental toxicity study in rabbits when administered via oral gavage. Female rabbits (6/dose) were exposed to the test material at doses of 0, 500, 600, 700, or 800 mg/kg/day. Animals were dosed via oral gavage once daily during Gestation Days 7–28. The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, macroscopic examinations, intrauterine growth and survival, and external fetal morphology. The analyzed dosing formulations were within the protocol-specified range of target concentrations for suspensions and were homogeneous. Test substance-related moribundity/mortality was observed at ≥ 600 mg/kg/day as evidenced by 1, 1, and 3 females, respectively, in the 600, 700, and 800 mg/kg/day groups that were found dead, aborted, or euthanized in extremis during Gestation Days 11–24, all with similar manifestation of toxicity. Clinical observations noted for these females included decreased fecal output, thin body condition, labored respiration, splayed hindlimbs, decreased activity, lying on side, and/or body cold to touch, as well as body weight losses and reduced food consumption leading up to death/abortion/euthanasia. Macroscopic findings of gallbladder with dark, green contents, dark, red foci in the lungs, fluid filled cysts in the oviduct, and/or accessory spleen were noted in 2 females at 800 mg/kg/day and enlarged gallbladder with gritty contents was noted in the female at 600 mg/kg/day at necropsy examination. Based on the moribundity/abortion/mortality and mean body weight losses with reduced food consumption for the majority of the surviving females at 800 mg/kg/day, this level was considered to have exceeded the maximum tolerated dose in rabbits and the remaining animals in this group were euthanized due to early group termination on Gestation Day 22, precluding further evaluation at this dose level. All other animals survived to the scheduled necropsy on Gestation Day 29. In surviving animals, decreased fecal output was noted for 1 female each in the 500, 600, and 700 mg/kg/day groups. Lower mean body weight gains and/or body weight losses, with correspondingly lower mean food consumption, were noted in the 700 mg/kg/day group during Gestation Days 7–20. As a result, mean absolute body weights were 9.9% lower than the control group on Gestation Day 20. Mean body weight gains in this group were higher during Gestation Days 20–29 while mean food consumption remained lower than the control group, resulting in a slightly lower (5.3%) mean absolute body weight on Gestation Day 29. Slightly lower mean body weight gains and food consumption were also noted in the 500 and 600 mg/kg/day groups, compared to the control group, generally throughout the treatment period (Gestation Days 7–29); mean absolute body weights in these groups were generally comparable to the control group throughout the study. Mean gravid uterine weights, adjusted body weights, and adjusted body weight gains were lower compared to the control group at 500, 600, and 700 mg/kg/day but did not occur in a clear dose-responsive manner. At the scheduled necropsy on Gestation Day 29, the only remarkable macroscopic finding was dark, red foci in the lungs of a single female in the 700 mg/kg/day group. Mean fetal body weights in the 500, 600, and 700 mg/kg/day groups were lower (10.31% to 13.25%) than the control group. However, the differences in mean fetal weights were not dose-responsive and all groups, including controls, were below the minimum mean value in the historical control data for definitive studies. As such, mean fetal body weights were deemed inconclusive in terms of assisting with dose level selection for the definitive study. Intrauterine survival was unaffected by test substance administration in the 500, 600, and 700 mg/kg/day groups, and there were no external fetal abnormalities noted in fetuses in this study. Based on moribundity, mortality, abortion, clinical observations, and maternal body weight losses with corresponding severely reduced food consumption at 800 mg/kg/day and mortality, maternal body weight losses with reduced food consumption at 700 mg/kg/day, dose levels of 100, 350, and 650 mg/kg/day were selected for a definitive prenatal developmental toxicity study of the test substance when administered orally by gavage to time-mated New Zealand White rabbits.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Purity: 98.3%
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation (Gestation Day 0): 11 to 13 weeks
- Weight at study initiation (Gestation Day 0): 229 to 289 g
- Animal Identification: Each animal was identified using a subcutaneously implanted electronic identification chip
- Housing: Individual; Solid bottom cages containing appropriate bedding material (Bed-O-Cobs® or other suitable material)
- Cage Identification: Individual (color-coded) cage cards were affixed to each cage and displayed at least the animal number(s), group number, dose level, study number, and sex of the animal
- Animal Enrichment: For enrichment, animals were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities
- Diet (e.g. ad libitum): Ad libitum; PMI Nutrition International, LLC Certified Rodent LabDiet® 5002; Results of analysis for nutritional components and environmental contaminants is provided by the supplier; It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Ad libitum, via an automatic watering system. Water bottles were provided, if required; Municipal tap water, treated by reverse osmosis and ultraviolet irradiation Periodic analyses of the water are performed; It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: Arrival to GD6; GD0 - GD6; Time-mated rats were received by Charles River on Gestation Days 1, 2, 3 and 4. The day on which confirmation of mating was made was designated Gestation Day 0.
- Animal Disposition: Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights.

JUSTIFICATION FOR TEST SYSTEM & NUMBER OF ANIMALS
-Test System: The Crl:CD(SD) rat has been recognized to be appropriate for developmental toxicity studies. Charles River has historical data on the background incidence of fetal malformations and developmental variations in this species. This animal model has been proven to be susceptible to the effects of developmental toxicants.
- Number of Animals: The number of animals is based on the US EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Development Toxicity Study, Aug 1998, and the OECD Guidelines for the Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, Jan 2018, which recommend evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or treatment-related moribundity and/or mortality, 25 females/group is an appropriate number of animals to obtain a sample size of 20 at termination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68°F to 77°F (20°C to 25°C)
- Humidity (%): 30% to 70%
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: From: 19 Jul 2021 To: 06 Aug 2021
Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose (400 cps) and 0.1% Cremophor EL in deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dose formulations were divided into aliquots where required to allow them to be dispensed on each dosing occasion. Dosing formulations were prepared (approximately weekly) at appropriate concentrations to meet dose level requirements. The prepared formulations were not adjusted for purity. Storage conditions were set to maintain a target temperature of 5°C

VEHICLE: 0.5% methylcellulose (400 cps) and 0.1% Cremophor EL in deionized water
- Concentration in vehicle: 0.5% methylcellulose (400cps); 0.1% Cremophor EL
- Amount of vehicle (if gavage): 10 mL/kg
- Storage Conditions: Set to maintain a target temperature of 5°C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Doses were analyzed via high performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV).

The analyzed dosing formulations contained 77.1% to 105.9% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85% to 115%) and were homogeneous, with the following exceptions. On the first day of formulation sample analysis (16 Jul 2021), Group 2 and 3 formulations failed to meet protocol specified acceptability criteria for concentration. However, further investigations of formulations prepared on 19 Jul 2021 revealed that additional stirring time during formulation preparation resulted in concentrations that met acceptance criteria. Furthermore, analysis of stored aliquots from the 16 Jul 2021 formulations, for Groups 2 and 3, following increased stir time, met protocol specified acceptance criteria for concentration acceptability thereby confirming that the initial low results was due to inadequate stirring of the bulk formulations. For all subsequent formulations during the study, the stir time was increased to 2 hours before sample collection, analysis and preparation of aliquots for dose administration. All subsequent formulations met protocol-specified acceptance criteria for concentration acceptability.
Details on mating procedure:
Time-mated rats were received by Charles River on Gestation Day 1, 2, 3 or 4. The day on which confirmation of mating was designated Gestation Day 0.
Duration of treatment / exposure:
GD 6 - 20
Frequency of treatment:
Single daily dose
Duration of test:
Terminal sacrifice on GD 21
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
350 mg/kg bw/day
Dose / conc.:
650 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral route of exposure was selected because this a possible route of human exposure. In a GLP-compliant study conducted according to OECD Test Guideline 422, at doses of 30, 100, 300, and 1000 mg test substance/kg/day, via oral gavage, clear evidence of maternal and developmental toxicity was evidenced at the highest dose (1000 mg/kg/day) as reduced maternal body weights and increased fetal toxicity. At 100 and 300 mg/kg/day, maternal effects were minimal, and there were no effects on embryo/fetal or pup survival or pup body weights. The results obtained for the test substance were consistent with other acute and repeated-dose studies and were hence considered to be representative of the different grades of the test substance.
In a previous dose range-finding prenatal developmental toxicity study conducted at Charles River, the test substance was administered via oral gavage to pregnant female rats at dose of 500, 600, 700 and 800 mg/kg/day. Test substance related clinical signs, maternal body weight losses and reduced food consumption, and a significant reduction in mean fetal weights, without concurrent effects on external fetal morphology were noted at 800 mg/kg/day. At lower doses, similar effects were observed, however, with much lower severity.
Based on these data, dosage levels of 100, 350 and 650 mg/kg/day were selected for the current study. It is anticipated that the high dose will show some maternal effects but not produce an incidence of fatalities that would prevent a meaningful evaluation of test substance related effects. The lower dosage levels were selected to identify potential dose response relationships.

- Rationale for animal assignment (if not random): Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily; conducted prior to dosing on days of scheduled dosing; approximately 1 hr post-dose

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Gestation Days: 5, 6, 9, 12, 15, 18, 21

MORTALITY: Yes
-Time schedule: At least twice daily (morning and afternoon), beginning upon arrival through termination/release; (except on days of receipt and necropsy where frequency was at least once daily)

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation Days: 0 (by supplier), 5, 6, 9, 12, 15, 18, and 21 (not collected from animals found dead)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Method of Euthanasia: Animals euthanized for humane reasons or at study termination were euthanized by carbon dioxide inhalation.
- Unscheduled Deaths: If necessary for humane reasons, animals were euthanized as per Testing Facility SOPs. These animals underwent necropsy, and specified tissues were retained. The number and location of implantation sites, corpora lutea, and viable fetuses were recorded. Recognizable fetuses were examined externally, euthanized (if necessary), and preserved in 10% neutral buffered formalin.
- Scheduled Euthanasia: Main study animals surviving until scheduled euthanasia were weighed and euthanized by carbon dioxide inhalation.
- Sacrifice on gestation day # 21
- Organs examined: Liver and thyroid gland were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals euthanized in poor condition or in extremis. Representative samples of the liver and thyroid gland (gross lesions when possible) were collected from all animals and preserved in 10% neutral buffered formalin. Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible. The thyroid and liver from all animals in all groups were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. The thyroid and liver were examined microscopically from all females in all groups.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
Thyroid Sample Hormone Collection:
- Method/Route of Collection: Jugular vein using the hand-held restraint method
- Target Volume: Approximately 1.0 mL/time point collected
- Special Requirements: Collected prior to noon on each day of collection, around the same time each day, and within a 2-hour window on each collection day, to reduce variability due to normal diurnal variations in physiological levels of thyroid hormones. Blood collections were performed in animal ante room, the necropsy laboratory, or as far away from live animals as possible to minimize stress-induced hormone fluctuations.
Thyroid Hormone Sample Processing: Samples were allowed to clot at ambient temperature for a minimum of 30 minutes before centrifugation. All samples were centrifuged (approximately 3000 rpm [approximately 2056xg] for approximately 10 min) at approximately 4°C. The maximum amount of serum was recovered and transferred into 2 new, uniquely labeled polypropylene tubes. Approximately 150 µL of the resultant serum was placed in 1 tube to be used for total T3 and T4 hormone analysis and approximately 100 µL was placed in a separate tube for TSH analysis. Samples were stored in a freezer set to maintain a target of -70°C.

Thyroid Hormone Sample Analysis: Samples to determine total T3 and T4 concentrations were analyzed using a validated UHPLC/MS/MS assay. Samples for TSH determination were analyzed using a validated Luminex Bead-Based (TSH) assay.
Fetal examinations:
FETAL EXAMINATIONS: Fetal examinations were conducted without knowledge of treatment group. External, internal, and skeletal fetal findings were recorded as either developmental variation (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal), malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life), or incidental (minor changes in coloration, mechanical damage to specimen, etc.). Representative photographs of all malformations, as appropriate, were included in the Study Records. Corresponding low magnification photographs, depicting both the malformed fetus and a comparison control fetus or normal littermate, were also included in the Study Records as needed and as appropriate for comparison, when possible.

- External examinations: Yes: [all per litter]
Each viable fetus was examined in detail, sexed, weighed, tagged, and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. Following euthanasia, anogenital distance was measured for all viable fetuses. The absolute and normalized (relative to the cube root of fetal body weight) values were reported. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed (if possible), and the tissue was discarded.

- Soft tissue examinations: Yes: [approximately half per litter]
The sex of all fetuses was confirmed by internal examination. Approximately one-half of the fetuses in each litter were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. Following examination, the carcasses and cephalic slices were discarded.

- Skeletal examinations: Yes: [approximately half per litter]
The remaining fetuses (approximately one-half from each litter, excluding any carcasses without heads) were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, fetuses were macerated in potassium hydroxide and stained with Alizarin Red S and Alcian Blue. The skeletal examination was made following this procedure.

- Head examinations: Yes: [approximately half per litter]
The heads from these fetuses examined for visceral alterations were removed and placed in Harrison’s fixative for subsequent processing and soft-tissue examination using the Wilson sectioning technique.
Statistics:
Numerical data and clinical and necropsy observations data were summarized by sex and occasion or by litter. Body Weight Gains were calculated between each scheduled interval as well as between GD 6-21. Food Consumption was calculated for each corresponding body weight interval.

Means, standard deviations, percentages, numbers, and/or incidences were reported, as appropriate by dataset. Data from nongravid females were excluded from calculation of means and from comparative statistics.

All statistical tests were conducted at the 5% significance level. All pairwise comparisons (vs. control) were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted. Body weight, body weight gains, food consumption, thyroid hormone data, gravid uterine weight and adjusted maternal body weights, organ weights, litter means, and anogenital distance were analyzed by the parametric/non-parametric methods listed below. Ovarian and uterine content and litter % of fetuses with gross/external/visceral/skeletal abnormalities were analyzed by the non-parametric methods listed below.

Parametric/Non-Parametric: Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Non-Parametric: The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.
Indices:
The following parental indices and litter calculations were included, where applicable:

- Pre-Implantation Loss = ((No. of corpora lutea - No. of implants)/No. of corpora lutea) x 100

- Post-Implantation Loss = ((No. of implants - No. of live fetuses)/No. of implants) x 100

- Sex Ratio (% Males) = (No. of male fetuses/Total No. of fetuses) x 100

- Litter % of Fet. with Abnor. = (No. of fet. in litter with a finding/No. of fet. in litter examine) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
There were no significant test substance-related clinical observations noted at any dose level. Observations noted in the test substance-treated groups, including fur staining or thin fur cover on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female in the 650 mg/kg/day group was euthanized in extremis on Gestation Day 12 following body weight losses (26.4% of Gestation Day 6 body weight), and severely reduced food consumption (≤ 1 g/animal/day during Gestation Days 6-12) and clinical observations of hunched posture (Gestation Days 9 and 12), erected fur (Gestation Day 12), decreased activity (Gestation Days 9 and 10), and thin (Gestation Days 9 and 12). At necropsy, there were no gross observations, and the female was gravid. Other tissues were not examined microscopically given the lack of gross findings. A microscopic cause of moribundity and subsequent euthanasia could not be determined. All other animals survived to scheduled euthanasia.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, statistically significant mean maternal body weight losses were noted in the 650 mg/kg/day group immediately following the initiation of treatment (Gestation Day 6-9). Thereafter, mean body weight gains were generally comparable to or slightly lower than the control group throughout the remainder of the treatment period (Gestation Days 9-21). Consequently, mean body weight gains were significantly lower than the control group when the overall treatment period (Gestation Days 6-21) was evaluated and mean absolute body weights in the 650 mg/kg/day group were 8.8% lower than the control group at scheduled termination on Gestation Day 21; differences from the control group attained statistical significance generally throughout the treatment period. Test substance-related lower mean gravid uterine weight, adjusted body weight, and adjusted body weight gain were noted for females in this group; the differences in adjusted body weight and adjusted body weight gain were statistically significant. Mean maternal body weights, body weight gains, adjusted body weights, adjusted body weight gains, and gravid uterine weights in the 100 and 350 mg/kg/day groups were unaffected by test substance administration. Other differences from the control group were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean maternal food consumption, evaluated as g/animal/day, in the 650 mg/kg/day group was statistically significantly lower than the control group following the initiation of dose administration (Gestation Days 6-9). This was considered test substance-related and corresponded to the mean body weight loss noted for these females. Thereafter, mean food consumption in this group was generally comparable to or slightly lower than the control group throughout the remainder of the dosing period (Gestation Days 9-21). Consequently, mean food consumption was significantly lower than the control group when the overall treatment period (Gestation Days 6-21) was evaluated.
Mean maternal food consumption, evaluated as g/animal/day, in the 100 and 350 mg/kg/day groups was unaffected by test substance administration. Mean food consumption in the 350 mg/kg/day group was statistically significantly lower than the control group during Gestation Days 6-9; however, this was transient and did not affect mean body weight gains. Other differences from the control group were slight and not statistically significant.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related changes in serum T4; however, serum T3 levels were 9.2%, 9.0%, and 15.5% lower than the control group in the 100, 350, and 650 mg/kg/day groups. Correspondingly, TSH levels were 54.4% and 49.8% higher than the control group in the 350 and 650 mg/kg/day groups. The lower T3 levels are consistent with microsomal enzyme induction due to hypertrophy in the liver. Given the lack of a clear dose response, and the lack of any differences in T4 levels, these differences were also considered adaptive and non-adverse.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related changes were observed in the liver and thyroid in the 650 mg/kg/day group. Mean absolute liver and thyroid/parathyroid weights were slightly elevated in the 650 mg/kg/day group when compared to the control group. The percent differences for liver and thyroid/parathyroid in all test substance-treated groups from the control group did not show a clear dose-response. However, there was a test substance-related statistically significantly lower mean body weight in the 650 mg/kg/day group. Both liver and thyroid weights show a linear relationship to body weight and should mirror changes in body weight, therefore, liver and thyroid weights relative to body weight are the best endpoint and slight elevations in absolute values would be toxicologically relevant. The rats were pregnant at the time of necropsy, so calculating organ weights relative to body weight would have had too much variability. The liver and thyroid/parathyroid weight elevations in the 650 mg/kg/day group in the face of body weight loss with correlating hepatocellular hypertrophy (liver) and follicular cell hypertrophy (thyroid) were considered test substance related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat and, therefore, were considered unrelated to administration of the test substance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related findings were observed in the liver of the 350 and 650 mg/kg/day groups and the thyroid of 100, 350, and 650 mg/kg/day groups.

The liver finding of centrilobular hepatocellular hypertrophy was noted in the 350 and 650 mg/kg/day group with a dose-response in incidence and severity. Minimal (350 and 650 mg/kg/day) to mild (650 mg/kg/day) hypertrophy was characterized by enlargement of hepatocytes with glassy, eosinophilic cytoplasm that was confined to the centrilobular area in minimal hypertrophy and extended into the midzonal area in mild hypertrophy. Hypertrophy correlated with higher absolute liver weight in the 650 mg/kg/day group.

In the thyroid, follicular cell hypertrophy was noted in the 100 (minimal to mild), 350 (minimal to moderate), and 650 (minimal to mild) mg/kg/day groups. Minimal to mild changes were characterized by enlargement of thyroid follicular cells with cytoplasmic vacuolation with cellular enlargement greater in mild findings. Moderate follicular cell hypertrophy was noted in a single female in the 350 mg/kg/day group with enlarged, vacuolated follicular cells that often-showed piling. Follicular cell hypertrophy correlated with higher absolute thyroid/parathyroid weight in the 650 mg/kg/day group.

Hypertrophy in the liver and/or thyroid gland was consistent with microsomal enzyme induction and was considered an adaptive response and non-adverse given the severity of changes and lack of test substance-related hepatocellular injury.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean fetal weights (male/female/combined) in the 650 mg/kg/day group were 6.476% to 6.740% lower than the control group; differences were statistically significant. Mean values were below the ranges of values in the historical control data and were hence considered substance related but non-adverse, based on the lower magnitude of the difference versus controls.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Intrauterine survival in the 650 mg/kg/day group was unaffected by test substance administration. A slightly higher (not statistically significant) mean litter proportion of post implantation loss was noted for females in this group; however, the difference was partially attributed to a single female with 80% post implantation loss and the mean value was within the range of values in the historical control data.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The numbers of fetuses (litters) available for morphological evaluation were 297(25), 299(24), 294(25), and 248(23) in the control, 100, 350, and 650 mg/kg/day groups, respectively.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
Mean anogenital distance in the 650 mg/kg/day group was comparable to the control group.
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related external malformations were noted at any dose level. Omphalocele (portion of intestine protruded with remnants of a membranous sac) was noted for one fetus in the 100 mg/kg/day group. However, this was only noted for a single fetus and was not noted in a dose-related manner.
No external developmental variations were observed in fetuses in this study.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the historical control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the historical control data.
Key result
Dose descriptor:
NOAEL
Effect level:
650 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no






































































































































































































Summary of Maternal Performance and Mortality



Sex: Female


 


Days(s) Relative to Mating (Litter: A)



0 mg/kg/day


 


Group 1



100 mg/kg/day


 


Group 2



350 mg/kg/day


 


Group 3



650 mg/kg/day


 


Group 4



Group Size - Females



 



25



25



25



25



Number of Females Pregnant



N+ve



25



24



25



24



 



%



100.0



96.0



100.0



96.0



Females with Live Fetuses



N+ve



25



24



25



24



 



%



100.0



100.0



100.0



100.0



Female with Resporptions



N+ve



8



9



13



9



 



%



32.0



37.5



52.0



37.5



Female with all Nonviable



N+ve



0



0



0



0



 



%



0.0



0.0



0.0



0.0



Placenta exam Normal



N+ve



25



23



25



23



 



%



100.0



95.8



100.0



100.0



Terminal Euthanasia



N+ve



25



25



25



24



 



%



100.0



100.0



100.0



96.0



Unscheduled Death/Euthanasia



N+ve



0



0



0



1



 



%



0.0



0.0



0.0



4.0



Found Dead



N+ve



0



0



0



0



 



%



0.0



0.0



0.0



0.0



Unscheduled Euthanasia



N+ve



0



0



0



1



 



%



0.0



0.0



0.0



4.0



Aborted



N+ve



0



0



0



0



 



%



0.0



0.0



0.0



0.0



Delivered



N+ve



0



0



0



0



 



%



0.0



0.0



0.0



0.0



 



















































































































































































































































































































































































































































































































































































































































































Summary of Ovarian and Uterine Examinations and Litter Observations



Sex: Female


 


Days(s) Relative to Mating (Litter: A)



0 mg/kg/day


 


Group 1



100 mg/kg/day


 


Group 2



350 mg/kg/day


 


Group 3



650 mg/kg/day


 


Group 4



Females with Live Fetuses



N+ve



25



24



25



23



 



%



100.0



100.0



100.0



100.0



Number of Corpora Lutea [k]



Mean



15.5



15.7



14.5



15.0



 



SD



2.7



2.5



2.8



2.9



 



N



25



24



25



23



 



%Diff



-



1.5



-6.2



-3.4



Number of Implantations [k]



Mean



12.3



13.3



12.6



11.6



 



SD



2.4



2.2



2.3



3.3



 



N



25



24



25



23



 



%Diff



-



8.2



1.9



-5.8



Pre-implantation Loss (%) [k]



Mean



18.69



14.14



11.69*



22.81



 



SD



17.10



12.39



15.57



15.94



 



N



25



24



25



23



 



%Diff



-



-24.38



-37.49



22.03



Total Number of Resorptions [k]



Mean



0.4



0.9



0.8



0.8



 



SD



0.7



1.6



0.8



1.3



 



N



25



24



25



23



 



%Diff



-



98.9



72.7



87.7



Number of Early Resorptions [k]



Mean



0.4



0.9



0.7



0.8



 



SD



0.7



1.6



0.8



1.3



 



N



25



24



25



23



 



%Diff



-



98.9



63.6



87.7



Number of Late Resorptions [k]



Mean



0.0



0.0



0.0



0.0



 



SD



0.0



0.0



0.2



0.0



 



N



25



24



25



23



 



%Diff



-



-



-



-



Total Number of Fetuses [k]



Mean



11.9



12.5



11.8



10.8



 



SD



2.3



2.6



2.3



3.8



 



N



25



24



25



23



 



%Diff



-



4.9



-0.7



-9.2



Number of Live Fetuses [k]



Mean



11.9



12.5



11.8



10.8



 



SD



2.3



2.6



2.3



3.8



 



N



25



24



25



23



 



%Diff



-



4.9



-0.7



-9.2



Number of Live Male Fetuses [k]



Mean



5.8



6.4



6.4



5.3



 



SD



2.2



2.5



2.5



2.4



 



N



25



24



25



23



 



%Diff



-



11.4



11.1



-8.7



Number of Live Female Fetuses [k]



Mean



6.1



6.0



5.4



5.5



 



SD



2.0



2.6



2.4



2.6



 



N



25



24



25



23



 



%Diff



-



-1.3



-11.8



-9.8



Number of Dead Fetuses [k]



Mean



0.0



0.0



0.0



0.0



 



SD



0.0



0.0



0.0



0.0



 



N



25



24



25



23



 



%Diff



-



-



-



-



Post-implantation Loss (5) [k]



Mean



3.29



7.10



6.01



9.42



 



SD



5.24



14.19



6.47



18.50



 



N



25



24



25



23



 



%Diff



-



115.96



82.77



186.69



Live Male Fetus/Litter (5) [k]



Mean



48.24



51.33



54.26



47.50



 



SD



14.20



17.57



18.23



18.79



 



N



25



24



25



23



 



%Diff



-



6.39



12.47



-1.53



Mean Fetal Weight all (g) [G]



Mean



5.920



5.958



5.863



5.537*



 



SD



0.257



0.380



0.395



0.493



 



N



25



24



25



23



 



%Diff



-



0.647



-0.970



-6.476



Mean Fetal Weight males (g) [G]



Mean



6.099



6.151



6.003



5.700*



 



SD



0.258



0.369



0.475



0.567



 



N



25



24



25



22



 



%Diff



-



0.861



-1.564



-6.541



Mean Fetal Weight females (g) [G1]



Mean



5.756



5.775



5.699



5.368**



 



SD



0.273



0.409



0.391



0.468



 



N



25



24



25



23



 



%Diff



-



0.321



-0.987



-6.740



Mean Fetal AGD males (mm) [G]



Mean



2.32



2.32



2.21



2.22



 



SD



0.17



0.15



0.16



0.17



 



N



25



24



25



22



 



%Diff



-



0.26



-4.41



-4.14



Mean Fetal AGD females (mm) [G]



Mean



0.89



0.92



0.87



0.90



 



SD



0.08



0.11



0.14



0.11



 



N



25



24



25



23



 



%Diff



-



4.08



-2.25



1.23



Mean Normalized Fetal AGD m [G]



Mean



1.268



1.269



1.220



1.245



 



SD



0.092



0.082



0.082



0.083



 



N



25



24



25



22



Mean Normalized Fetal AGD f [G]



Mean



0.494



0.515



0.485



0.512



 



SD



0.041



0.067



0.074



0.058



 



N



25



24



25



23



[k] - Kruskal-Wallis & Dunn: * = p ≤ 0.05


[G] - Kruskal-Wallis & Dunn: * = p ≤ 0.05


[G1] - Anova & Dunnett: ** = p ≤ 0.05



 



































































































































Summary of Thyroid Hormone Values - Terminal Euthanasia (Gestation Day 21)



Group



1



2



3



4



Dose (mg/kg/day)



0



100



350



650



T3 (pg/mL)



 



 



 



 



  Mean



318.1



288.9



289.2



268.7



  SD



99.3



94.3



63.3



94.0



  N



25



24



25



23



  % Difference



-



-9.2%



-9.0%



-15.5%



T4 (pg/mL)



 



 



 



 



  Mean



15166.8



14503.8



16066.8



14647.4



  SD



4547.8



5100.4



4212.7



5909.7



  N



25



24



25



25



  % Difference



-



-4.4%



+5.9%



-3.4%



TSH (pg/mL)



 



 



 



 



  Mean



1692.1



1772.5



2613.1



2536.3



  SD



851.9



1011.3



1866.3



1907.5



  N



21



22



20



20



  % Difference



-



+4.8%



+54.4%



+49.9%



None statistically significant versus control.


Bold - Test substance-related


Conclusions:
Based on the effects on body weight, body weight gain, and food consumption at 650 mg/kg/day, a dosage level of 350 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when the test substance was administered orally by gavage to time-mated Crl:CD(SD) rats. Due to the absence of adverse effects at any dose level, a dose level of 650 mg/kg/day, the highest dose level evaluated, was considered to be the NOAEL for embryo/fetal development.
Executive summary:

The objective of this study was to determine the potential of the test substance to induce developmental toxicity after maternal exposure (via oral gavage) from implantation to one day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.



The study design was as follows:


Experimental Design













































Group No.Test SubstanceDose Levela (mg/kg bw/d)Dos Volumeb (mL/kg)Dose Concentration (mg/mL)Number of Females
1Vehicle010025
2DVB-HP100101025
3DVB-HP350103525
4DVB-HP650106525

a Not corrected for salt, purity, and water content


b Based on the most recent body weight measurement


 


Animals were dosed via oral gavage once daily during Gestation Days 6–20.


 


The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, thyroid hormones, organ weights, macroscopic and microscopic examinations, intrauterine growth and survival, fetal anogenital distance, and fetal morphology.


 


The analyzed dosing formulations were within the protocol-specified range of target concentrations for suspensions and were homogeneous.


 


One female in the 650 mg/kg/day group was euthanized in extremis on Gestation Day 12 following body weight losses and severely reduced food consumption and clinical observations of hunched posture, erected fur, decreased activity, and thin. A microscopic cause of moribundity and subsequent euthanasia could not be determined. None of the other females in the 650 mg/kg/day group were found dead or euthanized in extremis or noted with similar clinical findings. However, several females in this group (17 of 24 gravid females) lost weight following the initiation of treatment (Gestation Days 6-9), therefore, a relationship of to test substance administration cannot be ruled out. All other animals survived to scheduled euthanasia.


 


With the exception of the aforementioned clinical findings for the female euthanized in extremis in the 650 mg/kg/day group, there were no significant test substance-related clinical observations noted at any dose level.


 


A test substance-related initial mean body weight loss, with corresponding lower mean food consumption, was noted for females in the 650 mg/kg/day group during Gestation Days 6-9. Thereafter, mean body weight gains and food consumption in the 650 mg/kg/day group were generally comparable to or slightly lower than the control group throughout the remainder of the treatment period (Gestation Days 9-21). Consequently, mean body weight in this group was 8.8% lower than the control group on Gestation Day 21. Mean gravid uterine weight, adjusted body weight, and adjusted body weight gain in the 650 mg/kg/day group were lower than the control group. No test substance-related effects were noted on mean body weight, body weight gain, food consumption, gravid uterine weight, adjusted body weight, and adjusted body weight gain in the 100 and 350 mg/kg/day groups when compared to controls.


 


No test substance-related maternal gross findings were noted at any dose level.


 


Test substance-related higher absolute liver and thyroid/parathyroid weights were noted in the 650 mg/kg/day group when compared to the control group. These results correlated to the liver finding of centrilobular hepatocellular hypertrophy in the 350 and 650 mg/kg/day groups and the thyroid finding of follicular cell hypertrophy in the 100, 350, and 650 mg/kg/day groups. While there were no changes in maternal T4 at any dosage level, lower serum T3 levels were noted in the 100, 350 and 650 mg/kg/day groups, with correspondingly higher mean TSH levels in the 350 and 650 mg/kg/day groups. Hypertrophy in the liver and/or thyroid gland was consistent with microsomal enzyme induction and lower T3 levels and these findings were considered nonadverse and adaptive given the severity of changes and lack of test substance-related hepatocellular injury. No other test substance-related organ weight effects or microscopic finding were noted at any dose level.


 


Test substance-related lower (6.476% to 6.740%) mean fetal weights (male/female/combined) were noted in the 650 mg/kg/day group. These effects were considered nonadverse based on the lower magnitude of the difference versus controls. No other test substance-related effects were noted on intrauterine growth and survival or anogenital distance at any dose level.


 


No test substance-related fetal malformations or variations were noted for fetuses at any dose level.


 


Based on the effects on maternal body weight, body weight gain, and food consumption at 650 mg/kg/day, a dosage level of 350 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when the test substance was administered orally by gavage to time-mated Crl:CD(SD) rats. Due to the absence of adverse effects at any dose level, a dose level of 650 mg/kg/day, the highest dose level evaluated, was considered to be the NOAEL for embryo/fetal development.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Species:
rabbit
Quality of whole database:
GLP Guideline study
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

A rat developmental toxicity study was performed in accordance with OECD Guideline 414. Animals were dosed via oral gavage once daily during Gestation days 6-20 at doses of 0, 100, 350, or 650 mg/kg/day. The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, thyroid hormones, organ weights, macroscopic and microscopic examinations, intrauterine growth and survival, fetal anogenital distance, and fetal morphology. One female in the 650 mg/kg/day group was euthanized in extremis on Gestation Day 12 following body weight losses and severely reduced food consumption and clinical observations of hunched posture, erected fur, decreased activity, and thin. A microscopic cause of moribundity and subsequent euthanasia could not be determined. None of the other females in the 650 mg/kg/day group were found dead or euthanized in extremis or noted with similar clinical findings. However, several females in this group (17 of 24 gravid females) lost weight following the initiation of treatment (Gestation Days 6-9), therefore, a relationship of to test substance administration cannot be ruled out. A test substance-related initial mean body weight loss, with corresponding lower mean food consumption, was noted for females in the 650 mg/kg/day group during Gestation Days 6-9. Thereafter, mean body weight gains and food consumption in the 650 mg/kg/day group were generally comparable to or slightly lower than the control group throughout the remainder of the treatment period (Gestation Days 9-21). Mean gravid uterine weight, adjusted body weight, and adjusted body weight gain in the 650 mg/kg/day group were lower than the control group. Test substance-related higher absolute liver and thyroid/parathyroid weights were noted in the 650 mg/kg/day group when compared to the control group. These results correlated to the liver finding of centrilobular hepatocellular hypertrophy in the 350 and 650 mg/kg/day groups and the thyroid finding of follicular cell hypertrophy in the 100, 350, and 650 mg/kg/day groups. While there were no changes in maternal T4 at any dosage level, lower serum T3 levels were noted in the 100, 350 and 650 mg/kg/day groups, with correspondingly higher mean TSH levels in the 350 and 650 mg/kg/day groups. Hypertrophy in the liver and/or thyroid gland was consistent with microsomal enzyme induction and lower T3 levels and these findings were considered non-adverse and adaptive given the severity of changes and lack of test substance-related hepatocellular injury. Test substance-related lower (6.476% to 6.740%) mean fetal weights (male/female/combined) were noted in the 650 mg/kg/day group. These effects were considered non-adverse based on the lower magnitude of the difference versus controls. Based on the effects on maternal body weight, body weight gain, and food consumption at 650 mg/kg/day, a dosage level of 350 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when the test substance was administered orally by gavage to time-mated Crl:CD(SD) rats. Due to the absence of adverse effects at any dose level, a dose level of 650 mg/kg/day, the highest dose level evaluated, was considered to be the NOAEL for embryo/fetal development.


 


A rabbit developmental toxicity study was performed in accordance with OECD Guideline 414. Animals were dosed via oral gavage once daily during Gestation Days 7–28 at doses of 0, 100, 350, or 650 mg/kg/day. The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, macroscopic examinations, intrauterine growth and survival, and fetal morphology. Two females in the 650 mg/kg/day group aborted on Gestation Day 20 or 28. Based on similar evidence of toxicity in other females in the 650 mg/kg/day group, these abortions were considered test substance-related and adverse. Test substance-related clinical finding included excreta-related findings in the 350 and 650 mg/kg/day groups, which correlated with lower mean food consumption noted in these groups. Lower mean maternal body weight gains or body weight losses, with corresponding lower mean food consumption, were noted for females in the 650 mg/kg/day group generally throughout the study (Gestation Days 7-29). As a result, mean body weights in this group were lower (up to 6.8%) than the control group during Gestation Days 17-29. Based on the evidence of abortion in this group, these differences in mean body weight, body weight gain, and food consumption were considered test substance-related and adverse. In addition, lower mean adjusted body weights, adjusted body weight gains, and gravid uterine weights were noted for females in this group. In the 350 mg/kg/day group, slightly lower mean body weight gains, with corresponding lower mean food consumption, were generally noted during Gestation Days 17-29. Mean body weights in this group were slightly lower for much of the study but did not achieve statistical significance. The slightly lower mean body weights, body weight gains, and lower mean food consumption noted for these females were considered test substance-related but were not of a sufficient magnitude to cause statistically significant decreases in mean absolute maternal body weights, and hence were considered test substance-related but non-adverse. At 350 mg/kg/day, mean gravid uterine weight was also slightly decreased but did not achieve statistical significance. Lower mean fetal weights (males, females, and combined) were noted in a dose-related manner in the 350 and 650 mg/kg/day groups. These differences were considered test substance-related and adverse. Based on evidence of abortion and effects on body weight, body weight gain, and food consumption at 650 mg/kg/day, a dose level of 350 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Due to the lower fetal weights at 350 and 650 mg/kg/day, a dosage level of 100 mg/kg/day was considered to be the NOAEL for embryo/fetal development when the test substance was administered orally by gavage to time-mated New Zealand White rabbits.

Justification for classification or non-classification

Based on the reduced postnatal pup survival between birth and PND 4 in the extended 1-generation study in rats along with the reduced fetal weights in the rabbit developmental toxicity study, a classification of Reproduction Cat 2 (H361: Suspected of damaging fertility or the unborn child) is warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Additional information