ethyl 2-acetyl-4-methyltridec-2-enoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

comducted under GLP conditions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical control data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016, which recommends that evaluation of each group be initiated with at least 10 males and 12–13 females per group.
Females were evaluated for estrous cyclicity during the pretest period and any females that failed to exhibit normal 4–5 day estrous cycles (e.g., EDDDE), during the pretest period, were excluded from the study; therefore, extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test substance-related moribundity and/or mortality, 10 females per group was an appropriate number of animals to obtain a sample size of 8 females per group at termination.

Sex:

male/female

Details on test animals or test system and environmental conditions:

On 29 Sep 2020 and 13 Oct 2020, female and male Crl:CD(SD) rats, respectively, were received from Charles River Laboratories, Inc., Raleigh, NC. The animals were approximately 11 weeks old and weighed between 203 and 390 g at the initiation of dosing.

Each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Offspring were identified by tattoo markings applied to the digits after parturition.

After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing. The testes were palpated at least once for all males during acclimation.

Selection, Assignment, Replacement, and Disposition of Animals:
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 or not exhibiting normal, 4- to 5-day estrous cycles were not assigned to groups.
Before the initiation of dosing, any assigned animals considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
To reduce variability among the F1 litters, 8 pups/litter of equal sex distribution, if possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. The remaining offspring were euthanized by an intraperitoneal injection of sodium pentobarbital (following thyroid hormone blood collection for pups used for blood collection; see Section 4.8.2.6.) and discarded.
The disposition of all animals was documented in the Study Records.

Husbandry:
- Housing
On arrival, animals were group housed (up to 3 animals of the same sex) until cohabitation.
During cohabitation, animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study. 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. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory
Animals (National Research Council, 2011). The animal facilities at Charles River Ashland are accredited by AAALAC International.

- Environmental Conditions
Target temperatures of 68°F to 78°F (20°C to 26°C) with a relative target humidity of 30% to 70% were maintained. A 12-hour light/12-hour dark cycle was maintained, except when interrupted for designated procedures.
- Food
PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal) was provided ad libitum throughout the study.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility.
It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water
Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system. Water bottles were provided, if necessary. Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility.
It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Animal Enrichment
Animals were socially housed for psychological/environmental and were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities.
- Veterinary Care
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the Study Records and reviewed by the Study Director.

Route of administration:

oral: gavage

Details on route of administration:

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 dose levels were determined from results of previous studies and were provided by the Sponsor Representative after consultation with the Charles River Study Director. In a previous study, Scentaurus Clean was administered to male and female Sprague Dawley rats via oral gavage for 14 days at dose levels of 250, 500, 750, and 1000 mg/kg/day (Mesnard, 2021, 00810040). In that study, all animals survived to the scheduled necropsy, and no remarkable clinical observations were noted. Lower mean body weight gains and food consumption were noted for males at 750 and 1000 mg/kg/day during Study Days 0–4, resulting
in mean absolute body weights that were up to 6.1% and 6.2% lower, respectively, than the control group. There were no effects on body weight or food consumption in males at 250 and 500 mg/kg/day or in females at any dose level. Changes in organ weights (absolute, relative to final body weight, and/or relative to brain weight) included slightly higher liver weights for males at 500 and 1000 mg/kg/day and females at 500, 750, and 1000 mg/kg/day and lower spleen weights for males at 750 mg/kg/day.

Vehicle:

corn oil

Remarks:

Corn oil, NF Lot No 2IH0387, Expiration date: 31 Jul 2021, Storage conditions: Kept in controlled temperature area set to maintain 18°C to 24°C, protected from light

Details on oral exposure:

Test substance dosing formulations were prepared based on Sponsor instructions 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 refrigerated (target of 5°C), protected from light, under nitrogen, until use. The dosing formulations were stirred continuously during dosing. Details of the preparation and dispensing of the test substance have been retained in the Study Records.

The test substance and vehicle were administered as a single daily oral gavage dose. Males were dosed for 14 days prior to mating and continuing throughout mating for a minimum of 28 days.
Females were dosed for 14 days prior to mating and continuing throughout mating, gestation, and lactation until 1 day prior to scheduled euthanasia. All animals were dosed at approximately the same time each day.
The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses described below were performed by a gas chromatographic method with flame ionization detection using a validated analytical procedure (Akalkotkar, 2020, 00810039).
- Concentration analysis: Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration, with each individual sample concentration within ± 20% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.
- Homogeneity analysis: Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Homogeneity results were considered acceptable if the relative standard deviation of the mean value was ≤ 10% and if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.
- Stability and Resuspension homogeneity analysis: Test substance formulations have been previously shown to be stable and homogeneous over the range of concentrations used on this study for at least 8 days of refrigerated storage (Akalkotkar, 2020, 00810039). Therefore, stability and resuspension homogeneity of test substance formulations will not be assessed on this study.

Duration of treatment / exposure:

The test substance and vehicle were administered as a single daily oral gavage dose. Males were dosed for 14 days prior to mating and continuing throughout mating for a minimum of 28 days. Females were dosed for 14 days prior to mating and continuing throughout mating, gestation, and lactation until 1 day prior to scheduled euthanasia. All animals were dosed at approximately the same time each day.
The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.

Frequency of treatment:

The test substance and vehicle were administered as a single daily oral gavage dose.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1: Vehicle control

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 animals/sex/dose

The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016, which recommends that evaluation of each group be initiated with at least 10 males and 12–13 females per group.
Females were evaluated for estrous cyclicity during the pretest period and any females that failed to exhibit normal 4–5 day estrous cycles (e.g., EDDDE), during the pretest period, were excluded from the study; therefore, extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test substance-related moribundity and/or mortality, 10 females per group was an appropriate number of animals to obtain a sample size of 8 females per group at termination.

Control animals:

yes, concurrent vehicle

Positive control:

not performed

Observations and examinations performed and frequency:

F0 generation:
- 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 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 once daily throughout the study. During the dosing period, these observations were performed prior to dosing. On dosing days, clinical observations were also recorded 2 hours postdose During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.
- Body Weights
Animals were weighed individually twice weekly throughout the study. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 0 (when possible), 1, 4, 7, 10, and 13. A fasted weight was recorded on the day of necropsy.
- Food Consumption
Food consumption was quantitatively measured twice weekly until cohabitation. 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, 10, and 13.
- Estrous Cycles
For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed. The slides were microscopically examined to determine the stage of the estrous cycle. 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] for 14 consecutive days before cohabitation and 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 individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.
- Breeding Procedures
After a minimum of 14 days of dosing, the animals were paired on a 1:1 basis within each group. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Vaginal lavages were performed daily during the mating period until evidence of mating was observed. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.
- Parturition
The day parturition was completed was designated Lactation Day 0 (Postnatal Day [PND] 0 for pups). During the period of expected parturition, females were observed 3 times daily for initiation and completion of parturition and for dystocia 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.
- Neurobehavioral Testing
1) FOB Assessments
FOB assessments were recorded for 5 animals/sex/group during the last week of dosing (males) or on Lactation Day 13 (females). 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/Body Carriage, Convulsions, Stereotypy, Tremor, Palpebral Closure/Ptosis
Handling Observations: Ease of Removal, Handling Reactivity
Open Field Observations: Rearing, Arousal/Alertness, Gait/Mobility, Vocalizations, Tremor, Respiration, Urinationa/Defecation, Stereotypy, Convulsions, Appearance, Lacrimation, Salivation,Exophthalmus,Palpebral Closure/Ptosis,Erected Fur
Sensory Observations: Touch Response/Tactile Reflex,Startle Response,Tail Pinch Response,Pupil Response,Body Temperature
Neuromuscular Observations: Body Tone,Grip strength-hind and forelimb,Rotarod performance,Hindlimb foot splay,Air Righting Reflex
Physiological Observations: Catalepsy

2) Motor Activity
Motor activity was assessed for 5 animals/sex/group during the last week of dosing (males) or on Lactation Day 13 (females). 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).

- Clinical Pathology
1) Sample Collection
Animals were fasted overnight prior to blood collection. Blood samples for hematology and serum chemistry were collected from a jugular vein for males and from the retro-orbital sinus from females anesthetized with isoflurane. 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 serum chemistry were collected without anticoagulants.

2) Hematology
Blood samples were analyzed for the parameters specified below.

Hematology Parameters: Differential leukocyte counta,Erythrocyte count,Total hemoglobin,Hematocrit,Mean corpuscular hemoglobin,Mean corpuscular volume,Mean corpuscular hemoglobin concentration,Mean platelet volume,Platelet count,Red cell distribution width,Reticulocyte count,Total leukocyte count
All presented on individual tables if a manual differential was performed, and the manual data were accepted and reported instead of the automated differential data

3) Coagulation
Blood samples were processed for plasma, and the plasma was analyzed for the parameters specified below..
Coagulation Parameters: Activated partial thromboplastin time,Fibrinogen,Prothrombin time,Sample quality

4) Serum Chemistry
Blood samples were processed for serum, and the serum was analyzed for the parameters
specified below.
Serum Chemistry Parameters:
Alanine aminotransferase
Albumin
A/G ratio (calculated)
Alkaline phosphatase
Aspartate aminotransferase
Bile Acids
Calcium
Chloride
Creatinine
Gamma glutamyltransferase
Globulin (calculated)
Glucose
Phosphorus
Potassium
Sodium
Sorbitol dehydrogenase
Total bilirubin
Total cholesterol
Total protein
Triglycerides
Urea nitrogen
Sample quality

- Thyroid Hormone Analysis
1) Sample Collection
Blood samples for thyroid hormone analyses were collected from a jugular vein into tubes without anticoagulants.

Samples were collected according to the information below:
Group Nos 1-4, Sex: Males, Time point: Study day 28, Thyroid hormones sample collected
Group Nos 1-4 (samples not analyzed), Sex: Females, Time point: Lactation day 13, Thyroid hormones sample collected

2) Sample Processing
Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in
a refrigerated centrifuge and stored in a freezer set to maintain -70°C.
4.8.1.10.3. Sample Analysis
Blood samples were analyzed for the parameters specified below.
Thyroid Hormone Parameters: Triiodothyronine (T3),Thyroxine (Total T4), Thyroid Stimulating Hormone (TSH)

Samples to be analyzed for T3 and T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were performed using a validated UHPLC/MS/MS assay (Lucarell, 2017, 99764). Samples to be analyzed for TSH were transferred to the Charles River Ashland Immunotoxicology Department; analyses were performed using a validated Luminex Bead-Based assay (Castagnier, 2017, 3600258; Peachee, 2020, 00099827).

Sacrifice and pathology:

Terminal Procedures - F0 Generation

Terminal procedures are summarized below.
Group 1, 10 males and 10 females, scheduled euthanasia day: Study day 28 for Males and lactation day 14 for females, Necropsy procedures: Necropsy, tissue collection, organ weights, Histology: full tissues and target tissues, Histopathology: full tissues and target tissues
Group 2, 10 males and 10 females, scheduled euthanasia day: Study day 28 for Males and lactation day 14 for females, Necropsy procedures: Necropsy, tissue collection, organ weights, Histology: gross lesions and target tissues, Histopathology: gross lesions and target tissues
Group 3, 10 males and 10 females, scheduled euthanasia day: Study day 28 for Males and lactation day 14 for females, Necropsy procedures: Necropsy, tissue collection, organ weights, Histology: gross lesions and target tissues, Histopathology: gross lesions and target tissues
Group 4, 10 males and 10 females, scheduled euthanasia day: Study day 28 for Males and lactation day 14 for females, Necropsy procedures: Necropsy, tissue collection, organ weights, Histology: full tissues and target tissues, Histopathology: full tissues and target tissues

Full tissues: 5 animals/sex/group, see tissue collection and preservation list
Target tissues: all animals, adrenal glands (both sexes) and thymus (females only)
Lactation day 14 for females: Females that failed to deliver were euthanized on Postmating day 25

- Unscheduled Deaths
No animals died during the course of the study.
- Scheduled Euthanasia
All surviving animals, including females that failed to deliver, were euthanized by carbon dioxide inhalation.
- Necropsy
Animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of former implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. Postimplantation loss was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. Uteri of females without macroscopic evidence of implantation were opened and placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).
- Organ Weights
The organs identified in Text Table 19 were weighed at necropsy for all scheduled euthanasia animals (for exceptions, see Appendix 1). Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

Organs Weighed at Necropsy:
Adrenal glands
Brain
Epididymidesa
Heart
Kidneys
Liver
Ovaries with oviducts
Pituitary gland
Prostate gland
Seminal vesicle (with coagulating gland and fluid)
Spleen
Testes
Thymus gland
Thyroids with parathyroids

- Tissue Collection and Preservation
Representative samples of the tissues identified in the list below were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated (for exceptions, see Appendix 1 in the study report).

Tissue Collection and Preservation:
Adrenals (2)
Aorta
Bone with marrow (sternebrae)
Brain
Coagulating glands (2)
Eyes with optic nerve (2) a
Gastrointestinal tract
Esophagus
Stomach
Duodenum
Peyer’s Patches
Jejunum
Ileum
Cecum
Colon
Rectum
Heart
Kidneys (2)
Liver (sections of 2 lobes)
Lymph node
Axillary (2)
Mandibular (2)e
Mesenteric
Lungs (including bronchi, fixed by inflation with
fixative)
Ovaries and oviduct (2)
Pancreas
Peripheral nerve (sciatic)e
Pituitary
Prostate
Mandibular salivary glands (2)e
Seminal vesicles (2)
Skeletal muscle (quadriceps)
Skin with mammary gland b
Spinal cord (cervical)
Spleen
Testes with epididymides (2) c and vas deferens
Thymus
Thyroids (with parathyroids if present [2])
Trachea
Urinary bladder
Uterus d with cervix and vagina
All gross lesions (all groups)
a Placed in Davidson's solution.
b For females; a corresponding section of skin was taken from the same anatomic area for males.
c Placed in modified Davidson’s solution. Care was taken to ensure separation between the left and right organs.
d Any uterus that was placed in 10% ammonium sulfide solution for detection of implantation sites was discarded
and not preserved in 10% neutral-buffered formalin.
e Only 1 examined.

- Histology
Tissue trimming was performed at the Testing Facility. Tissues identified in Tissue Collection and Preservation list from 5 animals/sex in the control and high-dose groups, as well as gross lesions, adrenal glands, and the thymus (females only) from all animals in all groups, were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.
- Histopathology
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified in Tissue Collection and Preservation list for microscopic examination were evaluated from 5 animals/sex in the control and high-dose groups. Gross lesions, adrenal glands, and the thymus (females only) were examined from all animals in all groups.

Other examinations:

In life procedures, observations and measurements - F1 Litter parameters:
- Viability
Litters were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. A daily record of litter size was maintained. Animals were not removed from 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 on PND 1, 4, 7, 10, and 13.
- Sex Determination
Pups were individually sexed on PND 0 or 1, and PND 4 and 13.
- Body Weights
Pups were weighed individually on PND 1, 4 (before culling), 7, 10, and 13.
- Preweaning Developmental Landmarks
1) Anogenital Distance
The anogenital distance of all pups was measured on PND 1 and 4. Anogenital distance was defined as the distance from the cranial margin of the anus to the caudal margin of the genital tubercle (Gallavan et al., 1999).
2) Assessment of Areolas/Nipple Anlagen Retention
On PND 13, all male pups were evaluated for the presence of nipples/areolae (Ostby et al., 1999). The number of nipples was recorded.
- Thyroid Hormone Analysis
1) Sample Collection
Blood samples for thyroid hormone analyses were collected via cardiac puncture from animals anesthetized with isoflurane into tubes without anticoagulants.
Samples were collected according to the information below:
Group Nos 1-4 (samples were only collected from culled pups and were pooled by litter), No. of pups: at least 2/litter, Time point: Postnatal day 4, Thyroid hormones sample collected
Group Nos 1-4, No. of pups: 2/sex/litter, Time point: Postnatal day 13, Thyroid hormones sample collected
2) Sample Processing
Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain -70°C.
3)Sample Analysis
Blood samples were analyzed for the parameters specified below.
Thyroid Hormone Parameters: Triiodothyronine (T3),Thyroxine (Total T4), Thyroid Stimulating Hormone (TSH)

Samples to be analyzed for T3 and T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were performed using a validated UHPLC/MS/MS assay (Lucarell, 2017, 99764). Samples to be analyzed for TSH were transferred to the Charles River Ashland Immunotoxicology Department; analyses were performed using a validated Luminex Bead-Based assay (Castagnier, 2017, 3600258; Peachee, 2020, 00099827).

Terminal procedures - F1 Litter parameters:
Terminal procedures are summarized below.

Group 1, No. of animals: 80, Scheduled euthanasia day: Postnatal day 13, Necropsy procedures: Necropsy, tissue collection, organ weights
Group 2, No. of animals: 72, Scheduled euthanasia day: Postnatal day 13, Necropsy procedures: Necropsy, tissue collection, organ weights
Group 3, No. of animals: 71, Scheduled euthanasia day: Postnatal day 13, Necropsy procedures: Necropsy, tissue collection, organ weights
Group 4, No. of animals: 80, Scheduled euthanasia day: Postnatal day 13, Necropsy procedures: Necropsy, tissue collection, organ weights
Unscheduled deaths, Necropsy procedures: Necropsy, tissue collection

- Unscheduled Deaths
A necropsy was conducted for animals that died on study, and specified tissues were saved. Intact offspring that were found dead or euthanized for humane reasons during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). Findings were recorded as developmental variations or malformations, as appropriate. A gross necropsy was performed on any pup found dead after PND 4.
- Scheduled Euthanasia
On PND 13, surviving animals were euthanized via an intraperitoneal injection of sodium pentobarbital.
-Necropsy
On PND 13, 1 pup/sex/litter was subjected to a complete necropsy examination, with emphasis
on developmental morphology and organs of the reproductive system. All other animals were
discarded without examination.
- Organ Weights
The organs identified in below were weighed at necropsy from 1 pup/sex/litter at the scheduled euthanasia. Organ weights were not recorded for animals found dead.
Organs Weighed at Necropsy: Thyroid (with parathyroids, if present) (Weighed post-fixation)
- Tissue Collection and Preservation
Representative samples of the tissues identified in Organs Weighed at necropsy list were collected from 1 pup/sex/litter at the scheduled euthanasia and preserved in 10% neutral buffered formalin.

Statistics:

Data collected during the predose period were not tabulated, summarized, or statistically analyzed, unless applicable to analyses in the proceeding sections. All statistical analyses were performed within the respective study phase, unless otherwise noted. Clinical and necropsy observations data were summarized but no inferential statistical analysis was performed.
Numerical data collected on scheduled occasions were summarized and statistically analyzed as indicated below according to sex and occasion or by litter. Values may also be expressed as a percentage of pretreatment period or control values, or fold change of control values, when deemed appropriate. Calculated values on Provantis tables may not be reproducible from the individual values presented because all calculations were conducted using non-rounded values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Increased incidences of salivation or evidence thereof (wet fur around the mouth or muzzle) were noted for males and females in all test substance-treated groups at 2 hours postdosing; this finding began on Study Day 8 and continued throughout the dosing period and was likely attributable to the taste of the test substance. Ploughing (repetitive pushing of head into bedding material) was noted for 2 and 4 males in the 400 and 800 mg/kg/day groups, respectively, at 2 hours postdosing during Study Days 21-27. In females, this finding was noted for 1 female in the 200 mg/kg/day group and 6 females each in the 400 and 800 mg/kg/day groups during Gestation Days 7-21, and for 4, 7, and 10 females in the 200, 400, and 800 mg/kg/day groups, respectively, during lactation. This finding was also attributed to the taste of the test substance.
The aforementioned observations were not considered adverse.

No other test substance-related clinical findings were noted. Other clinical findings noted in the test substance-treated groups, including thin fur cover or fur staining on various body surfaces, were noted infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled necropsy. One 200 mg/kg/day group female (No. 2502) and one 400 mg/kg/day group female (No. 3507) failed to deliver and were euthanized on Postmating Day 25.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES:
Statistically significantly lower mean body weight gain was noted in the 800 mg/kg/day group males during Study Days 0-3. Body weight gains in this group were also slightly lower than the control group during Study Days 7-16; the difference was statistically significant during Study Days 7-10. As a result, mean body weight gains in this group were statistically significantly lower than the control group for the overall premating period (Study Days 0-13) and entire dosing period (Study Days 0-27), and mean absolute body weights were statistically significantly lower (7.7% to 7.8%) than the control group on Study Days 23 and 27.
These body weight effects at 800 mg/kg/day were considered test substance-related and adverse.

Mean body weights and body weight gains in the 200 and 400 mg/kg/day group males were unaffected by test substance administration throughout the study.

FEMALES:
- Weekly:
Mean body weight gains in the 800 mg/kg/day group females were generally higher than the control group throughout the premating dosing period (Study Days 0-13); differences were statistically significant during Study Days 3-7 and for the overall premating dosing period (Study Days 0-13). Mean absolute body weight in this group was 6.9% higher than the control group on Study Day 13 but did not achieve statistical significance.
Therefore, these changes were not considered adverse, as the direction of change is not toxicologically relevant.

Mean body weights and body weight gains in the 200 and 400 mg/kg/day group females were unaffected by test substance administration throughout the premating dosing period.

- Gestation:
Mean maternal body weights and body weight gains in the 200, 400, and 800 mg/kg/day groups were unaffected by test substance administration during gestation. Statistically significantly higher mean body weight gains noted in all test substance-treated groups during Gestation Days 11-14 were transient and had no effect on overall gestational body weight gain. Higher mean absolute body weights (4.2% to 7.5%) were noted in the 800 mg/kg/day group throughout gestation when compared to the control group but were due to the higher body weight gains noted during the premating dosing period.

- Lactation:
Mean body weights and body weight gains in the 200, 400, and 800 mg/kg/day groups were unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant. Higher mean absolute body weights (5.4% to 7.9%) were noted in the 800 mg/kg/day group throughout lactation when compared to the control group but were due to the higher body weight gains noted during the premating dosing period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MALES:
Mean food consumption in the 400 and 800 mg/kg/day group males was statistically significantly lower than the control group during Study Days 0-3, but comparable to the control group throughout the remainder of the premating dosing period. The lower food consumption at 800 mg/kg/day corresponded to a statistically significantly lower mean body weight gain during the same interval and was considered test substance-related and adverse. The lower food consumption at 400 mg/kg/day was transient, minimal in magnitude (approximately 4 g), and did not have a remarkable effect on mean body weight gain, and therefore was considered nonadverse.
Mean food consumption in the 200 mg/kg/day group males was similar to that in the control group throughout the premating dosing period.

FEMALES:
- Weekly:
Mean food consumption in the 800 mg/kg/day group females was slightly lower (not statistically significant) than the control group during Study Days 0-3, but comparable to or slightly higher than the control group during the remainder of the premating dosing period. The lower food consumption was transient, minimal in magnitude (approximately 3.5 g), and did not have a corresponding effect on mean body weight gain, and therefore was considered nonadverse.
Mean food consumption in the 200 and 400 mg/kg/day group females was similar to that in the control group throughout the premating dosing period.

- Gestation:
Mean maternal food consumption in the 200, 400, and 800 mg/kg/day groups was unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.

- Lactation
Mean food consumption in the 200, 400, and 800 mg/kg/day groups was unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related effects on hematology parameters were observed at any dosage level.
Mean absolute eosinophils in all test substance-treated males appeared lower (not statistically significant) than the control group because of a higher mean in controls due to 2 animals (Nos. 1003 and 1004) with unusually high values. The mean values in the test substance-treated groups were within the range of values in the Charles River Ashland historical control data (Version 3.6), whereas the control group value was above the maximum mean value in the Charles River historical control data. Statistically significantly higher mean white blood cell counts (WBC) and absolute basophil values were noted for females in the 400 mg/kg/day group; however, these values did not occur in a dose-related manner. Other differences from the control group were not statistically significant and/or did not occur in a dose-responsive manner.

No test substance-related effects on coagulation parameters were observed at any dosage level. A statistically significantly lower APTT was noted in the 800 mg/kg/day group females, but the value was within the range of values in the Charles River Ashland historical control data. Other differences from the control group were not statistically significant and/or did not occur in a dose-responsive manner.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related effects on serum chemistry parameters were noted for males at 400 and 800 mg/kg/day and for females in all test substance-treated group. Higher mean ALP values were noted in the 400 and 800 mg/kg/day group males and females compared to the control group; differences were statistically significant for the 800 mg/kg/day group females. In addition, statistically significantly higher mean ALT values were noted for the 400 and 800 mg/kg/day group males compared to the control group. Furthermore, higher (not statistically significant) mean GGT and bile acid values were noted for the 800 mg/kg/day group males and 400 and 800 mg/kg/day group females. Finally, higher SDH levels were noted for females in all test substance-treated groups, and a statistically significantly lower mean cholesterol value was noted for females in the 800 mg/kg/day group. These changes, which were indicative of effects on liver enzymes, were noted in the absence of any histopathological correlates in the liver, and therefore these changes in serum chemistry parameters were not considered adverse.
No other test substance-related effects were noted on serum chemistry parameters.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly lower mean Total T4 values were noted in the 200, 400, and 800 mg/kg/day groups. A corresponding slightly higher TSH value was noted in the 800 mg/kg/day group compared to the control group, although the difference was not statistically significant. Total T3 values were comparable across all groups. There were no corresponding organ weight changes or histopathology findings in the thyroid at any dosage level. In addition, all values in all groups were within the range of values in the Charles River Ashland historical control data. Therefore, these thyroid hormone changes were not considered adverse.

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test substance administration at all concentrations when evaluated on Study Day 27 (males) or Lactation Day 13 (females). Values obtained from the 6 subintervals evaluated (0–10, 11–20, 21–30, 31–40, 41–50 and 51–60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the Charles River Ashland historical control data, with the following exception. The mean number of total activity counts for males in the 400 mg/kg/day group when all intervals were combined was statistically significantly higher than the control group; however, this did not occur in a dose-related manner.
Other differences from the control group were slight, not statistically significant, within the Charles River Ashland historical control data ranges and/or did not occur in a dose/exposurerelated manner. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the F0 animals were evaluated on Study Day 27 (males) or Lactation Day 13 (females).

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher mean adrenal gland weights were noted in the 800 mg/kg/day group in both sexes and correlated with diffuse adrenal cortical hypertrophy. Higher mean liver weights were noted in the 800 mg/kg/day group females, but there was no microscopic correlate.
Although not statistically significant, lower mean thymus weights were noted in the 800 mg/kg/day group in both sexes. This finding correlated to a reduced cortico-medullary ratio in the thymus of the females.
No other test substance-related organ weight changes were noted.

There were other isolated organ weight values that were statistically different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, other organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to administration of the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test substance.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Neurobehavioral assessments were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group on Study Day 27 (males) or on Lactation Day 13 (females).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the adrenal gland, test substance-related diffuse cortical hypertrophy was characterized by enlarged cortical cells, particularly in the zona fasciculata, and correlated to the increased adrenal gland weights in the 800 mg/kg/day group.
In the thymus, decreased cortico-medullary ratio was noted in the 800 mg/kg/day group females and correlated to decreased mean thymus weights.
These findings were considered nonadverse given the minimal severity.

Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

F0 systemic toxicity

Effect level:

>= 800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no evidence of adverse toxicity for F0 females up to the highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

F0 systemic toxicity

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

F1 neonatal toxicity

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

no

Conclusions:

Under the conditions of this screening study, in the absence of any effects on reproductive parameters at any dosage level, a dosage level of 800 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity of Scentaurus Clean when administered orally by gavage to Crl:CD(SD) rats. Decrements in body weight and body weight gains were noted for F0 males at 800 mg/kg/day, but there was no evidence of adverse toxicity for F0 females; therefore, the NOAEL for F0 systemic toxicity was considered to be 400 mg/kg/day for F0 males and 800 mg/kg/day for F0 females. The NOAEL for F1 neonatal toxicity was 400 mg/kg/day based on the effects on pup body weights and body weight gains for males and females in the 800 mg/kg/day group.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test substance when administered to rats for 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behavior, and conception through day 13 of postnatal life.

The study design was as follows:

Group Number	Test Substance	Dosage Level (mg/kg/day)	Number of Males	Number of Females
1	Vehicle Control	0	5	5
2	Scentaurus clean	200	5	5
3	Scentaurus clean	400	5	5
3	Scentaurus clean	800	5	5

Animals were dosed via oral gavage once daily. Males were dosed for 14 days prior to mating and continuing throughout mating for a minimum of 28 days (Study Days 0–27). Females were dosed for 14 days prior to mating and continuing through Lactation Day 13. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, neurobehavior, locomotor activity, thyroid hormones, clinical pathology, macroscopic findings, organ weights, and microscopic examinations.

No test substance-related effects were noted on F0 survival, estrous cyclicity, reproductive performance, parturition, neurobehavior, motor activity, or macroscopic findings.

Test substance-related clinical observations included increased incidences of salivation or evidence thereof (wet fur around the mouth or muzzle) for males and females in all test substance-treated groups at 2 hours following dose administration; these findings were likely attributable to the taste of the test substance. In addition, ploughing (repetitive pushing of head into bedding material) was noted for males in the 400 and 800 mg/kg/day groups and females in the 200, 400, and 800 mg/kg/day groups at 2 hours following dose administration and was also attributable to the taste of the test substance. The aforementioned observations were not considered adverse.

Test substance-related, statistically significantly lower mean body weight gain was noted in the 800 mg/kg/day group F0 males for the overall dosing period (Study Days 0-27), with corresponding statistically significantly lower mean food consumption during Study Days 0-3.

As a result, mean absolute body weights were statistically significantly lower (7.7% to 7.8%) than the control group on Study Days 23 and 27. These body weight effects in the 800 mg/kg/day group were considered adverse. Statistically significantly lower mean food consumption, with no corresponding remarkable change in body weight gain, was noted in the 400 mg/kg/day group males at the start of dosing (Study Days 0-3). This test substance-related change was transient, and not considered adverse. There were no other test substance-related effects on body weight or food consumption parameters for males at 200 and 400 mg/kg/day.

For F0 females, test substance-related, statistically significantly higher mean body weight gain was noted in the 800 mg/kg/day group for the overall premating dosing period (Study Days 0-13), with corresponding slightly higher mean food consumption noted sporadically during the premating period. Mean absolute body weight in this group was 6.9% higher (not statistically significant) than the control group on Study Day 13. While mean absolute body weights remained slightly higher (4.2% to 7.9%) in the 800 mg/kg/day group females during gestation and lactation when compared to the control group, mean body weight gains and food consumption were unaffected by test substance administration during these periods. The higher mean body weights and body weight gains for the 800 mg/kg/day group females were not

considered to be adverse. There were no test substance-related effects on body weights, body weight gains, or food consumption for females at 200 and 400 mg/kg/day.

There were no test substance-related effects on hematology or coagulation parameters for F0 males or females at any dosage level. Test substance-related effects on serum chemistry parameters included higher mean ALP values in the 400 and 800 mg/kg/day group males and females, higher mean ALT values for the 400 and 800 mg/kg/day group males, higher mean GGT and bile acid values for the 800 mg/kg/day group males and 400 and 800 mg/kg/day group females, higher mean SDH levels for the 200, 400, and 800 mg/kg/day group females, and a lower mean cholesterol value for the 800 mg/kg/day group females. Despite higher liver weights for females at 800 mg/kg/day, there were no histopathological correlates in the liver, and therefore these changes in serum chemistry parameters were not considered adverse.

Statistically significantly lower mean Total T4 values were noted in the 200, 400, and 800 mg/kg/day group F0 males, with a corresponding higher (not statistically significant) TSH value in the 800 mg/kg/day group compared to the control group. Mean Total T3 values were comparable across groups, and there were no corresponding organ weight changes or histopathology findings in the thyroid at any dosage level. Therefore, these thyroid hormone changes were not considered adverse.

Test substance-related effects on organ weights included higher mean adrenal gland weights in the 800 mg/kg/day group males and females, higher mean liver weights in the 800 mg/kg/day group females, and lower mean thymus weights in the 800 mg/kg/day group males and females.

Microscopically, test substance-related diffuse cortical hypertrophy in the adrenal gland was noted for males and females in the 800 mg/kg/day group and correlated to increased adrenal gland weights. In the thymus, decreased cortico-medullary ratio was noted in the 800 kg/kg/day group females and correlated to decreased mean thymus weights. All findings were considered nonadverse given the minimal severity. There were no test substance-related organ weight changes or microscopic findings at 200 and 400 mg/kg/day.

No test substance-related effects were noted on F1 litter viability and survival, clinical observations, anogenital distance, areolae/nipple anlagen retention, thyroid hormone values (PND 4 or PND 13), macroscopic findings, and organ weights.

Mean pup body weight gains for F1 males and females in the 800 mg/kg/day group were lower than the control group during PND 1-13, with statistical significance achieved during PND 7-13.

As a result, mean absolute body weights in the 800 mg/kg/day group were statistically significantly lower (13.75% for males and 11.92% for females) than the control group on PND 13. The effects on pup body weight and body weight gain at 800 mg/kg/day were considered test substance-related and adverse. Despite these differences, mean pup body weights in all groups were within the range of values in the Charles River Ashland historical control data (version 2019.05). There were no test substance-related effects on pup body weights and body weight gains at 200 and 400 mg/kg/day.

Under the conditions of this screening study, in the absence of any effects on reproductive parameters at any dosage level, a dosage level of 800 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity of Scentaurus Clean when administered orally by gavage to Crl:CD(SD) rats. Decrements in body weight and body weight gains were noted for F0 males at 800 mg/kg/day, but there was no evidence of adverse toxicity for F0 females; therefore, the NOAEL for F0 systemic toxicity was considered to be 400 mg/kg/day for F0 males and

800 mg/kg/day for F0 females. The NOAEL for F1 neonatal toxicity was 400 mg/kg/day based on the effects on pup body weights and body weight gains for males and females in the 800 mg/kg/day group.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The objective of this study was to evaluate the potential toxic effects of the test substance when administered to rats for 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behavior, and conception through day 13 of postnatal life.
The design of this study is based on OECD Test Guideline 422.

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-acetyl-4-methyltridec-2-enoate

EC Number:

816-455-0

Cas Number:

960253-23-0

Molecular formula:

C18H32O3

IUPAC Name:

ethyl 2-acetyl-4-methyltridec-2-enoate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical control data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016, which recommends that evaluation of each group be initiated with at least 10 males and 12–13 females per group.
Females were evaluated for estrous cyclicity during the pretest period and any females that failed to exhibit normal 4–5 day estrous cycles (e.g., EDDDE), during the pretest period, were excluded from the study; therefore, extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test substance-related moribundity and/or mortality, 10 females per group was an appropriate number of animals to obtain a sample size of 8 females per group at termination.After a minimum of 14 days of dosing, the animals were paired on a 1:1 basis within each group.
Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the
presence of sperm in a vaginal lavage. Vaginal lavages were performed daily during the mating

Sex:

male/female

Details on test animals or test system and environmental conditions:

On 29 Sep 2020 and 13 Oct 2020, female and male Crl:CD(SD) rats, respectively, were received from Charles River Laboratories, Inc., Raleigh, NC. The animals were approximately 11 weeks old and weighed between 203 and 390 g at the initiation of dosing.

Each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Offspring were identified by tattoo markings applied to the digits after parturition.

After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing. The testes were palpated at least once for all males during acclimation.

Selection, Assignment, Replacement, and Disposition of Animals:
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 or not exhibiting normal, 4- to 5-day estrous cycles were not assigned to groups.
Before the initiation of dosing, any assigned animals considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
To reduce variability among the F1 litters, 8 pups/litter of equal sex distribution, if possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. The remaining offspring were euthanized by an intraperitoneal injection of sodium pentobarbital (following thyroid hormone blood collection for pups used for blood collection; see Section 4.8.2.6.) and discarded.
The disposition of all animals was documented in the Study Records.

Husbandry:
- Housing
On arrival, animals were group housed (up to 3 animals of the same sex) until cohabitation.
During cohabitation, animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study. 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. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory
Animals (National Research Council, 2011). The animal facilities at Charles River Ashland are accredited by AAALAC International.

- Environmental Conditions
Target temperatures of 68°F to 78°F (20°C to 26°C) with a relative target humidity of 30% to 70% were maintained. A 12-hour light/12-hour dark cycle was maintained, except when interrupted for designated procedures.
- Food
PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal) was provided ad libitum throughout the study.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility.
It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water
Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system. Water bottles were provided, if necessary. Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility.
It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Animal Enrichment
Animals were socially housed for psychological/environmental and were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities.
- Veterinary Care
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the Study Records and reviewed by the Study Director.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Corn oil, NF Lot No 2IH0387, Expiration date: 31 Jul 2021, Storage conditions: Kept in controlled temperature area set to maintain 18°C to 24°C, protected from light

Details on exposure:

Test substance dosing formulations were prepared based on Sponsor instructions 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 refrigerated (target of 5°C), protected from light, under nitrogen, until use. The dosing formulations were stirred continuously during dosing. Details of the preparation and dispensing of the test substance have been retained in the Study Records.

The test substance and vehicle were administered as a single daily oral gavage dose. Males were dosed for 14 days prior to mating and continuing throughout mating for a minimum of 28 days.
Females were dosed for 14 days prior to mating and continuing throughout mating, gestation, and lactation until 1 day prior to scheduled euthanasia. All animals were dosed at approximately the same time each day.
The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.

Details on mating procedure:

After a minimum of 14 days of dosing, the animals were paired on a 1:1 basis within each group.
Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Vaginal lavages were performed daily during the mating period until evidence of mating was observed. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses described below were performed by a gas chromatographic method with flame ionization detection using a validated analytical procedure (Akalkotkar, 2020, 00810039).
- Concentration analysis: Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration, with each individual sample concentration within ± 20% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.
- Homogeneity analysis: Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Homogeneity results were considered acceptable if the relative standard deviation of the mean value was ≤ 10% and if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.
- Stability and Resuspension homogeneity analysis: Test substance formulations have been previously shown to be stable and homogeneous over the range of concentrations used on this study for at least 8 days of refrigerated storage (Akalkotkar, 2020, 00810039). Therefore, stability and resuspension homogeneity of test substance formulations will not be assessed on this study.

Duration of treatment / exposure:

The test substance and vehicle were administered as a single daily oral gavage dose. Males were dosed for 14 days prior to mating and continuing throughout mating for a minimum of 28 days. Females were dosed for 14 days prior to mating and continuing throughout mating, gestation, and lactation until 1 day prior to scheduled euthanasia. All animals were dosed at approximately the same time each day.
The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.

Frequency of treatment:

The test substance and vehicle were administered as a single daily oral gavage dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1: vehicle control

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 animals/sex/dose

The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016, which recommends that evaluation of each group be initiated with at least 10 males and 12–13 females per group.
Females were evaluated for estrous cyclicity during the pretest period and any females that failed to exhibit normal 4–5 day estrous cycles (e.g., EDDDE), during the pretest period, were excluded from the study; therefore, extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test substance-related moribundity and/or mortality, 10 females per group was an appropriate number of animals to obtain a sample size of 8 females per group at termination.

Control animals:

yes, concurrent vehicle

Details on study design:

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 dose levels were determined from results of previous studies and were provided by the Sponsor Representative after consultation with the Charles River Study Director. In a previous study, Scentaurus Clean was administered to male and female Sprague Dawley rats via oral gavage for 14 days at dose levels of 250, 500, 750, and 1000 mg/kg/day (Mesnard, 2021, 00810040). In that study, all animals survived to the scheduled necropsy, and no remarkable clinical observations were noted. Lower mean body weight gains and food consumption were noted for males at 750 and 1000 mg/kg/day during Study Days 0–4, resulting in mean absolute body weights that were up to 6.1% and 6.2% lower, respectively, than the control group. There were no effects on body weight or food consumption in males at 250 and 500 mg/kg/day or in females at any dose level. Changes in organ weights (absolute, relative to final body weight, and/or relative to brain weight) included slightly higher liver weights for males at 500 and 1000 mg/kg/day and females at 500, 750, and 1000 mg/kg/day and lower spleen weights for males at 750 mg/kg/day.

Positive control:

not performed

Examinations

Parental animals: Observations and examinations:

F0 generation:
- 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 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 once daily throughout the study. During the dosing period, these observations were performed prior to dosing. On dosing days, clinical observations were also recorded 2 hours postdose During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.
- Body Weights
Animals were weighed individually twice weekly throughout the study. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 0 (when possible), 1, 4, 7, 10, and 13. A fasted weight was recorded on the day of necropsy.
- Food Consumption
Food consumption was quantitatively measured twice weekly until cohabitation. 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, 10, and 13.
- Estrous Cycles
For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed. The slides were microscopically examined to determine the stage of the estrous cycle. 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] for 14 consecutive days before cohabitation and 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 individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.
- Breeding Procedures
After a minimum of 14 days of dosing, the animals were paired on a 1:1 basis within each group. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Vaginal lavages were performed daily during the mating period until evidence of mating was observed. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.
- Parturition
The day parturition was completed was designated Lactation Day 0 (Postnatal Day [PND] 0 for pups). During the period of expected parturition, females were observed 3 times daily for initiation and completion of parturition and for dystocia 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.
- Neurobehavioral Testing
1) FOB Assessments
FOB assessments were recorded for 5 animals/sex/group during the last week of dosing (males) or on Lactation Day 13 (females). 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/Body Carriage, Convulsions, Stereotypy, Tremor, Palpebral Closure/Ptosis
Handling Observations: Ease of Removal, Handling Reactivity
Open Field Observations: Rearing, Arousal/Alertness, Gait/Mobility, Vocalizations, Tremor, Respiration, Urinationa/Defecation, Stereotypy, Convulsions, Appearance, Lacrimation, Salivation,Exophthalmus,Palpebral Closure/Ptosis,Erected Fur
Sensory Observations: Touch Response/Tactile Reflex,Startle Response,Tail Pinch Response,Pupil Response,Body Temperature
Neuromuscular Observations: Body Tone,Grip strength-hind and forelimb,Rotarod performance,Hindlimb foot splay,Air Righting Reflex
Physiological Observations: Catalepsy

2) Motor Activity
Motor activity was assessed for 5 animals/sex/group during the last week of dosing (males) or on Lactation Day 13 (females). 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).

- Clinical Pathology
1) Sample Collection
Animals were fasted overnight prior to blood collection. Blood samples for hematology and serum chemistry were collected from a jugular vein for males and from the retro-orbital sinus from females anesthetized with isoflurane. 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 serum chemistry were collected without anticoagulants.

2) Hematology
Blood samples were analyzed for the parameters specified below.

Hematology Parameters: Differential leukocyte counta,Erythrocyte count,Total hemoglobin,Hematocrit,Mean corpuscular hemoglobin,Mean corpuscular volume,Mean corpuscular hemoglobin concentration,Mean platelet volume,Platelet count,Red cell distribution width,Reticulocyte count,Total leukocyte count
All presented on individual tables if a manual differential was performed, and the manual data were accepted and reported instead of the automated differential data

3) Coagulation
Blood samples were processed for plasma, and the plasma was analyzed for the parameters specified below..
Coagulation Parameters: Activated partial thromboplastin time,Fibrinogen,Prothrombin time,Sample quality

4) Serum Chemistry
Blood samples were processed for serum, and the serum was analyzed for the parameters
specified below.
Serum Chemistry Parameters:
Alanine aminotransferase
Albumin
A/G ratio (calculated)
Alkaline phosphatase
Aspartate aminotransferase
Bile Acids
Calcium
Chloride
Creatinine
Gamma glutamyltransferase
Globulin (calculated)
Glucose
Phosphorus
Potassium
Sodium
Sorbitol dehydrogenase
Total bilirubin
Total cholesterol
Total protein
Triglycerides
Urea nitrogen
Sample quality

- Thyroid Hormone Analysis
1) Sample Collection
Blood samples for thyroid hormone analyses were collected from a jugular vein into tubes without anticoagulants.

Samples were collected according to the information below:
Group Nos 1-4, Sex: Males, Time point: Study day 28, Thyroid hormones sample collected
Group Nos 1-4 (samples not analyzed), Sex: Females, Time point: Lactation day 13, Thyroid hormones sample collected

2) Sample Processing
Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in
a refrigerated centrifuge and stored in a freezer set to maintain -70°C.
4.8.1.10.3. Sample Analysis
Blood samples were analyzed for the parameters specified below.
Thyroid Hormone Parameters: Triiodothyronine (T3),Thyroxine (Total T4), Thyroid Stimulating Hormone (TSH)

Samples to be analyzed for T3 and T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were performed using a validated UHPLC/MS/MS assay (Lucarell, 2017, 99764). Samples to be analyzed for TSH were transferred to the Charles River Ashland Immunotoxicology Department; analyses were performed using a validated Luminex Bead-Based assay (Castagnier, 2017, 3600258; Peachee, 2020, 00099827).

Oestrous cyclicity (parental animals):

For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed. The slides were microscopically examined to determine the stage of the estrous cycle. 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] for 14 consecutive days before cohabitation and 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 individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Sperm parameters (parental animals):

Not examined

Litter observations:

In life procedures, observations and measurements - F1 Litter parameters:
- Viability
Litters were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. A daily record of litter size was maintained. Animals were not removed from 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 on PND 1, 4, 7, 10, and 13.
- Sex Determination
Pups were individually sexed on PND 0 or 1, and PND 4 and 13.
- Body Weights
Pups were weighed individually on PND 1, 4 (before culling), 7, 10, and 13.
- Preweaning Developmental Landmarks
1) Anogenital Distance
The anogenital distance of all pups was measured on PND 1 and 4. Anogenital distance was defined as the distance from the cranial margin of the anus to the caudal margin of the genital tubercle (Gallavan et al., 1999).
2) Assessment of Areolas/Nipple Anlagen Retention
On PND 13, all male pups were evaluated for the presence of nipples/areolae (Ostby et al., 1999). The number of nipples was recorded.
- Thyroid Hormone Analysis
1) Sample Collection
Blood samples for thyroid hormone analyses were collected via cardiac puncture from animals anesthetized with isoflurane into tubes without anticoagulants.
Samples were collected according to the information below:
Group Nos 1-4 (samples were only collected from culled pups and were pooled by litter), No. of pups: at least 2/litter, Time point: Postnatal day 4, Thyroid hormones sample collected
Group Nos 1-4, No. of pups: 2/sex/litter, Time point: Postnatal day 13, Thyroid hormones sample collected
2) Sample Processing
Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain -70°C.
3)Sample Analysis
Blood samples were analyzed for the parameters specified below.
Thyroid Hormone Parameters: Triiodothyronine (T3),Thyroxine (Total T4), Thyroid Stimulating Hormone (TSH)

Samples to be analyzed for T3 and T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were performed using a validated UHPLC/MS/MS assay (Lucarell, 2017, 99764). Samples to be analyzed for TSH were transferred to the Charles River Ashland Immunotoxicology Department; analyses were performed using a validated Luminex Bead-Based assay (Castagnier, 2017, 3600258; Peachee, 2020, 00099827).

Postmortem examinations (parental animals):

Terminal Procedures - F0 Generation

Terminal procedures are summarized below.
Group 1, 10 males and 10 females, scheduled euthanasia day: Study day 28 for Males and lactation day 14 for females, Necropsy procedures: Necropsy, tissue collection, organ weights, Histology: full tissues and target tissues, Histopathology: full tissues and target tissues
Group 2, 10 males and 10 females, scheduled euthanasia day: Study day 28 for Males and lactation day 14 for females, Necropsy procedures: Necropsy, tissue collection, organ weights, Histology: gross lesions and target tissues, Histopathology: gross lesions and target tissues
Group 3, 10 males and 10 females, scheduled euthanasia day: Study day 28 for Males and lactation day 14 for females, Necropsy procedures: Necropsy, tissue collection, organ weights, Histology: gross lesions and target tissues, Histopathology: gross lesions and target tissues
Group 4, 10 males and 10 females, scheduled euthanasia day: Study day 28 for Males and lactation day 14 for females, Necropsy procedures: Necropsy, tissue collection, organ weights, Histology: full tissues and target tissues, Histopathology: full tissues and target tissues

Full tissues: 5 animals/sex/group, see tissue collection and preservation list
Target tissues: all animals, adrenal glands (both sexes) and thymus (females only)
Lactation day 14 for females: Females that failed to deliver were euthanized on Postmating day 25

- Unscheduled Deaths
No animals died during the course of the study.
- Scheduled Euthanasia
All surviving animals, including females that failed to deliver, were euthanized by carbon dioxide inhalation.
- Necropsy
Animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of former implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. Postimplantation loss was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. Uteri of females without macroscopic evidence of implantation were opened and placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).
- Organ Weights
The organs identified in Text Table 19 were weighed at necropsy for all scheduled euthanasia animals (for exceptions, see Appendix 1). Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

Organs Weighed at Necropsy:
Adrenal glands
Brain
Epididymidesa
Heart
Kidneys
Liver
Ovaries with oviducts
Pituitary gland
Prostate gland
Seminal vesicle (with coagulating gland and fluid)
Spleen
Testes
Thymus gland
Thyroids with parathyroids

- Tissue Collection and Preservation
Representative samples of the tissues identified in the list below were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated (for exceptions, see Appendix 1 in the study report).

Tissue Collection and Preservation:
Adrenals (2)
Aorta
Bone with marrow (sternebrae)
Brain
Coagulating glands (2)
Eyes with optic nerve (2) a
Gastrointestinal tract
Esophagus
Stomach
Duodenum
Peyer’s Patches
Jejunum
Ileum
Cecum
Colon
Rectum
Heart
Kidneys (2)
Liver (sections of 2 lobes)
Lymph node
Axillary (2)
Mandibular (2)e
Mesenteric
Lungs (including bronchi, fixed by inflation with
fixative)
Ovaries and oviduct (2)
Pancreas
Peripheral nerve (sciatic)e
Pituitary
Prostate
Mandibular salivary glands (2)e
Seminal vesicles (2)
Skeletal muscle (quadriceps)
Skin with mammary gland b
Spinal cord (cervical)
Spleen
Testes with epididymides (2) c and vas deferens
Thymus
Thyroids (with parathyroids if present [2])
Trachea
Urinary bladder
Uterus d with cervix and vagina
All gross lesions (all groups)
a Placed in Davidson's solution.
b For females; a corresponding section of skin was taken from the same anatomic area for males.
c Placed in modified Davidson’s solution. Care was taken to ensure separation between the left and right organs.
d Any uterus that was placed in 10% ammonium sulfide solution for detection of implantation sites was discarded
and not preserved in 10% neutral-buffered formalin.
e Only 1 examined.

- Histology
Tissue trimming was performed at the Testing Facility. Tissues identified in Tissue Collection and Preservation list from 5 animals/sex in the control and high-dose groups, as well as gross lesions, adrenal glands, and the thymus (females only) from all animals in all groups, were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.
- Histopathology
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified in Tissue Collection and Preservation list for microscopic examination were evaluated from 5 animals/sex in the control and high-dose groups. Gross lesions, adrenal glands, and the thymus (females only) were examined from all animals in all groups.

Postmortem examinations (offspring):

Terminal procedures - F1 Litter parameters:
Terminal procedures are summarized below.

Group 1, No. of animals: 80, Scheduled euthanasia day: Postnatal day 13, Necropsy procedures: Necropsy, tissue collection, organ weights
Group 2, No. of animals: 72, Scheduled euthanasia day: Postnatal day 13, Necropsy procedures: Necropsy, tissue collection, organ weights
Group 3, No. of animals: 71, Scheduled euthanasia day: Postnatal day 13, Necropsy procedures: Necropsy, tissue collection, organ weights
Group 4, No. of animals: 80, Scheduled euthanasia day: Postnatal day 13, Necropsy procedures: Necropsy, tissue collection, organ weights
Unscheduled deaths, Necropsy procedures: Necropsy, tissue collection

- Unscheduled Deaths
A necropsy was conducted for animals that died on study, and specified tissues were saved. Intact offspring that were found dead or euthanized for humane reasons during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). Findings were recorded as developmental variations or malformations, as appropriate. A gross necropsy was performed on any pup found dead after PND 4.
- Scheduled Euthanasia
On PND 13, surviving animals were euthanized via an intraperitoneal injection of sodium pentobarbital.
-Necropsy
On PND 13, 1 pup/sex/litter was subjected to a complete necropsy examination, with emphasis
on developmental morphology and organs of the reproductive system. All other animals were
discarded without examination.
- Organ Weights
The organs identified in below were weighed at necropsy from 1 pup/sex/litter at the scheduled euthanasia. Organ weights were not recorded for animals found dead.
Organs Weighed at Necropsy: Thyroid (with parathyroids, if present) (Weighed post-fixation)
- Tissue Collection and Preservation
Representative samples of the tissues identified in Organs Weighed at necropsy list were collected from 1 pup/sex/litter at the scheduled euthanasia and preserved in 10% neutral buffered formalin.

Statistics:

Data collected during the predose period were not tabulated, summarized, or statistically analyzed, unless applicable to analyses in the proceeding sections. All statistical analyses were performed within the respective study phase, unless otherwise noted. Clinical and necropsy observations data were summarized but no inferential statistical analysis was performed.
Numerical data collected on scheduled occasions were summarized and statistically analyzed as indicated below according to sex and occasion or by litter. Values may also be expressed as a percentage of pretreatment period or control values, or fold change of control values, when deemed appropriate. Calculated values on Provantis tables may not be reproducible from the individual values presented because all calculations were conducted using non-rounded values.

Reproductive indices:

The following parental indices and natural delivery/reproductive parameters were reported, as appropriate:
- Gestation Length = The gestation length is calculated from Gestation Day 0 to the day the first pup is observed.
- Female Mating Index = Number of Females with Evidence of Mating (or no confirmed mating date and pregnant) / Number of Females Paired
- Female Fertility Index = Number of Pregnant Females / Number of Females with Evidence of Mating (or no confirmed mating date and pregnant)
- Female Pregnancy Index = Number of Pregnant Females / Number of Females Paired
- Male Mating Index = Number of Males with Evidence of Mating (or female partner confirmed pregnant) / Number of Males Paired
- Male Fertility Index = Number of Males Impregnating a Female / Number of Males with Evidence of Mating (or female partner confirmed pregnant)
- Male Pregnancy Index = Number of Males Impregnating a Female / Number of Males Paired
- Gestation Index: Percentage of pregnancies that result in birth of live fetuses = (Number of Animals with Live Offspring / Number of Animals Pregnant) X 100

Offspring viability indices:

- Number of implantation sites
- Number of offspring per litter: Live and dead pups
- General condition of dam and litter during the postpartum period.
- Live Birth Index: Percentage of pups born alive = (Number of Live Newborn Pups / Number of Newborn Pups) x 100
- Viability Index: Percentage of pups born that survive 4 days postpartum = (Number of Live Pups on Day 4 Postpartum / Number of Liveborn Pups) x 100
- Survival Index: Percentage of pups that survive 13 days postpartum = (Number of Live Pups on Day 13 Postpartum / Number of Live Pups on Day 4 Postpartum) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Increased incidences of salivation or evidence thereof (wet fur around the mouth or muzzle) were noted for males and females in all test substance-treated groups at 2 hours postdosing; this finding began on Study Day 8 and continued throughout the dosing period and was likely attributable to the taste of the test substance. Ploughing (repetitive pushing of head into bedding material) was noted for 2 and 4 males in the 400 and 800 mg/kg/day groups, respectively, at 2 hours postdosing during Study Days 21-27. In females, this finding was noted for 1 female in the 200 mg/kg/day group and 6 females each in the 400 and 800 mg/kg/day groups during Gestation Days 7-21, and for 4, 7, and 10 females in the 200, 400, and 800 mg/kg/day groups, respectively, during lactation. This finding was also attributed to the taste of the test substance.
The aforementioned observations were not considered adverse.

No other test substance-related clinical findings were noted. Other clinical findings noted in the test substance-treated groups, including thin fur cover or fur staining on various body surfaces, were noted 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:

no mortality observed

Description (incidence):

All animals survived to the scheduled necropsy. One 200 mg/kg/day group female (No. 2502) and one 400 mg/kg/day group female (No. 3507) failed to deliver and were euthanized on Postmating Day 25.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES:
Statistically significantly lower mean body weight gain was noted in the 800 mg/kg/day group males during Study Days 0-3. Body weight gains in this group were also slightly lower than the control group during Study Days 7-16; the difference was statistically significant during Study Days 7-10. As a result, mean body weight gains in this group were statistically significantly lower than the control group for the overall premating period (Study Days 0-13) and entire dosing period (Study Days 0-27), and mean absolute body weights were statistically significantly lower (7.7% to 7.8%) than the control group on Study Days 23 and 27.
These body weight effects at 800 mg/kg/day were considered test substance-related and adverse.

Mean body weights and body weight gains in the 200 and 400 mg/kg/day group males were unaffected by test substance administration throughout the study.

FEMALES:
- Weekly:
Mean body weight gains in the 800 mg/kg/day group females were generally higher than the control group throughout the premating dosing period (Study Days 0-13); differences were statistically significant during Study Days 3-7 and for the overall premating dosing period (Study Days 0-13). Mean absolute body weight in this group was 6.9% higher than the control group on Study Day 13 but did not achieve statistical significance.
Therefore, these changes were not considered adverse, as the direction of change is not toxicologically relevant.

Mean body weights and body weight gains in the 200 and 400 mg/kg/day group females were unaffected by test substance administration throughout the premating dosing period.

- Gestation:
Mean maternal body weights and body weight gains in the 200, 400, and 800 mg/kg/day groups were unaffected by test substance administration during gestation. Statistically significantly higher mean body weight gains noted in all test substance-treated groups during Gestation Days 11-14 were transient and had no effect on overall gestational body weight gain. Higher mean absolute body weights (4.2% to 7.5%) were noted in the 800 mg/kg/day group throughout gestation when compared to the control group but were due to the higher body weight gains noted during the premating dosing period.

- Lactation:
Mean body weights and body weight gains in the 200, 400, and 800 mg/kg/day groups were unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant. Higher mean absolute body weights (5.4% to 7.9%) were noted in the 800 mg/kg/day group throughout lactation when compared to the control group but were due to the higher body weight gains noted during the premating dosing period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MALES:
Mean food consumption in the 400 and 800 mg/kg/day group males was statistically significantly lower than the control group during Study Days 0-3, but comparable to the control group throughout the remainder of the premating dosing period. The lower food consumption at 800 mg/kg/day corresponded to a statistically significantly lower mean body weight gain during the same interval and was considered test substance-related and adverse. The lower food consumption at 400 mg/kg/day was transient, minimal in magnitude (approximately 4 g), and did not have a remarkable effect on mean body weight gain, and therefore was considered nonadverse.
Mean food consumption in the 200 mg/kg/day group males was similar to that in the control group throughout the premating dosing period.

FEMALES:
- Weekly:
Mean food consumption in the 800 mg/kg/day group females was slightly lower (not statistically significant) than the control group during Study Days 0-3, but comparable to or slightly higher than the control group during the remainder of the premating dosing period. The lower food consumption was transient, minimal in magnitude (approximately 3.5 g), and did not have a corresponding effect on mean body weight gain, and therefore was considered nonadverse.
Mean food consumption in the 200 and 400 mg/kg/day group females was similar to that in the control group throughout the premating dosing period.

- Gestation:
Mean maternal food consumption in the 200, 400, and 800 mg/kg/day groups was unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.

- Lactation
Mean food consumption in the 200, 400, and 800 mg/kg/day groups was unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related effects on hematology parameters were observed at any dosage level.
Mean absolute eosinophils in all test substance-treated males appeared lower (not statistically significant) than the control group because of a higher mean in controls due to 2 animals (Nos. 1003 and 1004) with unusually high values. The mean values in the test substance-treated groups were within the range of values in the Charles River Ashland historical control data (Version 3.6), whereas the control group value was above the maximum mean value in the Charles River historical control data. Statistically significantly higher mean white blood cell counts (WBC) and absolute basophil values were noted for females in the 400 mg/kg/day group; however, these values did not occur in a dose-related manner. Other differences from the control group were not statistically significant and/or did not occur in a dose-responsive manner.

No test substance-related effects on coagulation parameters were observed at any dosage level. A statistically significantly lower APTT was noted in the 800 mg/kg/day group females, but the value was within the range of values in the Charles River Ashland historical control data. Other differences from the control group were not statistically significant and/or did not occur in a dose-responsive manner.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related effects on serum chemistry parameters were noted for males at 400 and 800 mg/kg/day and for females in all test substance-treated group. Higher mean ALP values were noted in the 400 and 800 mg/kg/day group males and females compared to the control group; differences were statistically significant for the 800 mg/kg/day group females. In addition, statistically significantly higher mean ALT values were noted for the 400 and 800 mg/kg/day group males compared to the control group. Furthermore, higher (not statistically significant) mean GGT and bile acid values were noted for the 800 mg/kg/day group males and 400 and 800 mg/kg/day group females. Finally, higher SDH levels were noted for females in all test substance-treated groups, and a statistically significantly lower mean cholesterol value was noted for females in the 800 mg/kg/day group. These changes, which were indicative of effects on liver enzymes, were noted in the absence of any histopathological correlates in the liver, and therefore these changes in serum chemistry parameters were not considered adverse.
No other test substance-related effects were noted on serum chemistry parameters.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly lower mean Total T4 values were noted in the 200, 400, and 800 mg/kg/day groups. A corresponding slightly higher TSH value was noted in the 800 mg/kg/day group compared to the control group, although the difference was not statistically significant. Total T3 values were comparable across all groups. There were no corresponding organ weight changes or histopathology findings in the thyroid at any dosage level. In addition, all values in all groups were within the range of values in the Charles River Ashland historical control data. Therefore, these thyroid hormone changes were not considered adverse.

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test substance administration at all concentrations when evaluated on Study Day 27 (males) or Lactation Day 13 (females). Values obtained from the 6 subintervals evaluated (0–10, 11–20, 21–30, 31–40, 41–50 and 51–60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the Charles River Ashland historical control data, with the following exception. The mean number of total activity counts for males in the 400 mg/kg/day group when all intervals were combined was statistically significantly higher than the control group; however, this did not occur in a dose-related manner.
Other differences from the control group were slight, not statistically significant, within the Charles River Ashland historical control data ranges and/or did not occur in a dose/exposurerelated manner. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the F0 animals were evaluated on Study Day 27 (males) or Lactation Day 13 (females).

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Neurobehavioral assessments were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group on Study Day 27 (males) or on Lactation Day 13 (females).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the adrenal gland, test substance-related diffuse cortical hypertrophy was characterized by enlarged cortical cells, particularly in the zona fasciculata, and correlated to the increased adrenal gland weights in the 800 mg/kg/day group.
In the thymus, decreased cortico-medullary ratio was noted in the 800 mg/kg/day group females and correlated to decreased mean thymus weights.
These findings were considered nonadverse given the minimal severity.

Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The mean number and lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE
No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. Single mating pairs in each of the 200 and 400 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. The mean number and lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.

NATURAL DELIVERY OBSERVATIONS
Mean gestation lengths and gestation index in the 200, 400, and 800 mg/kg/day groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.
The mean postimplantation loss (unaccounted-for sites) and implantation sites in the 200, 400, and 800 mg/kg/day groups were similar to the control group values.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

F0 reproductive toxicity

Effect level:

>= 800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed up to the highest dose tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance administration. Eight (3), 5 (2), 6 (6), and 6 (4) pups (litters) in the control, 200, 400, and 800 mg/kg/day groups, respectively, were found dead or euthanized in extremis. Two (2) and 2 (1) pups (litters) in the 400 and 800 mg/kg/day groups, respectively, were missing and presumed to have been cannibalized.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Eight (3), 5 (2), 6 (6), and 6 (4) pups (litters) in the control, 200, 400, and 800 mg/kg/day groups, respectively, were found dead or euthanized in extremis. 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. Macroscopic findings observed in the test substance-treated groups were noted infrequently and/or in a manner that was not dose-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean male and female birth weights (PND 1) were comparable across groups.
Mean pup body weight gains for males and females in the 800 mg/kg/day group were lower than the control group throughout the postnatal period (PND 1-13); differences were statistically significant during PND 7-13. As a result, mean absolute body weights in the 800 mg/kg/day group were 13.08% to 13.75% (males) and 9.90% to 11.92% (females) lower than the control group on PND 10 and 13; differences were generally statistically significant. The effects on pup body weight and body weight gain at 800 mg/kg/day were considered adverse. However, mean pup body weights in all groups, including the 800 mg/kg/day group, were within the range of values in the Charles River Ashland historical control data.
Mean male and female pup body weights and body weight changes in the 200 and 400 mg/kg/day groups were unaffected by test substance administration throughout the postnatal period (PND 1-13).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distances (absolute and relative to the cube root of pup body weight) in the 200, 400, and 800 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and/or not statistically significant.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen retention in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. There were no retained nipples in any group.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on thyroid/parathyroid weights in the F1 males and females at any dosage level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No internal findings that could be attributed to parental test substance administration were noted at the necropsy of pups euthanized on PND 13. No internal findings were noted in the test substance-treated groups.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone analysis:
- Postnatal Day 4 Culled Pups
There were no test substance-related effects on thyroid hormone values on PND 4 in the 200, 400, and 800 mg/kg/day groups. Differences from the control group were slight and not statistically significant, with the following exception. The mean TSH concentration in the 800 mg/kg/day group was statistically significantly higher than the control group for culled pups on PND 4. However, there were no corresponding effects on Total T3 and T4 concentrations in this group, and in fact, the mean Total T4 concentration was slightly higher than the control group, which is inconsistent with the effect on TSH. Furthermore, there was high variability in the TSH data amongst the 800 mg/kg/day group, with 3 values nearly 1.5 times those of the remaining litters in the group. Therefore, the higher TSH concentration was attributed to biological variability and not test substance-related.
- Postnatal Day 13 Pups
There were no test substance-related effects on thyroid hormone values on PND 13 for males or females in the 200, 400, and 800 mg/kg/day groups. However, some statistically significant differences were noted. In males, statistically significantly lower mean Total T3 concentrations were noted in the 400 and 800 mg/kg/day groups, while statistically significantly higher mean Total T4 concentrations were noted in the 200 and 800 mg/kg/day groups. Similarly, in females,
lower mean Total T3 concentrations and higher mean Total T4 concentrations were noted in all substance-treated groups; differences were generally statistically significant. These changes did not occur in a manner that is suggestive of a disruption in thyroid function, and occurred in the absence of any effect on TSH concentrations or thyroid weights (see Section 8.3.8.).
Furthermore, the changes in Total T4 concentrations were not clearly dose-responsive.
Therefore, these changes in T3 and T4 concentration amongst male and female F1 pups on PND 13 were considered spurious and unrelated to test substance administration.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Details on results (F1)

LITTER DATA AND POSTNATAL SURVIVAL
The total mean number of newborn pups, number of live newborn pups, and the percentage of males at birth in the 200, 400, and 800 mg/kg/day groups were similar to the control group values. Postnatal survival, including live birth index (survival on PND 0), viability index (survival from PND 0-4), and survival index (survival from PND 4-13) in the 200, 400, and 800 mg/kg/day groups were unaffected by test substance administration.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

neonatal toxicity

Generation:

F1

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this screening study, in the absence of any effects on reproductive parameters at any dosage level, a dosage level of 800 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity of Scentaurus Clean when administered orally by gavage to Crl:CD(SD) rats. Decrements in body weight and body weight gains were noted for F0 males at 800 mg/kg/day, but there was no evidence of adverse toxicity for F0 females; therefore, the NOAEL for F0 systemic toxicity was considered to be 400 mg/kg/day for F0 males and 800 mg/kg/day for F0 females. The NOAEL for F1 neonatal toxicity was 400 mg/kg/day based on the effects on pup body weights and body weight gains for males and females in the 800 mg/kg/day group.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test substance when administered to rats for 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behavior, and conception through day 13 of postnatal life.

The study design was as follows:

Group Number	Test Substance	Dosage Level (mg/kg/day)	Number of Males	Number of Females
1	Vehicle Control	0	5	5
2	Scentaurus clean	200	5	5
3	Scentaurus clean	400	5	5
3	Scentaurus clean	800	5	5

Animals were dosed via oral gavage once daily. Males were dosed for 14 days prior to mating and continuing throughout mating for a minimum of 28 days (Study Days 0–27). Females were dosed for 14 days prior to mating and continuing through Lactation Day 13. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, neurobehavior, locomotor activity, thyroid hormones, clinical pathology, macroscopic findings, organ weights, and microscopic examinations.

No test substance-related effects were noted on F0 survival, estrous cyclicity, reproductive performance, parturition, neurobehavior, motor activity, or macroscopic findings.

Test substance-related clinical observations included increased incidences of salivation or evidence thereof (wet fur around the mouth or muzzle) for males and females in all test substance-treated groups at 2 hours following dose administration; these findings were likely attributable to the taste of the test substance. In addition, ploughing (repetitive pushing of head into bedding material) was noted for males in the 400 and 800 mg/kg/day groups and females in the 200, 400, and 800 mg/kg/day groups at 2 hours following dose administration and was also attributable to the taste of the test substance. The aforementioned observations were not considered adverse.

Test substance-related, statistically significantly lower mean body weight gain was noted in the 800 mg/kg/day group F0 males for the overall dosing period (Study Days 0-27), with corresponding statistically significantly lower mean food consumption during Study Days 0-3.

As a result, mean absolute body weights were statistically significantly lower (7.7% to 7.8%) than the control group on Study Days 23 and 27. These body weight effects in the 800 mg/kg/day group were considered adverse. Statistically significantly lower mean food consumption, with no corresponding remarkable change in body weight gain, was noted in the 400 mg/kg/day group males at the start of dosing (Study Days 0-3). This test substance-related change was transient, and not considered adverse. There were no other test substance-related effects on body weight or food consumption parameters for males at 200 and 400 mg/kg/day.

For F0 females, test substance-related, statistically significantly higher mean body weight gain was noted in the 800 mg/kg/day group for the overall premating dosing period (Study Days 0-13), with corresponding slightly higher mean food consumption noted sporadically during the premating period. Mean absolute body weight in this group was 6.9% higher (not statistically significant) than the control group on Study Day 13. While mean absolute body weights remained slightly higher (4.2% to 7.9%) in the 800 mg/kg/day group females during gestation and lactation when compared to the control group, mean body weight gains and food consumption were unaffected by test substance administration during these periods. The higher mean body weights and body weight gains for the 800 mg/kg/day group females were not

considered to be adverse. There were no test substance-related effects on body weights, body weight gains, or food consumption for females at 200 and 400 mg/kg/day.

There were no test substance-related effects on hematology or coagulation parameters for F0 males or females at any dosage level. Test substance-related effects on serum chemistry parameters included higher mean ALP values in the 400 and 800 mg/kg/day group males and females, higher mean ALT values for the 400 and 800 mg/kg/day group males, higher mean GGT and bile acid values for the 800 mg/kg/day group males and 400 and 800 mg/kg/day group females, higher mean SDH levels for the 200, 400, and 800 mg/kg/day group females, and a lower mean cholesterol value for the 800 mg/kg/day group females. Despite higher liver weights for females at 800 mg/kg/day, there were no histopathological correlates in the liver, and therefore these changes in serum chemistry parameters were not considered adverse.

Statistically significantly lower mean Total T4 values were noted in the 200, 400, and 800 mg/kg/day group F0 males, with a corresponding higher (not statistically significant) TSH value in the 800 mg/kg/day group compared to the control group. Mean Total T3 values were comparable across groups, and there were no corresponding organ weight changes or histopathology findings in the thyroid at any dosage level. Therefore, these thyroid hormone changes were not considered adverse.

Test substance-related effects on organ weights included higher mean adrenal gland weights in the 800 mg/kg/day group males and females, higher mean liver weights in the 800 mg/kg/day group females, and lower mean thymus weights in the 800 mg/kg/day group males and females.

Microscopically, test substance-related diffuse cortical hypertrophy in the adrenal gland was noted for males and females in the 800 mg/kg/day group and correlated to increased adrenal gland weights. In the thymus, decreased cortico-medullary ratio was noted in the 800 kg/kg/day group females and correlated to decreased mean thymus weights. All findings were considered nonadverse given the minimal severity. There were no test substance-related organ weight changes or microscopic findings at 200 and 400 mg/kg/day.

No test substance-related effects were noted on F1 litter viability and survival, clinical observations, anogenital distance, areolae/nipple anlagen retention, thyroid hormone values (PND 4 or PND 13), macroscopic findings, and organ weights.

Mean pup body weight gains for F1 males and females in the 800 mg/kg/day group were lower than the control group during PND 1-13, with statistical significance achieved during PND 7-13.

As a result, mean absolute body weights in the 800 mg/kg/day group were statistically significantly lower (13.75% for males and 11.92% for females) than the control group on PND 13. The effects on pup body weight and body weight gain at 800 mg/kg/day were considered test substance-related and adverse. Despite these differences, mean pup body weights in all groups were within the range of values in the Charles River Ashland historical control data (version 2019.05). There were no test substance-related effects on pup body weights and body weight gains at 200 and 400 mg/kg/day.

Under the conditions of this screening study, in the absence of any effects on reproductive parameters at any dosage level, a dosage level of 800 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity of Scentaurus Clean when administered orally by gavage to Crl:CD(SD) rats. Decrements in body weight and body weight gains were noted for F0 males at 800 mg/kg/day, but there was no evidence of adverse toxicity for F0 females; therefore, the NOAEL for F0 systemic toxicity was considered to be 400 mg/kg/day for F0 males and

800 mg/kg/day for F0 females. The NOAEL for F1 neonatal toxicity was 400 mg/kg/day based on the effects on pup body weights and body weight gains for males and females in the 800 mg/kg/day group.