Reaction mass of 2-[[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]methyl]oxirane and 1,3-bis[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]propan-2-ol and 1-[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]-3-[4-[[4-[3-[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]-2-hydroxy-propoxy]-3,5-dimethyl-phenyl]methyl]-2,6-dimethyl-phenoxy]propan-2-ol

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Initiation of dosing: 14 September 2021 Completion of in-life: 05 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
Study designed as per ECHA communication/decision number CCH-D-2114575528-35-01/F.

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]propan-2-ol; 1-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-3-{4-[(4-{3-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-2-hydroxypropoxy}-3,5-dimethylphenyl)methyl]-2,6-dimethylphenoxy}propan-2-ol; 2-{[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]methyl}oxirane

EC Number:

941-357-0

Molecular formula:

Not applicable for multi-constituent substances

IUPAC Name:

1,3-bis[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]propan-2-ol; 1-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-3-{4-[(4-{3-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-2-hydroxypropoxy}-3,5-dimethylphenyl)methyl]-2,6-dimethylphenoxy}propan-2-ol; 2-{[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]methyl}oxirane

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The Crl:WI(Han) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical data for this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

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: (P); 6-7 weeks old
- Weight at study initiation: (P) Males: 157-239 g; Females: 100-158 g; (F1) Varied as commenced as pups
- Fasting period before study: NA
- Housing: On arrival (F0) or following weaning (F1), the F0 animals were group housed (2 to 3 animals of the same sex) until cohabitation. During cohabitation, the F0 animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Following weaning (F1), animals were group housed (2 to 3 animals of the same sex) until euthanasia. All offspring selected to constitute Cohort 2B were single-housed overnight, prior to euthanasia on PND 22. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve. Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dose level, and sex. Cages were arranged on the racks in group order.
- Use of restrainers for preventing ingestion (if dermal): NA
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal) was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system. Water bottles were provided, if required. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: A minimum of 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 25°C
- Humidity (%): 30% to 70%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained.
IN-LIFE DATES: From: To: 14 September 2021 to 5 April 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400, NF

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared approximately weekly, and an adequate amount of each formulation was dispensed into daily aliquots, which were stored at room temperature (18°C to 24°C) until use. The dosing formulations were stirred continuously during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard vehicle
- Concentration in vehicle: 15, 30 and 50 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): NA
- Purity: NA

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating.
- Proof of pregnancy: Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage.
- Further mating after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: Female No. 2992 in the 150 mg/kg/day group was paired with Male No. 3124 on 22 Nov 2021 and was found with evidence of mating on 06 Dec 2022. Following collection of the Gestation Day 0 data, this female was found paired with Male No. 3125, of the same group. Upon discovery, Female No. 2992 was single housed as required following mating. This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because the female was already positive for mating, and therefore, the mating was attributed to the correct male. Furthermore, the female was found with a male of the same treatment group, and was only in the cage for a short period of time. Animal health was not affected, nor was interpretation of the data.

Analytical verification of doses or concentrations:

yes

Remarks:

High performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV) using a validated analytical procedure

Details on analytical verification of doses or concentrations:

A high performance liquid chromatographic (HPLC) method with ultraviolet (UV) detection was used for the determination of the substance concentration in formulations containing polyethylene glycol 400 (PEG400), specific gravity 1.125. The method was validated in a previous study for the analysis of formulations ranging in test substance concentration from 10 to 220 mg/mL. Also in the previous study, test substance homogeneity and, following 8 days of room temperature storage, resuspension homogeneity were assessed and verified in formulations ranging in concentration from 10 to 220 mg/mL. Test substance stability following 8 days of room temperature and refrigerated storage in formulations ranging in concentration from 10 to 220 mg/mL was also established. In the present study, formulations used for dose administration were analyzed to verify test substance homogeneity and/or concentration acceptability. Homogeneity testing showed that the formulation technique used produced homogeneous preparations. The dose formulations were within specification for concentration acceptability.

Duration of treatment / exposure:

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 (for a minimum of 18 weeks). F0 females were dosed orally by gavage for a minimum of 70 consecutive days prior to mating and continuing throughout mating, gestation, and lactation, through the day prior to euthanasia. The offspring selected for the F1 generation began dosing following weaning until the day prior to euthanasia (PND 52 [Cohort 1 Surplus], PND 78 [Cohort 2A], PND 91 [Cohort 1A], and PND 98 [Cohort 1B]). All animals were dosed at approximately the same time each day. Animals assigned to Cohort 2B were not directly dosed with the test or control substances.

Frequency of treatment:

Once daily

Details on study schedule:

As per 'Duration of treatment/exposure'.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Doses / concentrations 2

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

Control: 25 males and females
75 mg/kg/day: 25 males and females
150 mg/kg/day: 25 males and females
250 mg/kg/day: 25 males and 28 females (Three females in the 250 mg/kg/day group were found dead on Study Day 7 with confirmed dosing errors; 3 additional females were added to study on Study Day 8).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The route of administration was oral (gavage) because this is a potential route of exposure to humans. Historically, this route has been used extensively for studies of this nature.

The doses were selected based on 4 previous studies, which included a 90-day toxicity study, a 28-day combined toxicity and reproductive/developmental study, a prenatal developmental toxicity study, and a lacteal transfer study in Wistar rats. In the 90-day study, dose levels were 100, 300, and 600 mg/kg/day. In the 28-day combined toxicity study, dose levels were 100, 300, and 1000 mg/kg/day. Doses of 1000 and 600 mg/kg/day were not well tolerated, resulting in unscheduled deaths. A dose of 300 mg/kg/day resulted in increased liver weights and corresponding minimal hepatocellular hypertrophy, as well as changes in clinical biochemistry. In the prenatal developmental toxicity and lacteal transfer studies, dose levels were 100, 300, and 600 mg/kg/day. A dose of 600 mg/kg/day resulted in reduced body weight gain during gestation (-20%) and lactation (-14%), as well as reduced gravid uterine weights (-7.8%), fetal weights (-6.3%), and offspring body weights (-9.1%). Higher incidences of several skeletal variations, including thin cartilaginous structures of the vertebra sacral arch were observed at 300 and 600 mg/kg/day. These data demonstrated that a dose level of 250 mg/kg/day meets the CLP Regulation (EC No. 1272/2008) and ECHA requirements (Jul 2021) for the highest possible dose level with the potential to elicit clear evidence of reproductive (Repr.1B; H360F) and developmental (Repr.1B; H360D) toxicity, while avoiding severe suffering or deaths in the parental generation. Based on these data, dose levels of 75, 150, and 250 mg/kg/day were selected for this study.

- 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. Animals at extremes of body weight range were not assigned to groups. To reduce variability among the F1 litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups.
For the F1 generation, 4 F1 pups/sex/litter from all available litters 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. If required, surplus pups were maintained on study (Cohort 1 Surplus) in order to assure that at least 3 F1 pups/sex/litter were examined for the attainment of postweaning developmental landmarks. These surplus pups were subjected to a gross necropsy following attainment of developmental landmarks. 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, and Cohort 2B.
- Fasting period before blood sampling for clinical biochemistry: All animals (including those not scheduled for clinical pathology assessment) were fasted overnight prior to blood collection.

Positive control:

Not a guideline requirement

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.
Cage-side observations were recorded daily, prior to dosing and at 2–4 hours following each dose administration. Cage-side observations were not conducted prior to dosing on days that detailed clinical observations were performed. During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were removed from the cage, and a detailed clinical observation was performed weekly, beginning with the first day of dose administration. During the dosing period, these observations were performed prior to dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually weekly throughout the study and prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 0 (when possible), 1, 4, 7, 14, and 21. A fasted weight was recorded on the day of necropsy. Terminal body weights were not collected from animals found dead.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was quantitatively measured weekly throughout the study, except during the mating period. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 14, and 21. Food efficiency (body weight gained as a percentage of food consumed) was calculated and reported.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

THYROID HORMONE ANALYSIS: Yes
- Blood samples for thyroid hormone analyses were collected (prior to 1200 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels) from the jugular vein into tubes without anticoagulants. Samples were taken from 10 animals/sex/group on week 18 for analysis of Triiodothyronine (T3), Thyroxine (Total T4) and Thyroid Stimulating Hormone (TSH).

CLINICAL PATHOLOGY: Yes
- All animals (including those not scheduled for clinical pathology assessment) were fasted overnight prior to blood collection. Urine was collected overnight using metabolism cages. Blood samples for hematology and 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 10 animals/sex/group on week 18 and analysed for hematology, coagulation, clinical chemistry and urinalysis.

Hematology parameters: Total leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hemoglobin distribution width (HDW), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (Platelet), Reticulocyte count percent (RETIC), Absolute (RETIC Absolute), Differential leukocyte count (Percent and absolute); Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC), Gated red cell distribution width (RDW), Platelet estimate, Red cell morphology (RBC Morphology) and Sample quality.

Coagulation parameters: Activated partial thromboplastin time (APTT) and Prothrombin time (PT).

Clinical chemistry parameters: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total BILI), Urea nitrogen, Bile Acids, 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), Direct bilirubin (Direct BILI), Indirect bilirubin (Indirect BILI) and Appearance.

Urinalysis parameters: 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).

Oestrous cyclicity (parental animals):

Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each F0 female for 14 days prior to cohabitation and continuing until evidence of mating was observed or until the end of the mating period. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P], beginning 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 based on standard criteria.

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:

Percent motile (or progressively motile) sperm = (Number of motile (or progressively motile) sperm/Total number of sperm counted) x 100

The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. 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 F1 litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups.

For the F1 generation, 4 F1 pups/sex/litter from all available litters 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. If required, surplus pups were maintained on study (Cohort 1 Surplus) in order to assure that at least 3 F1 pups/sex/litter were examined for the attainment of postweaning developmental landmarks. These surplus pups were subjected to a gross necropsy following attainment of developmental landmarks. 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, and Cohort 2B.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Live births, still births, mean live litter size, presence of gross anomalies (examined on PND 0, 4, 14 and 21), postnatal survival (between birth and PND 0/4), postnatal survival for all other intervals, physical or behavioural abnormalities, number and sex of pups (sexed on PND 0, 4, 14 and 21), weight gain (weighed on PND 0, 4, 7, 14 and 21), anogenital distance (AGD; PND 1), assessment of Areolas/Nipple Anlagen Retention (PND 13) and thyroid hormone analysis (PND 4 and 21). F1 post weaning developmental landmarks included balanopreputial Separation (PND 35) and vaginal patency assessments (PND 25).

The day parturition was initiated was designated Lactation Day 0 (Postnatal Day [PND] 0 for pups). During the period of expected parturition, females were observed twice daily for initiation and completion of parturition and for dystocia (prolonged or difficult labor) or other difficulties. All females were allowed to deliver naturally. Beginning on the day parturition was initiated, the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery was first observed. The dam and litter remained together until weaning on PND 21.

GROSS EXAMINATION OF DEAD PUPS:
A necropsy was conducted for animals that died on study. If necessary for humane reasons, animals were euthanized as per Testing Facility SOPs. These animals underwent necropsy. Intact offspring that were found dead 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 euthanized for humane reasons after PND 4.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: An auditory startle response test was performed on animals assigned to Cohort 2A on PND 24 (± 1 day). Auditory startle response testing was performed in a room equipped with a white-noise generation system. Each test session consisted of a 5-minute acclimation period with a 68 dB to 72 dB broadband background white noise. The startle stimulus for each trial was a 110 dB to 120 dB mixed-frequency noise burst stimulus, approximately 20 ms in duration. Responses were recorded during the first 100 ms following the onset of the startle stimulus for each trial. Each test session consisted of 50 trials, with 8-seconds between each trial. Startle response data were analyzed in 5 blocks of 10 trials each. Startle response measurements obtained were PEAK (peak response amplitude) and Tpeak (latency to PEAK).

FOB findings were recorded for all Cohort 2A animals on PND 65. The FOB used at Charles River is based on previously developed protocols. Testing was performed by the same trained technicians, when possible, who did not know the animal’s group assignment and was performed at approximately the same time each day. The FOB was performed in a sound-attenuated room equipped with a white noise generator. All animals were observed for the following parameters as described below.
Home cage observations: Posture, Convulsions/tremors, Feces consistency, Biting and Palpebral (eyelid) closure.
Handling Observations: Ease of removal from cage, Lacrimation/chromodacryorrhea, Piloerection, Palpebral closure, Eye prominence, Red/crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/eye/skin color and Muscle tone.
Open Field Observations (recorded over a 2 minute time period): Mobility, Rearing, Convulsions/tremors, Grooming, Bizarre/stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/defecation, Gait score and Backing.
Sensory Observations: Approach response, Touch response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Tail pinch response, Eyeblink response, Hindlimb extension and Olfactory orientation.
Neuromuscular observations: Hindlimb extensor strength, Hindlimb foot splay, Grip strength-hind and forelimb and Rotarod performance.
Physiological observations: Catalepsy, Body temperature and Body weight.

Motor activity was assessed on animals assigned to Cohort 2A on PND 65. 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).

F1 generation (post weaning):
Viability: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.

Observations: The animals were removed from the cage, and a detailed clinical observation was performed weekly, beginning with the first day of dose administration. During the dosing period, these observations were performed prior to dosing. Cage-side observations were recorded daily, prior to dosing and at 2–4 hours following each dose administration. Cage-side observations were not conducted prior to dosing on days that detailed clinical observations were performed. During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

Bodyweights: Animals were weighed individually weekly following weaning.

Food consumption: Food consumption was quantitatively measured weekly following weaning with the exception of pups assigned to Cohort 2B which were scheduled for euthanasia on PND 22.

Estrous cyclicity: 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 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 female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.

Thyroid hormone analysis (cohort 1A): Blood samples for thyroid hormone analyses were collected (prior to 1200 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels) from the jugular vein into tubes without anticoagulants. Samples were collected on PND 91 from all groups (10 animals/sex/group) and analysed for T3, Total T4 and TSH.

Clinical pathology (Cohort 1A): Animals were fasted overnight prior to blood collection. Urine was collected overnight using metabolism cages. Blood samples for hematology and 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 on PND 91 for haematology, coagulation, clinical chemistry and urinalysis parameters from all groups (10 animals/sex/group) for the same parameters as the F0 generation.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not required

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed between study days 126 and 129.
- Maternal animals: All surviving animals were sacrificed between study days 126 and 129.

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 implantation 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.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights: The organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead. Paired organs were weighed together, unless otherwise noted. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
Organs weighed included: Adrenal glands, Brain, Epididymidesa (total and cauda), Heart, Kidneys, Liver, Ovaries, Pituitary gland, Prostate gland, Seminal vesicles with coagulating glands (with accessory fluids), Spleen, Testes, Thyroids with parathyroids, Thymus gland and Uterus with oviducts and cervix.

Histology: Tissues identified from all animals/sex in the control and high-dose groups and from all animals found dead, 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. The following regions of the epididymis were embedded in paraffin: caput, corpus, and cauda; the vas deferens was examined when possible. Five (5) sections were taken approximately 100 μm apart from the inner third of each ovary from any F0 females 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. In addition, a single section was taken from remaining F0 females for a qualitative bilateral evaluation of each ovary. For females found dead, a single section from each ovary was qualitatively evaluated.

Histopathology: 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. Histopathological examination of the testis included a qualitative assessment of the stages of spermatogenesis. 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).
For any F0 female 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, a quantitative histopathologic evaluation of multiple sections 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.
Tissues examined microscopically: Adrenal glands (2), Aorta, Bone with marrow (sternebrae), Brain, Coagulating glands (2), Eyes with optic nerve (2), 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], and mesenteric), Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary, Prostate, Submandibular salivary gland (2), Seminal vesicles (2), Skeletal muscle (quadriceps), Skin with mammary gland, Spinal cord (cervical), Spleen, Testes with epididymides (2) and vas deferens (1), Thymus, Thyroids with (with parathyroids if present [2]), Trachea, Urinary, bladder, Uterus with cervix and vagina and All gross lesions (all groups).
Groups: Full tissues were examined for Group 1 and 4 with gross lesions and selected tissues for groups 2 and 3. Full tissues were also examined for all unscheduled deaths.

Postmortem examinations (offspring):

- F1 litters: Scheduled euthanasia occurred on PND 4 and 21 (unselected pups).

Unscheduled deaths: A necropsy was conducted for animals that died on study. If necessary for humane reasons, animals were euthanized as per Testing Facility SOPs. These animals underwent necropsy. Intact offspring that were found dead 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 euthanized for humane reasons after PND 4.

Scheduled euthanasia: 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, nonselected pups were euthanized by exsanguination (those pups used for blood/thyroid collection) or by carbon dioxide inhalation.

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, nonselected pups were subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system.

Organ weights: The organs identified were weighed at necropsy from up to 10 non-selected F1 pup/sex/group on PND 21. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated. The organs weighed included; brain, thymus, liver, spleen and thyroid.

- F1 generation (Cohort 1): Scheduled euthanasia occurred on PND 52, 91 and 98.
Unscheduled deaths: A necropsy was conducted for F1 animals that died on study, and specified tissues were saved. If necessary for humane reasons, F1 animals were euthanized as per Testing Facility SOPs. These animals underwent necropsy, and specified tissues were retained.

Scheduled euthanasia: 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 (for exceptions, see Appendix 1). The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported:
Percent motile (or progressively motile) sperm = (Number of motile (or progressively motile) sperm/Total number of sperm counted) x 100

The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique (Linder et al., 1992). Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and cauda epididymis from all 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.

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: The organs identified were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together, unless otherwise noted. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated. The organs weighed included; Adrenal glands, Brain, Epididymides (total and cauda), Heart, Kidneys, Liver, Lymph nodes (iliac and mandibular), Ovaries, Pituitary gland, Prostate gland, Seminal vesicles with coagulating glands (with accessory fluids), Spleen, Testes, Thyroids with parathyroids, Thymus gland and Uterus with oviducts and cervix.

Histology: The same procedure as used for the F0 generation was utilised.

- Histopathology (Cohort 1A):
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. 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 was conducted. This examination included enumeration of the total number of primordial follicles.

- 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.
Leucocyte subsets and phenotypes included:
Total T lymphocytes, CD45+ CD3+ (abbreviated as CD3+ for reporting)
Helper T lymphocytes, CD45+ CD3+ CD4+ (abbreviated as CD3+ CD4+ for reporting)
Cytotoxic T lymphocytes, CD45+ CD3+ CD8+ (abbreviated as CD3+ CD8+ for reporting)
B lymphocytes, CD45+ CD3- CD45RA+ (abbreviated as CD3- CD45RA+ for reporting)
NK cells, CD45+ CD3- CD161a+ (abbreviated as CD3+ CD161a+ for reporting)
NK-T cells, CD45+ CD3+ CD161a+ (abbreviated as CD3- CD161a+ for reporting)
Monocytes, CD45+ CD3- CD11bc+ (abbreviated as CD3- CD11bc+ for reporting)

Immunophenotyping results were expressed as relative frequency (%) of total spleen leukocytes for each subset. The absolute lymphocyte count (cells/organ) of each subset was calculated by multiplying each leukocyte subset frequency (%) with the total leukocyte count.

- F1 Neuropathology:
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.).

Scheduled euthanasia: All animals were deeply anesthetized by intraperitoneal injection of sodium pentobarbital and perfused in situ with fixative (4% paraformaldehyde solution in 0.1M phosphate buffer).

Necropsy: 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.

Organ weights: The whole brains were removed (including olfactory bulbs), weighed, and the dimensions (length [excluding olfactory bulbs] and width) were recorded.

Histology: 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.

Neuropathology: 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. These included; Brain (olfactory bulbs, cerebral cortex, hippocampus/dentate gyrus, basal ganglia, thalamus, hypothalamus, midbrain, cerebellum, pons, and medulla oblongata (full coronal section was prepared)), Spinal cord (at cervical swellings C3 – C7 and at lumbar swellings T13 – L4), Trigeminal ganglion/nervesa, Lumbar dorsal root ganglion with lumbar dorsal root fibers at T13 – L4, Lumbar ventral root fibers at T13 – L4 (individually dissected ventral roots were blocked separately), Cervical dorsal root ganglia with cervical dorsal root fibers at C3 – C7, Cervical ventral root fibers at C3 – C7 (individually dissected ventral roots were blocked separately),, Pituitary gland, Caudal nerves (2), Sciatic nerves (2), Sural nerves (2), Tibial nerves (2), Peroneal nerves (2), Nasal tissue with olfactory epithelium, Optic nerves, Eyes, and Skeletal muscle (anterior tibial and gastrocnemius).

Morphometric analysis (Cohort 2A): Histopathological examination included a simple morphometric analysis for Cohort 2A animals on PND 78. Simple linear morphometric measurements were obtained from homologous sections of the brain at 3 levels. Level 1 was a coronal section obtained just rostral to the genu of the corpus callosum and contained the following major structures: cerebral cortex, corpus callosum, basal ganglia (caudate putamen, nucleus accumbens), and lateral ventricles. Two bilateral measurements were obtained at this level: the vertical height of the hemisphere just medial to the lateral ventricle, and the vertical height of the cerebral cortex from the apex of the corpus callosum/cingulum to the dorsal surface of the hemisphere. Level 3 was a coronal section obtained approximately at the level of the optic chiasm, just rostral to the pituitary, and contained the following major structures: cerebral cortex, corpus callosum, basal ganglia, hippocampus/dentate hilus, thalamus, hypothalamus, and lateral and third ventricles. Four bilateral measurements were taken at this level: the radial thickness of the cortex, the vertical height between the layers of the hippocampal pyramidal neurons along a line that passed through the termination of the dorsal limb of the dentate hilus, the vertical height of the dentate hilus measured at the termination of the ventral limb, and the length of the ventral limb of the dentate hilus taken along a straight line from the crest to the termination of the ventral limb. Level 5 was a sagittal section of the cerebellum and medulla oblongata, taken slightly lateral to the midline, and contained the following major structures: cerebellum, medulla oblongata (pyramidal tract, trapezoid body, medial longitudinal fasciculus), and fourth ventricle. Single measurements of the dorso-ventral thickness of the medulla and of the thickness of the base of cerebellar lobule no. 9 were obtained at this level. For purposes of reporting, the measurements from the right and left hemispheres, where appropriate, were combined to obtain a mean overall measurement for that structure.

Statistics:

As per 'Any other information on materials and methods incl. tables.'

Reproductive indices:

Mating, fertility, copulation, and conception indices were calculated. Mean gestation length was also calculated.

Offspring viability indices:

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) were calculated. Total litter loss was determined when the last pup in the litter was found dead or euthanized in extremis prior to the scheduled euthanasia.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related clinical findings were noted during the generation at the daily examinations or at 2–4 hours following dosing. 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.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three females (Nos. 2967, 3000, and 3002) in the 250 mg/kg/day group were found dead on Study Day 7; all 3 females were noted with a perforated esophagus at necropsy. These deaths were attributed to the dosing procedure, and not considered test substance-related. Three additional females were added to study on Study Day 8 to take the place of these females. All other F0 males and females 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 body weights and body weight gains were noted in the 75, 150, and 250 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). Any statistically significant differences from the control group were transient, had no impact on mean body weights, and/or did not present in a dose-related manner.

During gestation: Mean maternal body weights and body weight gains were unaffected by test substance administration during gestation. Differences between the control, 75, 150, and 250 mg/kg/day groups were slight and not statistically significant.

During lactation: Mean maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance administration during lactation. Differences between the control, 75, 150, and 250 mg/kg/day groups were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Weekly: Food consumption and food efficiency in the 75, 150, and 250 mg/kg/day groups was unaffected by test substance administration. The values in the test substance-treated groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males). Any statistically significant differences from the control group were transient, had no impact on mean body weights, and/or did not present in a dose-related manner.

During gestation: Mean maternal food consumption and food efficiency were unaffected by test substance administration during gestation. Differences between the control, 75, 150, and 250 mg/kg/day groups were slight and not statistically significant.

During lactation: Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency were unaffected by test substance administration during lactation. Differences between the control, 75, 150, and 250 mg/kg/day groups were slight and not statistically significant.

Food efficiency:

no effects observed

Description (incidence and severity):

No effect of treatment on food efficiency during the weekly, gestation and lactation checks.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on hematology parameters in F0 males at any dose level. Statistically significant differences compared to the control group were attributed to single animals, did not present in a dose-responsive manner, and/or were within range of the Charles River Ashland historical control data (version 3.6). There were no test substance-related effects on hematology parameters in F0 females at any dose level. Differences from controls were slight and not statistically significant.

In regard to coagulation parameters, there were no test substance-related effects on coagulation parameters in F0 males or females at any dose level. Differences from controls were slight and not statistically significant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on clinical chemistry parameters in F0 males at any dose level. Statistically significant differences from control were attributed to single animals, did not present in a dose-responsive manner, and/or were within range of the Charles River Ashland historical control data There were no test substance-related effects on clinical chemistry parameters in F0 females at any dose level. Differences from controls were slight, not statistically significant, and/or did not occur in a dose-responsive manner.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly lower total T4 values were noted in all test substance-treated groups compared to the control group for F0 males and females. Values were 28.4% to 65.4% and 21.2% to 48.4% lower for males and females, respectively, and presented in a dose-responsive manner. There were no remarkable changes in mean TSH or total T3 values for F0 males and females at any dose level. The changes in the T4 levels did not correlate with any other endpoints. There were no microscopic changes in the thyroid gland and no changes in the thyroid gland weights (absolute or relative), nor was there the expected negative feedback that stimulates TSH production; the absence of changes in thyroid gland weights and microscopic findings was consistent with the normal serum TSH levels. The effect in T4 levels was therefore not considered adverse.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on urinalysis parameters in F0 males or females at any dose level. Differences from controls were slight and not statistically significant.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related microscopic findings in any of the dose groups of F0 males or females. Any microscopic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. F0 animals suspected of reduced fertility (i.e., those that failed to mate, conceive, sire, or deliver healthy offspring) were subjected to histopathological evaluation. In males, 0/20, 2/20 (Animal Nos. 3098 and 3115), 3/20 (Animal Nos. 3124, 3125 and 3145) and 1/20 (Animal No. 3073) in the 0, 75, 150 and 250 mg/kg/day groups, respectively, were evaluated. In females, 0/20, 2/20 (Animal Nos. 2987 and 2993), 3/20 (Animal Nos. 2992, 2995 and 3049), and 1/20 (Animal No. 2972) animals in the 0, 75, 150 and 250 mg/kg/day groups, respectively, were evaluated. There were no microscopic findings to explain failure to mate, conceive, sire, or deliver.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The mean lengths of estrous cycles in these groups were also similar to the control group value with the following exception. A statistically significantly lower mean estrous cycle length was noted for the 150 mg/kg/day group (3.8 days) compared to the concurrent control group (4.6 days). However, the value was within range of the Charles River Ashland historical control data and did not present in a dose-related manner and was therefore not attributed to test substance administration.

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 (Male Nos. 3098 and 3115 and Female Nos. 2987 and 2993) and 2 (Male Nos. 3124 and 3125 and Female Nos. 2992 and 2995) mating pairs in the 75 and 150 mg/kg/day groups, respectively, did not produce a litter. 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. Mean F0 gestation lengths in the test substance-treated groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the 75, 150, and 250 mg/kg/day groups were 21.7, 21.5, and 21.4 days, respectively, compared to mean gestation lengths of 21.5 days in the concurrent control group and 21.8 days in the Charles River Ashland historical control data. No signs of dystocia were noted at any dose level.

Details on results (P0)

In the F0 generation, 3 females in the 250 mg/kg/day group were found dead on Study Day 7 with a perforated esophagus noted at necropsy. These deaths were attributed to the dosing procedure, and not considered test substance-related. Three additional females were added to study on Study Day 8 to take the place of these females. All other F0 males and females survived to the scheduled necropsy. Statistically significantly lower total T4 values (21.2% to 65.4%) were noted in all test substance-treated groups compared to the control group for F0 males and females and were considered test substance-related. The changes in the T4 levels did not correlate with any relevant endpoints. There were no microscopic changes in the thyroid gland and no changes in the thyroid gland weights (absolute or relative), nor was there the expected negative feedback that stimulates TSH production; the absence of changes in thyroid gland weights and microscopic findings was consistent with the normal serum T3 and TSH levels. Therefore, the changes in T4 levels were considered nonadverse. Test substance-related higher kidney weights (absolute and relative) in the 150 and 250 mg/kg/day F0 females and higher liver weights (absolute and relative) in the 75, 150 and 250 mg/kg/day F0 females were noted. The changes were considered nonadverse due to the lack of microscopic correlation to the weight changes.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

F1 prior to weaning: The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by test substance administration.

F1 generation following weaning: No test substance-related clinical findings were noted during the F1 generation at the daily examinations or at the 2–4 hours following dosing observations. 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.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1 prior to weaning: The mean number of pups born, live litter size, percentage of males per litter at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0–1, 1–4 (pre-selection), 4 (post-selection)–7, 7–14, 14–21, and from birth to PND 4 (pre-selection) and PND 4 (post-selection)–21 were unaffected by the test substance at all dose levels. Differences from the control group were slight and not statistically significant.

Six (3), 7(5), 1(1), and 2(1) pups (litters) in the control, 75, 150, and 250 mg/kg/day groups, respectively, were found dead or euthanized in extremis. Three (3), 1(1), 4(3), and 6(6) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.

F1 generation following weaning: Male Nos. 3009-03 and 2983-01 in the 75 mg/kg/day group were found dead on PND 21. Female No. 2948-02 in the 75 mg/kg/day group and Female No. 2965-10 in the 150 mg/kg/day group were found dead on PND 22 and 42, respectively. There were no remarkable clinical findings or body weight effects noted for any of these animals prior to death. Male No. 3009-06 in the 75 mg/kg/day group was euthanized in extremis on PND 41 following clinical signs of gasping, increased respiration rate and labored respiration. At necropsy, Female No. 2965-10 was noted with oviduct cysts; there were no other findings noted at necropsy for the unscheduled deaths. Based on the lack of findings or deaths in the 250 mg/kg/day animals, these deaths were not attributed to the test substance. All other animals assigned to the F1 generation survived to the scheduled euthanasia.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

F1 prior to weaning: Mean male and female pup body weights and body weight changes in the 75, 150, and 250 mg/kg/day groups were unaffected by test substance administration throughout the pre-weaning period. No statistically significant differences from the control group were noted.

F1 generation following weaning: No test substance-related effects on mean body weights and body weight gains were noted in the 75, 150, and 250 mg/kg/day groups during the postweaning period. Differences from the control group were transient, did not occur in a dose-related manner, and/or were not statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

F1 generation following weaning: Food consumption in the 75, 150, and 250 mg/kg/day groups were unaffected by test substance administration. The values in the test substance-treated groups were generally similar to the control group values during the postweaning period. Differences from the control group were transient, did not occur in a dose -related manner, and/or were not statistically significant.

Food efficiency:

no effects observed

Description (incidence and severity):

F1 generation following weaning: Food efficiency in the 75, 150, and 250 mg/kg/day groups were unaffected by test substance administration. The values in the test substance-treated groups were generally similar to the control group values during the postweaning period. Differences from the control group were transient, did not occur in a dose -related manner, and/or were not statistically significant.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematology (Cohort 1A): There were no test substance-related effects on hematology parameters in F1 males and females. Differences from controls were slight and not statistically significant.

Coagulation (Cohort 1A): There were no test substance-related effects on coagulation parameters in F1 males and females. Statistically significantly lower activated partial thromboplastin time in F1 males at 250 mg/kg/day was within the reference range of the Charles River Ashland historical control data. Other changes from control were slight and/or did not occur in a dose-responsive manner.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical chemistry (Cohort 1A): There were no test substance-related effects on clinical chemistry parameters in F1 males and females. Any statistically significant differences from control were of minimal magnitude, did not present in a dose-responsive manner, and/or were within the range of the Charles River Ashland historical control data.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on urinalysis parameters in F1 males and females. Statistically significant lower urobilinogen was noted in all test substance-treated F1 males, but was within the range of the Charles River Ashland historical control data and not considered test substance-related.

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. The mean ages of attainment of balanopreputial separation were 44.9, 43.6, and 45.9 days in the 75, 150, and 250 mg/kg/day groups, respectively, when compared to 45.3 in the control group. Mean body weights at the age of attainment were 197.8 g, 192.0 g, and 198.5 g in the same respective groups compared to
195.8 g in the control group. Differences from the control group were slight, did not occur in a dose-responsive manner, and/or were not 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. The mean ages of attainment of vaginal patency were 32.9, 33.6, and 33.1 days in the 75, 150, and 250 mg/kg/day groups, respectively, when compared to 33.8 days in the control group. Mean body weights at the age of attainment were 105.8 g, 108.3 g, and 103.5 g in the same respective groups compared to 107.4 g in the control group. None of the differences from the control group were statistically significant.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance (absolute and relative to the cube root of pup body weight) was unaffected by test substance administration. Statistically significantly lower mean anogenital distances (absolute and relative to the cube root of pup body weight) was noted for females in all test substance-treated groups and males in the 250 mg/kg/day group. However, all values (including controls) were below the minimum mean values in the Charles River Ashland historical control data (version 2020.02). Additionally, lower values in females are not considered and androgen related effect, and therefore is not considered toxicologically relevant.

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. There were no retained areolas/nipples noted in F1 males at any dose level.

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. None of the differences from the control group were statistically significant.

Organ Weights (Cohorts 1A and 1B):
Cohort 1A: There were test substance-related organ weight changes including lower prostate gland and seminal vesicles/coagulating gland/accessory gland (SV/CG/ACC) weights in 250 mg/kg/day F1 males; higher kidney and thyroid gland/parathyroid gland in 75, 150 and 250 mg/kg/day F1 females. The changes were considered test substance-related because the changes followed a dose response profile. The mean organ weight changes were considered to be non-adverse and toxicologically irrelevant.

There were additional statistically significant changes in mean organ weights, but the changes did not follow a dose response profile, and did not have microscopic correlations. Therefore, these changes were considered to represent biological variability unrelated to the test substance.
These organ weight changes included:
- higher liver weights in the 150 mg/kg/day F1 males (absolute and relative to body and brain weight); and higher liver weight relative to body weight in the 250 mg/kg/day F1 males. The changes in liver weights in the males did not follow a dose response profile and were considered to likely represent biological variability unrelated to test substance administration.
- Higher kidney weight relative to brain weight in the 150 mg/kg/day F1 males (p<0.05); The change showed no dose response profile and was considered unrelated to test substance administration.
- Higher adrenal gland weight (relative to brain weight) in the 250 mg/kg/day F1 females; Given that the change affected only the relative weight, it was considered unrelated to test substance administration.

Cohort 1B: No test substance-related organ weight changes were noted. Any variation in mean organ weights between treatment groups were considered incidental and/or unrelated to test substance administration.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Unscheduled deaths: Six (3), 7(5), 1(1), and 2(1) pups (litters) in the control, 75, 150, and 250 mg/kg/day groups, respectively, were found dead or euthanized in extremis from PND 0 through the selection of the F1 generation. 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: No internal findings were noted at the necropsy of F1 culled pups euthanized on PND 4.
PND 21 Non-selected Pups: No internal findings were noted at the necropsy of F1 pups euthanized on PND 21.
PND 21 Pups Selected for Organ Weights: At the PND 21 necropsy of F1 weanlings selected for organ weights, no internal findings were observed at any dose level.

Macroscopic Pathology (Cohorts 1A and 1B): No test substance-related macroscopic findings were noted. Any and all macroscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to test substance administration.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic Evaluations (Cohorts 1A and 1B): There were no test substance-related microscopic findings in any dose group of F1 males or females.
Any microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to test substance administration. There were no test substance-related effects in follicle counts in F1 females from Cohort 1A. Any variation in mean counts was due to biological variability and/or variation in tissue sectioning.

Immunophenotyping (Cohort 1A): No test substance-related splenic immunophenotyping changes were noted at any dose level. All 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.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormone analysis:
PND 4 culled pups: There were no test substance-related effects on T4, T3, and TSH values in PND 4 F1 culled pups at any dose level. Differences from controls were slight and not statistically significant.

PND 21 Pups Selected for Hormone Analysis: There were lower serum total T4 in the 150 mg/kg/day PND 21 male pups, and lower total T4 in the 150 and 250 mg/kg/day female pups. There was statistically significant higher (p<0.05) total T3 in the 250 mg/kg/day PND 21 male pups. There were no statistically significant test substance-related changes in the serum TSH levels in male or female pups at PND 21. The changes in the total T4 showed no clear dose response profile in males or in females, and occurred in a direction opposite to the higher T3 in the 250 mg/kg/day males. Given the dose response profile for serum T4 in F0 males and females, the changes in T4 at PND 21 were considered to be test substance-related. The higher total serum T3 in the 250 mg/kg/day males was considered to be incidental.

PND 0 Litter Data and Postnatal Survival: The mean number of pups born, live litter size, percentage of males per litter at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0–1, 1–4 (pre-selection), 4 (post-selection)–7, 7–14, 14–21, and from birth to PND 4 (pre-selection) and PND 4 (post-selection)–21 were unaffected by the test substance at all dose levels. Differences from the control group were slight and not statistically significant.

Thyroid hormone analysis (Cohort 1A): Statistically significantly lower total T4 values were noted in all test substance-treated groups compared to the control group for F1 males on PND 91. Values were 33.4% to 41.9% lower and presented in a dose-responsive manner. There were no remarkable changes in mean T4 values for F1 females or in mean TSH or serum total T3 values for F1 males and females at any dose level. The lower T4 in males was not associated with any changes in thyroid gland weight or microscopic findings in the male thyroid gland and were therefore not considered adverse. The absence of such findings was consistent with normal levels of serum TSH.

Estrous Cycle Data (Cohort 1A): The mean ages at the first occurrence of estrus in the 75, 150, and 250 mg/kg/day groups (34.4, 35.3, and 34.5 days, respectively) were generally comparable to the control group (35.4 days). In addition, the duration from vaginal opening to first estrus in these same respective groups (2.8, 3.1, and 2.5 days) was generally comparable to the control group (2.5 days). None of the differences were statistically significant.
The mean lengths of estrous cycles in the test substance-treated groups from PND 75–91 were also similar to the control group value. None of these differences were statistically significant.

Sperm Evaluations (Cohort 1A): No test substance-related effects were observed on F1 sperm parameters (mean epididymal sperm numbers and sperm motility, progressive motility, and morphology) in males at any dose level. Differences from the control group were slight and were not statistically significant.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory Function and Neurobehavior Testing (Cohort 2A)
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 the test substance at 75, 150, and 250 mg/kg/day had no significant effect on auditory startle responsiveness. The PEAK and Tpeak values for all trials combined were similar in the F1 males and females among all groups. No statistically significant differences from the control group were noted. No effects were noted in the pattern of the habituation response over the entire 50 block test session.

Functional observational battery
Home Cage Observations: Home cage 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.
Handling observations: Handling 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.
Open field observations: Open field parameters were unaffected by test substance administration. Any statistically significant differences noted for the test substance-treated males and females on PND 65 did not occur in a dose-responsive manner.
Sensory observations: Sensory 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.
Neuromuscular observations: Neuromuscular parameters were unaffected by test substance administration. Any statistically significant differences noted for the test substance-treated males and females on PND 65 did not occur in a dose-responsive manner.
Physiological observations: 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 test substance administration at all dose levels when evaluated 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. Differences from the control group were slight and not statistically significant. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the F1 animals were evaluated at PND 65.

Neuropathology (Cohorts 2A and 2B)
Macroscopic examination: No test substance-related macroscopic findings were noted. Any and all macroscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to test substance.

Brain Weights and Measurements: No test substance-related organ weight changes were noted in Cohort 2A (PND 78). Any variation in mean organ weights between treatment groups were considered incidental and/or unrelated to test substance administration. There was statistically significant lower brain weight in the 150 mg/kg/day males (p<0.01), but the change did not follow a dose response profile and had no correlation to other changes in brain measurements (gross or microscopic measurements). Therefore, the isolated statistical finding was considered to be unrelated to test substance administration. No test substance-related organ weight changes were noted in Cohort 2B (PND 22). Any variation in mean organ weights between treatment groups were considered incidental and/or unrelated to administration of the test substance. There was statistically significant lower brain width in the 150 mg/kg/day females (p<0.05), but the change did not follow a dose response profile and had no correlation to other changes in brain measurements (weight, other gross measurements, any microscopic measurements). Therefore, the isolated statistical finding was considered to be unrelated to test substance administration.

Microscopic examination: There were no test substance-related microscopic findings in the nervous system tissues evaluated.

Morphometric Analysis (Cohort 2A): There were no test substance-related effects on morphometric measurements in any dose groups of F1 males or females. There was a statistically significant increase in the S5 (hippocampus thickness) and S6 (cerebellum thickness) in the females in the 250 mg/kg/day group, when compared to the control group. These changes had no microscopic, organ weight, or gross brain measurement correlates, and were in a direction opposite to that normally expected for a developmental effect. Therefore, these 2 statistically significant changes were considered unrelated to test substance administration and were a result of biological variability.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

In F1 pups prior to weaning, there were lower serum total T4 in the 150 mg/kg/day male pups and lower total T4 in the 150 and 250 mg/kg/day female pups on PND 21. The changes in the total T4 showed no clear dose response profile in males or females. However, given the dose response profile for serum T4 in F0 males and females, the changes in T4 at PND 21 were considered to be test substance-related but nonadverse.
In the F1 generation, 2 males in the 75 mg/kg/day group were found dead on PND 21, 1 female each in the 75 and 150 mg/kg/day groups were found dead on PND 22 and 42, respectively. There were no remarkable clinical findings or body weight effects noted for any of these animals prior to death. An additional male in the 75 mg/kg/day group was euthanized in extremis on PND 41 following clinical signs of gasping, increased respiration rate and labored respiration. At necropsy, the female in the 150 mg/kg/day group was noted with oviduct cysts. There were no other macroscopic findings in the unscheduled deaths. Based on the lack of findings or deaths in the 250 mg/kg/day animals, these deaths were not attributed to the test substance. All other animals assigned to the F1 generation survived to the scheduled euthanasia. Statistically significantly lower total T4 values (33.4% to 41.9%) were noted in all test substance-treated group males compared to the control group on PND 91 and were considered test substance related. The lower T4 in males was not associated with any changes in thyroid gland weight or microscopic findings in the male thyroid gland. The changes were therefore not considered adverse. On PND 91, test substance-related organ weight changes, including lower prostate gland and seminal vesicles/coagulating gland/accessory gland (SV/CG/ACC) weights in 250 mg/kg/day F1 males and higher kidney and thyroid gland/parathyroid gland in 75, 150 and 250 mg/kg/day F1 females, were noted. The changes were considered test substance-related because the changes followed a dose response profile, however, were considered to be non-adverse and toxicologically irrelevant.
With the exception of the above-mentioned effects on F0 and F1 T4 levels and organ weights, no other test substance-related effects were noted on this study. Reproductive endpoints in the F0 and F1 generations and immunotoxicity and neurotoxicity endpoints in the F1 generation were unaffected by administration of the substance orally (via gavage) to Crl:WI(Han) rats at 75, 150, and 250 mg/kg/day. Based on the lack of any equivocal effects on the general categories of triggers for the production of a second-generation, a second generation was not assessed on this study.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1A)

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1B)

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

3

Dose descriptor:

NOAEL

Generation:

F1 (cohort 2A)

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

4

Dose descriptor:

NOAEL

Generation:

F1 (cohort 2B)

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

5

Dose descriptor:

NOAEL

Generation:

other: F1 surplus (used for post-weaning developmental landmarks)

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Summary or organ weight data (F0 generation)

Males				Females
Group	2	3	4	2	3	4
Dose (mg/kg/day)	75	150	250	75	150	250
No. animals per group	25	25	25	25	25	25
Liver (No. weighed)	25	25	25	25	25	25
Absolute value	13.56	13.52	13.62	9.48*	9.72**	10.03**
% of bodyweight	3.219	3.321**	3.408**	3.854*	3.893**	4.063**
% of brain weight	669.533	678.125	683.962	515.639*	520.026*	542.632**
Kidney (No. weighed)	25	25	25	25	25	25
Absolute value	2.99	2.99	2.97	1.92	1.99**	1.98**
% of bodyweight	0.711	0.735**	0.747**	0.784	0.798**	0.802**
% of brain weight	147.549	149.827	149.539	104.454	106.618**	106.958**

Based upon statistical analysis of group means, values with asterisk are significantly different from control group – P ≤ 0.05; refer to data tables for actual significance levels and tests used.

Bold – Test substance related

Results of F0 Reproductive Performance

Parameter	Dose level (mg/kg/day)				CRL HC Mean (Range)^a
	0	75	150	250
Male Mating Index (%)	100	96	100	100	99.4 (94.7-100)
Female Mating Index (%)	100	96	100	100	99.8 (96-100)
Male Fertility Index (%)	100	92	92	100	94.2 (84 -100)
Female Fertility Index (%)	100	92	92	100	94.4 (84 -100)
Male Copulation Index (%)	100	95.8	92	100	94.8 (84-100)
Female Conception Index (%)	100	95.8	92	100	94.5 (84 - 100)
Estrous Cycle Length (days)	4.6	4.2	3.8**	4.1	4.2 (3.6 – 4.8)
Pre-Coital Interval (days)	2.4	3.9	2.8	3.0	2.7 (1.9 – 3.6)

a - Charles River Ashland historical control data (version 2020.02).

* = Statistically significant at 0.05 compared to the control group.

** = Statistically significant at 0.01 compared to the control group.

Summary or organ weight data (F1 generation (Cohort 1A))

Males				Females
Group	2	3	4	2	3	4
Dose (mg/kg/day)	75	150	250	75	150	250
No. animals per group	20	20	20	20	20	20
Prostate (No. weighed)	20	20	20	-	-	-
Absolute value	0.57	0.57	0.52*	-	-	-
% of bodyweight	0.162	0.162	0.155**	-	-	-
% of brain weight	28.800	29.336	26.205**	-	-	-
SV/CG/ACC (No. weighed)	20	20	20	-	-	-
Absolute value	1.35	1.32	1.21**	-	-	-
% of bodyweight	0.386	0.372	0.361	-	-	-
% of brain weight	68.661	67.235	60.996**	-	-	-
Kidney (No. weighed)	20	20	20	20	20	20
Absolute value	2.87	2.95	2.74	1.74*	1.73*	1.78**
% of bodyweight	0.819	0.828	0.817	0.850**	0.842*	0.863**
% of brain weight	145.776	151.611*	138.021	95.108**	94.833**	99.451**
Thyroid/parathyroid (No. weighed)	20	20	20	20	20	20
Absolute value	0.0183	0.0189	0.0183	0.0153*	0.0155*	0.0156**
% of bodyweight	0.005	0.005	0.005	0.008*	0.008*	0.008*
% of brain weight	0.931	0.971	0.922	0.840*	0.850*	0.873**

Based upon statistical analysis of group means, values with asterisk are significantly different from control group – P ≤ 0.05; refer to data tables for actual significance levels and tests used.

Bold – Test substance related

Summary of Brain Morphometry - F1 Generation Cohort 2A (PND 78)

	Males		Females
Group	1	4	1	4
Dose level (mg/kg/day)	0	250	0	250
Level 1^a	10^b	10	10	10
S1 - Thickness Frontal Cortex^a	2.0450	2.0982	2.0665	2.0231
(SD)	0.0881	0.0818	0.1253	0.0982
S2 - Thickness Parietal Cortex	1.9995	2.0169	1.9529	1.9756
(SD)	0.0823	0.0747	0.1016	0.1166
S3 - Width Caudate-Putamen	4.2303	4.3213	4.2247	4.2031
(SD)	0.2150	0.1587	0.1414	0.2044
S4 - Thickness Corpus Callosum	0.3719	0.3918	0.3768	0.3678
(SD)	0.0562	0.0547	0.0546	0.0562
Level 2^a	10	10	10	10
S5 - Thickness Hippocampus	1.5315	1.5062	1.4519	1.5347*
(SD)	0.0762	0.0651	0.0509	0.0569
Level 3^a	10	10	10	10
S6 - Height Cerebellum	6.1396	6.2900	5.6663	6.2581*
(SD)	0.2885	0.3101	0.4259	0.2244

a - All values expressed as mean measurement in mm.

b - Number of animals measured

Values highlighted in bold are significantly different from control group – P ≤ 0.05; refer to data tables for actual significance levels and tests used.

Applicant's summary and conclusion

Conclusions:

Based on the lack of adverse findings noted for F0 and F1 males and females and F1 litters at all dose levels tested, 250 mg/kg/day (the highest level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for F0 and F1 systemic toxicity, F0 reproductive toxicity, F1 neonatal toxicity, and F1 immunotoxicity and brain morphometry for the test substance when administered via oral gavage to male and female Crl:WI(Han) rats.