Zinc bis(dibenzyldithiocarbamate)

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Apr 2021 to 10 Mar 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD guidance document supporting OECD test guideline 443 on the extended onegeneration reproductive toxicity test, No 151

Version / remarks:

2013

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.56 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

2014

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS :

- Premating exposure duration for parental (P0) animals: 10 weeks

- Basis for dose level selection: The dose levels were selected based on the results of a preliminary reproductive toxicity study (Simplified reproduction/developmental toxicity screening test) with oral exposure of the test item in rats), and in an attempt to produce graded responses to the test item. In this study, no treatment-related changes were noted in any of the parameters investigated and a parental, reproduction and developmental no-observed-adverse-effect level (NOAEL) of the test item of at least 1000 mg/kg/day was established. The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

-Exclusion of extension of Cohort 1B

- Exclusion of developmental neurotoxicity Cohorts 2A and 2B

- Exclusion of developmental immunotoxicity Cohort 3

- Route of administration: Oral, gavage

- Other considerations: The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for reproduction and developmental toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical 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 total number of animals to be used in this study is considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Test material

Test material information

Constituent 1

Reference substance name:

Zinc bis(dibenzyldithiocarbamate)

EC Number:

238-778-0

EC Name:

Zinc bis(dibenzyldithiocarbamate)

Cas Number:

14726-36-4

Molecular formula:

C30H28N2S4Zn

IUPAC Name:

zinc bis(dibenzyldithiocarbamate)

Details on test material:

Zinc dibenzyldithiocarbamate (ZBEC)
solid
Mw= 610g/mol

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
-Females nulliparous and non-pregnant: yes
-Age/weight at dosing: F0-animals: approximately 6 weeks/ F0-males: 140 to 230 g; F0-females: 110 to 160 g.
-Housing: On arrival, prior to mating and during the post-weaning period, animals were grouped housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Makrolon type IV; height 18 cm or type 2000P; 61x43.5x21.5 cm depending on body weight). During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (type III; height 18 cm). During the post-mating phase, males were housed in Makrolon type IV or type 2000P cages (61x43.5x21.5 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (type III, height 18 cm). During the lactation phase, females were housed in Makrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination (unscheduled deaths, spares, and pups of Cohort Surplus) or until weaning on PND 21 (Cohorts 1A, 1B, 1C). The cages contained appropriate bedding and were equipped with water bottles. Animals were separated during designated procedures/activities.
-Diet: Pelleted rodent diet ad libitum
-Water: Municipal tap water, provided via water bottles ad libitum
-Acclimatisation period: At least 5 days

ENVIRONMENTAL CONDITIONS:
-Temperature: 18 to 24°C
-Humidity: 40 to 70%
-Air changes: At least 10 air changes per hour.
-Photoperiod: 12-hours light and 12-hours dark (may be interrupted for designated procedures).

IN-LIFE DATES: from 14 Apr 2021 to 10 Mar 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
-Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment were made for specific gravity of the vehicle. No correction were made for the purity/composition of the test item. Any residual volumes were discarded.

VEHICLE
- Amount of vehicle: 4 mL/kg
- Concentration in vehicle: 25, 75, 250 mg/mL
- The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 consecutive days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- Further matings after unsuccessful attempts: After 14 days, females who have not shown evidence of mating were separated from their males without further opportunity of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and concentration were determined based on Zinc content since there is no
better, feasible analytical method available for this type of molecule. Dose formulation samples were collected for analysis in week 1, 8, 15 and 22 of treatment.

-Sample for analysis: Duplicate middle samples for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 4 (concentration and homogeneity analysis).
-Sample volume: Approximately 500 mg accurately weighed.
-Acceptance criteria: For concentration, the criteria for acceptability were mean sample concentration results within or equal to ± 10% for solutions or ±15% for suspensions of target concentration. For homogeneity, the criteria for acceptability were a coefficient of variation (CV) of concentrations of ≤ 10% for each group.

Stability analysis of the test item in the vehicle was not determined since the available analytical method is only capable of measuring the zinc content in the test substance and, therefore, unable to measure the test item itself. During the current study, the dosing formulations containing the test item were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item. In addition, to limit the impact, the test item preparation were performed with approved procedures and documented in detail. This GLP exception was therefore considered as being minor with no impact on the outcomes and the integrity and the achievement of the objective of the study.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week. F0-males were treated for a minimum of 11 weeks, including 10 weeks prior to mating (with the objective of covering at least one spermatogenic cycle) and during the mating period, up to and including the day before scheduled necropsy. F0-females were treated for a minimum of 16 weeks, including 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females were not dosed during littering. Prior to weaning, pups were not treated directly but could potentially be exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces. From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B and 1C were dosed up to and including the day before scheduled necropsy. The F1-animals of Cohort Surplus and Spares (not assigned to one of the cohorts) were not dosed. The dose volume for each animal were based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The first day of dosing were designated as Day 1 (exception: alternate animals used for replacement after Day 1 assumed the day of the animal being replaced). The dosing formulations were stirred continuously during dose administration. A dose control system (DCS) were used as additional check to verify the dosing procedure according to Standard Operating Procedures

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4

No. of animals per sex per dose:

F0 Animals: 25
F1 Animals:
-Cohort 1A: 20
-Cohort 1B: 20
-Cohort 1C: 20
-Surplus: 10 (The F1-animals of Cohort Surplus were not dosed)

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected based on the results of a preliminary reproductive toxicity study (Simplified reproduction/developmental toxicity screening test) with oral exposure of the test item in rats), and in an attempt to produce graded responses to the test item. In this study, no treatment-related changes were noted in any of the parameters investigated and a parental, reproduction and developmental no-observed-adverse-effect level (NOAEL) of the test item of at least 1000 mg/kg/day was established. The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily throughout the study. Animals are observed for general health/mortality and moribundity. Animals are not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
- Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

CLINICAL OBSERVATIONS
- Time schedule: at least twice daily, up to the day prior to necropsy. Conducted prior to dosing and after dosing

ARENA OBSERVATIONS
-Tim schedule: once before the first administration of the test item and at weekly intervals during the treatment period.

BODY WEIGHT
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

FOOD CONSUMPTION
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0-4, 4-7, 7-11, 11-14, 17, and 17-20 post-coitum and during lactation on PND 1-4, 4-7, 7-14, 14 and 14-21.

WATER CONSUMPTION
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

CLINICAL PATHOLOGY - F0-Generation
Blood of 10 selected animals/sex/group of F0-animals was collected on the day of scheduled necropsy. Samples were collected from the retro-orbital sinus under anaesthesia using isoflurane in the animal facility. The selected F0-animals were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available.
- Haematology parameters assessed: White Blood Cell Count (WBC), Reticulocytes (absolute), Neutrophils (absolute), Red Blood Cell Distribution Width Gated (RDWG), Lymphocytes (absolute), Hemoglobin, Monocytes (absolute), Hematocrit, Eosinophils (absolute), Mean corpuscular volume (MCV), Basophils (absolute), Mean corpuscular hemoglobin (MCH), Large unstained cells (LUC) (absolute), Mean corpuscular hemoglobin concentration (MCHC), Red Blood Cell Count (RBC), Platelets
- Coagulation assessed: Prothrombin time (PT) and Activated partial thromboplasting time (APTT)
- Clinical chemistry parameters assessed: Alanine aminotransferase (ALT), Creatinine, Aspartate aminotransferase (AST), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos)
- Thyroid hormone: Blood samples at a target volume of 1.0 mL. After clotting and centrifugation, serum of F0-animals was used for measurement of both T4 and TSH. Serum samples retained for possible future analysis were maintained by the Test Facility in the freezer (≤-75°C). Under these storage conditions, samples are stable for 6 months.
- Urinalysis: Urine was collected into a specimen vial from the 10 selected animals/sex/group of F0-animals housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available. Parameters assessed: Volume, Sediment, Specific gravity, White blood cells (WBC-sed.), Clarity, Red blood cells (RBC-sed.), Colour, Casts, pH, Epithelial cells, Blood, Crystals, Leukocyte esterase, Bacteria, Bilirubin, Protein, Ketones, Glucose, Other.

Oestrous cyclicity (parental animals):

Estrous stages were determined by examining the cytology of vaginal lavage samples.
Daily vaginal lavage was performed beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of scheduled necropsy, a vaginal lavage was also taken. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.

Sperm parameters (parental animals):

Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with hematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.
One epididymis (right) was removed, placed in labeled bags, and kept in the freezer at ≤ 15°C. After thawing, the right epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 post-partum: yes
- To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) is acceptable. In case less than 20 litters with at least 8 live pups/litter are available, litters may be culled to a size which is aimed to be adequate for allocation of a sufficient number of pups into the respective cohorts.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

F1-Generation until Weaning (PND 21):
- Mortality/Moribundity Checks: Pups were observed twice daily for general health/mortality, simultaneously with the mortality/moribundity check of the dam. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
- Clinical observations: were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.
- Body Weights: Live pups were weighed individually on PND 1, 4, 7, 13 and 21
- Sex: was externally determined for all pups on PND 1,4 and 13
- Anogenital Distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/Nipple Retention: All male pups in each litter were examined for the number of areola/nipples on PND 13.

F1-Generation from Weaning (PND 21) onward (Cohort 1A, 1B, 1C):
- Mortality/Moribundity Checks: Throughout the study, animals were observed for general health/mortality and moribundity at least twice daily. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
- Clinical Observations: Clinical observations were performed at least twice daily, up to the day prior to necropsy. These observations were at least conducted prior to dosing and after dosing. Animals were observed for specific clinical signs.
- Arena Observations: Animals were observed for specific clinical signs in a standard arena once on the first Day 8 of weaning and thereafter at weekly intervals during the treatment period
- Body Weights: Animals were weekly weighed. This started on a specific date on which all pups were at least at PND 21. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation.
- Food Consumption: Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy.
- Water Consumption: Water consumption was monitored by visual inspection of the water bottles on regular basis throughout the study.
- Vaginal Patency: Vaginal patency (vaginal opening) was monitored daily for all females from PND 25 onwards until vaginal patency was present, by visual inspection of the vaginal area.
- Balanopreputial Separation: Balanopreputial separation (prepuce opening) was monitored daily for all males from PND 35 onwards until balanopreputial separation was present, by visual inspection of the genital area. Body weight was recorded on the day of acquisition of balanopreputial separation.

F1-Generation from Weaning (PND 21) onward (Cohort 1A and 1B):
- Stage of Estrus Determination: Estrous stages were determined by examining the cytology of vaginal lavage samples. A vaginal lavage was taken on the day of scheduled necropsy for all females, except for females that have to be euthanized in extremis or died
spontaneously.

Cohort specific investigations: Cohort 1A
Estrous Cycle Determination: Estrous stages were determined by examining the cytology of vaginal lavage samples, taken during two periods.
During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrus was determined, in order to determine the time interval between these two events. During the second period, daily vaginal lavage was performed from PND 75 to 88.

Clinical Pathology -F1-animals, PND 4 pups
On PND 4 at culling, blood was collected from two surplus pups per litter by decapitation, between 7.00 and 10.30 a.m. in the necropsy room, and samples were pooled per litter.

Clinical Pathology – Cohort 1A
Blood of 10 selected animals/sex/group of Cohort 1A animals was collected on the day of scheduled necropsy. The selected Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water will be available. Isoflurane was used as anaesthesia. The following parameters were assessed:
- Haematology: White blood cells (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Large unstained cells (LUC) (absolute), Red Blood Cell Count, Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW), Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets.
-Coagulation: Prothrombin time (PT), Activated partial thromboplastin time (APTT)
- Clinical Chemistry: Alanine aminotransferase (ALT), Creatinine, Aspartate aminotransferase (AST), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos)
- Thyroid hormone: Thyroxine (T4), Thyroid Stimulating Hormone (TSH). Blood samples at a target volume of 1.0 mL (Cohort 1A).
-Urinalysis: Volume, Specific gravity, Clarity, White blood cells (WBC-sed.), Clarity Red blood cells (RBC-sed.), Colour, Casts, pH, Epithelial cells, Blood, Crystals, Leukocyte esterase, Bacteria, Bilirubin, Protein, Ketones, Glucose, Other

Postmortem examinations (parental animals):

SACRIFICE
A necropsy was conducted on animals that died on study, and specified tissues were saved, but not weighed. If necessary, the animal were refrigerated to minimize autolysis. Animals euthanized for humane reasons were deeply anesthetized using isoflurane and subsequently exsanguinated. Specified tissues were retained, but not weighed. Dams with no surviving pups were euthanized within 24 hours after the last pup is found dead or missing. Females were not fasted before necropsy. The terminal body weight was recorded and specified tissues were weighed and retained.
Animals surviving until scheduled euthanasia were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. All animals surviving to scheduled necropsy were fasted overnight with a maximum of approximately 24 hours before necropsy. Water was available.
Scheduled necropsies:
-Males which sire: After successful mating and a minimum of 10 weeks of treatment.
-Males which fail to sire: At the end of the mating period and after a minimum of 10 weeks of treatment.
-Females which deliver: LD 23-25.
-Females which fail to deliver: With evidence of mating: Post-coitum Days 25-27.
Without evidence of mating: Approximately 24-26 days after the last day of the mating period.

GROSS NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs identified in Table 1 were weighed at necropsy for all scheduled euthanasia animals and females with total litter loss. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. Organ weight as a percent of body weight (using the terminal body weight) were calculated.
Representative samples of the tissues identified in Table 1 were collected from all animals and preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated. For females which failed to deliver a complete litter, uterine contents (i.e. any fetuses, placenta and implantation sites) were fixed (if applicable), but were not examined histopathologically in first instance.

Postmortem examinations (offspring):

SACRIFICE
- Unscheduled Deaths until weaning:
Recognizable fetuses of females that die spontaneously or are euthanized in extremis were examined externally and sexed (both externally and internally, if possible). Live fetuses were euthanized by decapitation. Pups that are sacrificed in extremis, younger than 7 days, were euthanized by decapitation. Pups sacrificed in extremis on or after PND 7 were euthanized by an intraperitoneal injection of sodium pentobarbital.
Pups found dead during the weekend were fixed in identified containers containing 70%
ethanol if not being subjected to necropsy on the same day.
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology. For pups found dead from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally, if possible). Descriptions of all abnormalities were recorded. Abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Culled Pups (PND 4): On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation.
GROSS NECROPSY
For all animals, necropsy procedures was performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. Tissues were preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution) unless otherwise indicated. Additional tissue samples may be collected to elucidate abnormal findings. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. Organ weight as a percent of body weight (using the terminal body weight) were calculated.

- Unscheduled Deaths weaning onwards:
Necropsy was conducted on animals that died during the study within 24 hours. If necessary, the animal were refrigerated to minimize autolysis. Animals that were euthanized for humane reasons as per Test Facility SOPs were deeply anesthetized using isoflurane and subsequently exsanguinated.
Spare F1-animals which were not assigned to one of the Cohorts were sacrificed between PND 22-24 by intraperitoneal injection of sodium pentobarbital. Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex will be determined (both externally and internally, if possible). Descriptions of all external abnormalities were recorded. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director.

HISTOPATHOLOGY / ORGAN WEIGTHS
The same tissues indicated in Table 1-5 were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin (HE).

For Cohort 1A animals of Groups 1 and 4, HE stained step sections of both ovaries and
corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine
section) were prepared. One of the ovaries was quantitatively evaluated for follicles (primordial and small growing follicles counted together), as well as corpora lutea initially.

Cohort specific terminal procedures - Cohort 1A
Scheduled Deaths, Cohort 1A: Scheduled necropsy of Cohort 1A was conducted on PND 89-95. Cohort 1A animals surviving to scheduled necropsy were deprived of food overnight (with a maximum of 24 hours) before necropsy, but water was available. The animals were weighed and deeply anesthetized using isoflurane and subsequently exsanguinated.
The organs identified for weighing and representative samples of the tissues mentioned in the
Tissue Collection and Preservation Table 2 were weighed and collected.
For all surviving males of Cohort 1A, the following assessments were performed:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with hematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples. One epididymis (right) was removed, placed in labeled bags, and kept in the freezer at ≤ 15°C. After thawing, the right epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.
- Splenic Lymphocyte Subpopulation Analysis: From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. If possible, one pup (male or female) was selected per litter (20 litters in total). One half of the spleen was kept on ice until splenic lymphocytes were isolated using 70 µm cell strainers. The other half of the spleen was preserved for histopathological evaluation. Splenocytes were counted with the Coulter Counter Z1. The following subpopulations were determined in isolated splenic lymphocytes using the BD FACSCanto™ flow cytometer system on the day of necropsy: T-cells, T-helper cells, T-cytotoxic cells, B-cells, NK-cells, Ratio T-helper cells/ T-cytotoxic cells (Th/Tc). The % lymphoid cells of peripheral blood mononuclear cells (PBMC) were determined using the Forward Scatter and Side Scatter.

Cohort specific terminal procedures - Cohort 1B
- Scheduled Deaths - Cohort 1B: Scheduled necropsy of Cohort 1B were conducted on ≥ PND 97. Cohort 1B animals were not deprived of food overnight before necropsy. These animals had a terminal body weight recorded and were deeply anesthetized using isoflurane and subsequently exsanguinated (See Table 3).

Cohort specific terminal procedures - Cohort 1C
- Scheduled Deaths - Cohort 1C: Scheduled necropsy of Cohort 1C were conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals were not deprived of food overnight before necropsy. Terminal body weight were not recorded. The animals were deeply anesthetized using isoflurane and subsequently exsanguinated. (See Table 4)

Cohort specific terminal procedures - Cohort Surplus
- Scheduled Deaths – Cohort Surplus: Scheduled necropsy of Cohort Surplus were conducted on PND 22-24. Cohort Surplus animals were not deprived of food overnight before necropsy. Terminal body weight was recorded. The animals were deeply anesthetized using isoflurane and subsequently exsanguinated. Descriptions of all macroscopic abnormalities were recorded. The organs identified for weighing and representative samples of the tissues mentioned in the Tissue Collection and Preservation Table 5 were weighed and collected.

Statistics:

Statistics for data collected/processed in ToxData:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
-Parametric:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
-Non-Parametric:
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
-Incidence:
An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons was conducted using Fisher’s exact test whenever the overall test is significant

Statistics for data collected/processed in Provantis:
-Inferential Statistical Methods:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted.
-Parametric/Non-parametric:
Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F test or Kruskal Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
-Incidence:
Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Reproductive indices:

See Table 6

Offspring viability indices:

See Table 6

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were noted during daily detailed clinical observations. No findings were noted during the weekly arena observations in this study. Salivation and reflux were seen after dosing among a few animals and was considered not toxicologically relevant, taking into account the nature and minor severity of the effect. Incidental findings that were noted included alopecia, focal erythema, scales, scabs, wounds, salivation, an enlarged eye, exophthalmos, chromodacryorrhea, opacity of the eye, piloerection, hunched posture, and red staining of the genital region. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment with the test item.
Any statistically significant increases in body weight gain in males were considered to be unrelated to treatment with the test item since no trend was apparent regarding dose and/or duration of treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was considered to have been unaffected by treatment with the test item.
Any statistically significant changes in food consumption after correction for body weight in females were considered to be unrelated to treatment with the test item since no trend was apparent regarding dose and/or duration of treatment.

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day the following differences were noted that were considered to be related to the test item: A higher mean white blood cell count (WBC; not statistically significant; 1.12x of control) which was mainly caused by a higher mean lymphocyte count (not statistically significant; 1.15x of control) in males; a higher mean white blood cell count (WBC; not statistically significant; 1.13x of control) which was mainly caused by a higher mean neutrophil count (not statistically significant; 1.31x of control) in females. The lower mean corpuscular hemoglobin level (MCH) with concurrent lower mean corpuscular hemoglobin concentration (MCHC) in females at 1000 mg/kg/day, was considered unrelated to treatment with the test item. These changes were minimal and there was a great overlap in individual values between the groups. See Tables 7 and 8.
A test item-related increase in mean prothrombin time (PT) of 1.11x of control was noted in males at 1000 mg/kg/day. At the individual level, one male with a particular long prothrombin time (23.6 sec) was noted. This result could not be verified (not sufficient material for repetition) and should therefore be interpreted with caution. However, also after exclusion of this value, prothrombin time of 5/9 high dose males were above the concurrent control range. A longer mean activated partial thromboplastin time (APPT) of 1.10x of control (not statistically significant) was noted in males at 1000 mg/kg/day. As all individual values were within the range of the controls, the slightly longer mean APPT was considered unrelated to treatment with the test item. Coagulation parameters of females were considered not affected by treatment with the test item. See Table 9.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical parameters were considered unaffected by treatment with the test item. The higher mean glucose concentration noted in males at 1000 mg/kg/day (1.22x of control) was considered a minimal change for this parameter and therefore not to be the result of treatment with the test item. Any other observed changes, in clinical chemistry parameters were considered to be unrelated to treatment with the test item as these were minimal only, occurred in the absence of a dose-related trend and/or were caused by general overlap and variability in individual values. In males at 300 and 1000 mg/kg/day, T4 levels were decreased when compared to concurrent controls (0.69x and 0.84x, respectively). Mean values remained within the historical control range (Historical control data for Wistar Han rats: F0-animals (2017-2021) Total T4 (μg/dL) - males: mean = 5.92; P5 – P95 = 4.195-7.920 (n=120)).

Endocrine findings:

not specified

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis parameters were considered to be unaffected by treatment with the test item. At 1000 mg/kg/day, the urine of 4/10 males was positive for leukocyte esterase (LEUK EST) and the urine of 1/10 males contained a moderate amount of red blood cells (RBC). In absence of white blood cells or other signs of infection and/or at isolated incidence, the presence of leukocyte esterase or red blood cells in these animals were considered not test item-related. For a control male animal, a lower specific gravity (1.009) was noted which was considered to be caused by the relatively high volume of urine (23.0 mL) excreted by this animal. Since this is a control animal, there was no relationship with treatment with the test item.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related microscopic findings. All of the recorded microscopic findings (including the microscopic findings in the liver of males) were within the range of background pathology encountered in rats of this age and strain. There was no test material related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item. Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for one female animal (control) and one test Group animal (1000 mg/kg/day) (both with normal litter). Given their incidental nature, absence of a dose-related incidence and absence of a correlation to pregnancy status, these findings did not indicate a relationship with treatment with the test item.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Sperm motility, concentration and morphology parameters were considered unaffected by treatment with the test item. A lower number of spermatozoa with a coiled tail was recorded for males at 300 mg/kg/day. Since a dose-related trend was absent and an opposite effect would be expected in case of target organ toxicity this was considered not to be related to treatment with the test item.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating Index:
Mating index was unaffected by treatment with the test item. All females showed evidence of mating.

Precoital Time:
Precoital time was unaffected by treatment with the test item. All females were mated within the first 6 days of the mating period, except for two females at 100 mg/kg/day for which it took 13 and 14 days before mating could be confirmed. In the absence of a dose-related incidence, this was considered not to be related to treatment with the test item.

Number of Implantation Sites:
Number of implantation sites was considered not to be affected by treatment with the test item.

Fertility Index:
Fertility index was unaffected by treatment with the test item. All mated females were pregnant
.
Histopathological Evaluation of Reproductive Performance:
All couples delivered healthy offspring.
The only finding of note was a retained placenta, correlating with a thickened implantation site of one uterus horn, in one 1000 mg/kg/day female. This was considered to be an incidental finding. Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Gestation Index and Duration:
Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were unaffected by treatment with the test item. All pregnant females had live offspring. One female in the control and 1000 mg/kg/day group each had a gestation duration of 23 days while a gestation duration of 21 or 22 days would be expected for rats of this age and strain. In the absence of a dose-related incidence, this slightly longer duration of gestation was considered not to be related to treatment with the test item.

Parturition/Maternal Care – F0-Generation:
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Post-Implantation Survival Index:
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item. Post-implantation survival index was 93, 95, 90 and 89% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. For two females at 100 mg/kg/day the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 23-25 days of lactation. No toxicological relevance was attached to this finding in the current study.

Litter Size:
Litter size was considered not to be affected by treatment with the test item. Live litter sizes were 11.7, 11.3, 10.9 and 10.4 for the control, 100, 300 and 1000 mg/kg/day groups, respectively. A (not statistically significant) trend towards lower mean number of live litter size may be observed. However, at individual level there was a high overlap in litter size. The slightly lower mean number of live pups at first litter check at 1000 mg/kg/day (mean = 10.4 vs. 11.7 for control) was caused by the relatively small size of two litters (4 or 5 pups). Excluding these litters resulted in a mean of 10.9 live pups at first litter check which is considered within normal range.

Sex Ratio:
Sex ratio was considered unaffected by treatment with the test item.

Live Birth Index:
The number of live offspring on Day 1 after littering compared to the total number of offspring born was unaffected by treatment with the test item. The live birth indices were 99, 99, 100 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. A total of six pups were found dead at first litter check, of which two pups in the control group, three pups at 100 mg/kg/day and one pup at 1000 mg/kg/day. For two pups at 100 mg/kg/day, no milk was noted at necropsy and one pup was cannibalized. Advanced autolysis was noted for the dead pup at 1000 mg/kg/day. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Viability Index:
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was unaffected by treatment with the test item. Viability indices were 100, 100, 99 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. One pup of the control group and one pup at 300 mg/kg/day were missing on PND 2 or 3. Pups missing were most likely cannibalized. One pup at 100 mg/kg/day was sacrificed in extremis on PND 1 due to a blue discoloration of the abdomen and an abnormal posture and was noted to have misshapen head at necropsy. One pup at 300 mg/kg/day was found dead on PND 3. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Weaning Index:
The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was unaffected by treatment with the test item. The weaning indices were 100, 100, 99 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. One pup at 300 mg/kg/day was found dead on PND 15. At necropsy, a blue snout, alopecia, a pale discoloration of the liver, thymus reduced in size, lungs grown together with the stomach and the stomach, spleen and pancreas in the thoracic cavity were observed. No toxicological relevance was attributed to this dead pup since the mortality incidence did not show a dose-related trend.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

General Toxicity

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred during lactation among pups that were considered to be related to treatment with the test item. One pup at 100 mg/kg/day had a missing eye from PND 20 and another pup of this litter had a misshapen head. These findings were considered incidental and unrelated to treatment with the test-item. Other clinical signs noted in pups were blue discoloration of the snout or abdomen, a wound, scabs, alopecia and a bent tail apex. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age and were therefore considered not to be toxicologically relevant. No test item-related clinical signs were noted during daily detailed clinical observations in the F1 Generation. No findings were noted during the weekly arena observations in this study. Reflux was seen after dosing among a few animals at control and 100 mg/kg/day and was considered not toxicologically relevant, taking into account the nature and minor severity of the effect. Incidental findings that were noted included alopecia, scabs, wounds, a bent or broken tail apex, salivation, rales, exophthalmos and hunched posture. These findings occurred in the control group only (salivation and rales) and/or were within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these clinical signs were considered to be unrelated to treatment with the test item.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was unaffected by treatment with the test item. Viability indices were 100, 100, 99 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. One pup of the control group and one pup at 300 mg/kg/day were missing on PND 2 or 3. Pups missing were most likely cannibalized. One pup at 100 mg/kg/day was sacrificed in extremis on PND 1 due to a blue discoloration of the abdomen and an abnormal posture and was noted to have misshapen head at necropsy. One pup at 300 mg/kg/day was found dead on PND 3. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. No mortality occurred during the study period that was considered the result of treatment with the test item. Two female animals (one dosed 100 mg/kg/day, Cohort 1A and one dosed 300 mg/kg/day, Cohort 1A) were found dead or sacrificed for humane reasons, respectively, on treatment Day 35. Shortly before their deaths, clinical signs included labored respiration, quick breathing, gasping, hunched posture and/or piloerection. Macroscopic and microscopic examination revealed evidence of gavage-related incidents in the form of gray-white and/or reddish, gelatinous, watery cloudy contents of the thoracic cavity (macroscopic finding) and acute inflammation of capsule of the thymus, pleura of the lung and epicard of the heart. One male (300 mg/kg/day, Cohort 1B) was found dead in its cage on treatment Day 35. At necropsy, findings noted included perforation of the esophagus and gray-white, watery-clear, oil like contents of the thoracic cavity and therefore, the death of this animal was considered related to the gavage administration procedure. One male (100 mg/kg/day, Cohort 1C) died on treatment Day 18 and one female (300 mg/kg/day, Cohort 1B) on treatment Day 32 due to a procedure-related incident.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups during lactation were considered unaffected by treatment with the test item. Body weights and body weight gain of the F1 Generation were considered to have been unaffected by treatment with the test item. Any statistically significant differences in mean body weight / body weight gain in treated males and/or females when compared to controls were considered to be unrelated to treatment with the test item since no trend was apparent regarding dose and/or duration of treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was considered to have been unaffected by treatment with the test item. Any statistically significant changes in food consumption before or after correction for body weight in females were considered to be unrelated to treatment with the test item since no trend was apparent regarding dose and/or duration of treatment.

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological parameters of treated Cohort 1A rats were considered affected by treatment with the test item at 1000 mg/kg/day. In males at 1000 mg/kg/day, a higher mean white blood cell count (WBC; 1.22x of control) which was mainly caused by a higher mean lymphocyte count (LYMPH; 1.23x of control) was observed. The lower mean corpuscular hemoglobin level (MCH) with concurrent lower mean corpuscular volume (MCHC) in females at 1000 mg/kg/day, was considered unrelated to treatment with the test item. These changes were minimal only and there was a great overlap in individual values between the groups. The change in red blood cell distribution width gated (RDWG) at 100 mg/kg/day was considered to be unrelated to treatment with the test item in the absence of a dose-related trend. See Table 10 and Table 11.
Coagulation parameters were considered unaffected by treatment with the test item. The longer mean activated partial thromboplastin time (APPT) of 1.11x of control (not statistically significant) noted in males at 1000 mg/kg/day was considered to be not related to treatment with the test item as 9/10 individual values were within the range of the control group. The longer APPT at 100 mg/kg/day was considered unrelated to treatment with the test item in absence of a dose-related trend. At 300 mg/kg/day, one female had a relatively high value for APPT (31.2 sec). This result could not be verified (not sufficient material for repetition) and should therefore be interpreted with caution.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Serum T4 levels in male and female pups, culled at PND 4 and of Cohort Surplus at PND 22 were considered not to be affected by treatment with the test item. At 1000 mg/kg/day, higher mean TSH levels were recorded in male and female pups of Cohort Surplus at PND 22 (1.26 and 1.19x of control, respectively; not statistically significant). Mean values remained within the historical control data range (Historical control data for Wistar Han rats: F1-animals (2017-2021); TSH (μIU/mL) - males: mean = 0.081; P5 – P95 = 0.0245-0.2065 (n=100); TSH (μIU/mL) - females: mean = 0.076; P5 – P95 = 0.0245-0.1765 (n=100)).
The following clinical chemistry parameters were considered affected by treatment with the test item at 1000 mg/kg/day in males Cohort 1A animals: a higher mean cholesterol concentration (CHOL; 1.22x of control); a higher mean urea concentration (1.17x of control). See Table 12. Any remaining changes in clinical chemistry parameters were considered to be unrelated to treatment with the test item as these were minimal only, occurred in the absence of a dose-related trend, and/or were caused by general overlap and variability in individual values.
Serum T4 levels in males and females and TSH levels in males were considered unaffected by treatment with the test item. Higher TSH levels of 1.42, 2.88 and 2.26x of control were noted in females at 100, 300 and 1000 mg/kg/day, respectively, without reaching statistical significance . As in this type of study the variability of the individual values of TSH is large, the higher mean value at 100 mg/kg/day was considered to be unrelated to treatment with the test item. However, considering the magnitude of change at 300 and 1000 mg/kg/day, a test item-related effect was suspected. Mean and most individual TSH levels remained within the historical control range (Historical control data for Wistar Han rats: F1-animals (2017-2021); TSH (μlU/mL) - females: mean = 0.075; P5 – P95 = 0.0130-0.2025 (n=120)).

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis parameters were considered not to be affected by treatment with the test item. The statistically significantly lower mean specific gravity recorded for females at 1000 mg/kg/day was considered not related to treatment with the test item, given the minor difference compared to means of other dose groups, including the control group. Blood noted in the urine of two females at 1000 mg/kg/day was not supported by any morphological changes in the urinary tract. Also, since this was recorded for two out of ten animals only, this finding was considered not test item-related. At 300 mg/kg/day, an increase in mean urine volume was observed which was considered the result of one female with a particularly high urine volume (25.0 mL). For this animal, a slightly lower specific gravity (1.007) was noted which was considered to be caused by the high urine volume of this animal. As this was an isolated finding, a possible relation to treatment with the test item was not indicated.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

The age at attainment of balanopreputial separation and vaginal opening, body weight at attainment, age at first estrus and time between attainment of vaginal opening and first estrus were considered to have been unaffected by treatment with the test item. The interval between day of vaginal opening and day of first estrus was not significantly different between the groups. All individual values remained within the concurrent control range (1-7 days), except for two female animals (1000 mg/kg/day) for which it took slightly longer (11 days). At the isolated incidence and based on the fact that also one control female animal had an interval of 11 days, this finding was considered to be not related to treatment with the test item.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered unaffected by treatment with the test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment with the test item had no effect on areola/nipple retention. For none of the examined male pups nipples were observed on PND 13.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (PND 89-95): There were no test material-related alterations in organ weights. Statistically significant higher organ weights compared to the control group, were present in male reproductive organs of Cohort 1A. This accounted for the testes weight at 100 mg/kg/day (absolute) and 300 mg/kg/day (absolute and relative to body weight), prostate gland weight at 300 and 1000 mg/kg/day (absolute and relative to body weight), and epididymides weight at 300 mg/kg/day (absolute and relative to body weight). There was no dose response or microscopic correlate. Therefore, these weight changes were regarded unrelated to the treatment with the test material. Any other differences in organ weights of males and females of Cohort 1A, were considered not to be test material-related due to the lack of a dose-related pattern, lack of a test material-related microscopic correlate and/or general overlap and variability in individual values (including higher absolute brain weight at 300 mg/kg/day and higher liver to body weight ratio at 1000 mg/kg/day in males).
Cohort 1B (≥ PND 90): There were no test material-related alterations in organ weights. Statistically significant higher organ weights compared to the control group, were noted for adrenals weight at 100 mg/kg/day (absolute and relative to body weight) and prostate gland weight at 300 mg/kg/day (absolute and relative to body weight) and 1000 mg/kg/day (relative to body weight). There was no dose response and therefore, these weight changes were regarded unrelated to the treatment with the test material.
Cohort Surplus (PND 22-24): No changes were noted in brain, thymus (absolute and relative) and terminal body weight in PND 22-24 animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 pups until weaning: no macroscopic findings were noted among pups that either died preterm or survived until scheduled necropsy at the end of the lactation period that were considered to be related to treatment with the test item. For several pups found dead at first letter check no milk, cannibalism or advanced autolysis was noted. For one pup at 300 mg/kg/day which was found dead on PND 15, the lungs were grown together with the stomach. In addition, the stomach, spleen and pancreas were observed in the thoracic cavity and a pale discoloration of the liver and the thymus reduced in size were noted at necropsy. For one pup at 100 mg/kg/day surviving until scheduled necropsy, a missing left eye and a small right eye were noted and for one pup at 1000 mg/kg/day a scab in the throat region was noted at necropsy. At the incidence observed and in absence of a dose-related response, these findings were considered unrelated to treatment with the test item.
Cohort 1A (PND 89-95): there were no test material-related gross observations. All recorded macroscopic findings at the end of the treatment period were within the range of background gross observations encountered in rats of this age and strain.
Cohort 1B (≥ PND 90): macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item.
Cohort 1C (males: ≥ PND 35; females: ≥ PND 25): Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item.
Cohort Surplus (PND 22-24): No alterations were noted at necropsy that were considered to have arisen as a result of treatment with the test item.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic findings. All of the recorded microscopic findings (including the microscopic findings in the liver of males) were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. Spermatogenesis: stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present. Ovarian Follicle Counts: there were no test material-related effects on the ovarian follicle counts in the 1000 mg/kg/day group females when compared to control group females. Any variations between group mean counts represented biological variability and statistically significance was not present.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Estrous Cycle (Cohort 1A): Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for one female at 1000 mg/kg/day. For one female of the control group and two females at 100 mg/kg/day, cycle regularity could not be determined as these females had only one complete estrous cycle (of 4 or 5 days) during the 14 days observation period. At the isolated incidence (irregular cycle) or in absence of a dose-related trend, these findings were considered to be unrelated to treatment with the test item.

Sperm Analysis (Cohort 1A): at 300 and 1000 mg/kg/day, the mean number of spermatozoa with a detached head was higher (7 and 6 cells vs. 3 in the control group; not statistically significant at 300 mg/kg/day). Although both group means remained within the range considered normal, a total of 2/20 and 4/20 animals had values that exceeded the control range (0-10 cells) at 300 and 1000 mg/kg/day, respectively. One of these males at 300 mg/kg/day also had the highest number of cells with an abnormal neck. Also, at 1000 mg/kg/day, the incidence of animals showing at least one spermatozoa with an abnormal neck appeared higher than the control incidence (12/20 animals at 1000 mg/kg/day vs. 4/20 animals in the control group). Sperm morphology at 100 mg/kg/day and sperm motility and concentration parameters at all dose levels were considered unaffected by treatment with the test item.

Splenic Lymphocyte Subpopulation (Cohort 1A): Splenic lymphocyte subpopulations were considered unaffected by treatment with the test item. A slightly higher Th/Tc ratio was observed for males at 100 mg/kg/day. As this shift was slight of nature and occurred in the absence of a dose-related trend, this was considered to represent biological variability and as such regarded as unrelated to treatment with the test item.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental Toxicity - until weaning

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental Toxicity - post weaning

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Dose Formulation Analyses

Accuracy: The concentrations analyzed in the formulations of Groups 2, 3 and 4 prepared for use in Weeks 8, 15 and 22 were in agreement with target concentrations (i.e., mean accuracies between 85 and 115%). For the formulation of Groups 3 and 4 prepared for use in Week 1, the mean accuracy was above the target concentration (i.e., 153 and 118% of target). This was most likely due to problems with the analytical system. Small responses at m/z 66 (isotope of quantification of the test item) were detected in the Group 1 formulations prepared for use in Weeks 1, 8, 15 and 22. In Group 1 formulations of Weeks 1 and 8, this response had a negative value, which was due to the fact that the response was below calibration curve intercept. In Group 1 formulations of Weeks 15 and 22 the calculated maximum contribution to Group 2 samples was 0.031 and 0.017%, respectively, taking the dilution factor into account. The observed response in Group 1 formulations was not considered to derive from the test item since similar responses were obtained in the analytical blanks and was therefore considered to have no impact on the study.

Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e., coefficient of variation ≤ 10%), except for the formulation of Group 4 prepared for use in Week 1 (i.e., coefficient of variation 14%). The out of specification result was obtained most likely due to problems with the analytical system.

 

Table 7: Summary of Hematology values (males, P0 Generation)

Sex: Male		Reporting Hematology
		WBC (10^9/L) [G]	NEUT (10^9/L) [G]	LYMPH (10^9/L) [G]	MONO (10^9/L) [G]	EOS (10^9/L) [G]	BASO (10^9/L) [G1]	LUC (10^9/L) [G]	RBC (10^12/L) [G]	RETIC (10^9/L) [G]	RDWG (%) [G]	HGB (g/L) [G]	HCT (L/L) [G]	MCV (fL) [G]	MCH (pg) [G]	MCHC (g/L) [G]	PLT (10^9/L) [G]
0 mg/kg/day Group 1	Mean	5.420	0.932	4.295	0.100	0.067	0.006	0.016	8.369	192.22	12.07	153.1	0.4417	52.85	18.34	346.7	647.2
	SD	0.651	0.225	0.563	0.024	0.027	0.007	0.005	0.472	21.95	0.38	5.2	0.0153	1.90	0.99	7.1	97.7
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
100 mg/kg/day Group 2	Mean	5.312	0.921	4.216	0.085	0.068	0.005	0.013	8.506	177.22	12.10	152.2	0.4427	52.08	17.91	344.1	659.9
	SD	1.082	0.262	1.073	0.025	0.023	0.005	0.007	0.374	19.17	0.37	4.4	0.0166	1.25	0.53	8.3	55.1
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	tCtrl	0.98	0.99	0.98	0.85	1.01	0.83	0.81	1.02	0.92	1.00	0.99	1.00	0.99	0.98	0.99	1.02
300 mg/kg/day Group 3	Mean	5.481	0.904	4.351	0.095	0.099	0.008	0.020	8.648	207.23	11.96	154.2	0.4536	52.47	17.88	340.4	667.7
	SD	0.727	0.294	0.695	0.032	0.042	0.004	0.008	0.413	40.23	0.49	4.5	0.0179	1.40	0.69	7.3	62.7
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	tCtrl	1.01	0.97	1.01	0.95	1.48	1.33	1.25	1.03	1.08	0.99	1.01	1.03	0.99	0.97	0.98	1.03
1000 mg/kg/day Group 4	Mean	6.071	0.926	4.940	0.104	0.082	0.003	0.017	8.371	197.82	11.92	151.0	0.4392	52.48	18.07	343.9	668.0
	SD	1.278	0.272	1.041	0.030	0.039	0.005	0.007	0.277	35.86	0.53	3.9	0.0130	1.24	0.50	5.2	78.6
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	tCtrl	1.12	0.99	1.15	1.04	1.22	0.50	1.06	1.00	1.03	0.99	0.99	0.99	0.99	0.99	0.99	1.03

[G] - Anova & Dunnett: * = p ≤ 0.05
[G1] - Kruskal-Wallis & Dunn

 

Table 8: Summary of Hematology values (females, P0 Generation)

Sex: Female		Reporting Hematology
		WBC (10^9/L) [G]	NEUT (10^9/L) [G]	LYMPH (10^9/L) [G]	MONO (10^9/L) [G]	EOS (10^9/L) [G]	BASO (10^9/L) [G1]	LUC (10^9/L) [G]	RBC (10^12/L) [G]	RETIC (10^9/L) [G]	RDWG (%) [G]	HGB (g/L) [G]	HCT (L/L) [G]	MCV (fL) [G]	MCH (pg) [G]	MCHC (g/L) [G]	PLT (10^9/L) [G]
0 mg/kg/day Group 1	Mean	4.278	1.163	2.819	0.161	0.100	0.010	0.025	8.239	142.43	11.73	166.4	0.4747	57.67	20.24	350.9	865.4
	SD	1.273	0.383	0.900	0.076	0.056	0.005	0.014	0.523	70.30	0.81	8.0	0.0249	2.27	0.95	7.5	100.6
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
100 mg/kg/day Group 2	Mean	4.562	1.184	3.119	0.146	0.074	0.011	0.029	8.467	146.68	11.58	165.6	0.4839	57.16	19.56	342.3	839.8
	SD	0.948	0.317	0.680	0.047	0.016	0.003	0.016	0.343	44.35	0.94	5.6	0.0188	1.32	0.60	7.8	75.3
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	tCtrl	1.07	1.02	1.11	0.91	0.74	1.10	1.16	1.03	1.03	0.99	1.00	1.02	0.99	0.97	0.98	0.97
300 mg/kg/day Group 3	Mean	4.305	1.424	2.632	0.154	0.076	0.005	0.017	8.356	170.79	11.64	162.5	0.4753	56.99	19.53	342.4	850.3
	SD	0.656	0.434	0.374	0.065	0.024	0.005	0.008	0.466	57.18	0.83	4.7	0.0189	2.75	1.12	10.7	103.8
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	tCtrl	1.01	1.22	0.93	0.96	0.76	0.50	0.68	1.01	1.20	0.99	0.98	1.00	0.99	0.96	0.98	0.98
1000 mg/kg/day Group 4	Mean	4.841	1.527	3.024	0.165	0.090	0.007	0.024	8.735	145.86	11.77	165.1	0.4845	55.49	18.93 **	340.9 *	888.3
	SD	1.673	0.804	0.835	0.082	0.045	0.005	0.018	0.369	69.35	0.78	4.7	0.0161	1.49	0.65	6.8	108.3
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	9
	tCtrl	1.13	1.31	1.07	1.02	0.90	0.70	0.96	1.06	1.02	1.00	0.99	1.02	0.96	0.94	0.97	1.03

[G] - Anova & Dunnett: * = p ≤ 0.05
[G1] - Kruskal-Wallis & Dunn

 

Table 9: Summary of Coagulation Values (males, P0 Generation)

Sex: Male		Reporting Coagulation
		PT (sec) [G]	APTT (sec) [G]
0 mg/kg/day Group 1	Mean SD N	16.07 0.72 9	16.66 5.58 9
100 mg/kg/day Group 2	Mean	16.40	16.04
	SD	0.49	2.19
	N	10	10
	tCtrl	1.02	0.96
300 mg/kg/day Group 3	Mean	16.31	15.09
	SD	0.65	1.99
	N	10	10
	tCtrl	1.02	0.91
1000 mg/kg/day Group 4	Mean	17.86 **	18.31
	SD	2.15	3.66
	N	10	10
	tCtrl	1.11	1.10

[G] - Anova & Dunnett: ** = p ≤ 0.01

 

Table 10: Summary of Hematology values (males, F1 Generation, Cohort 1A)

Sex: Male		Reporting Hematology
		WBC (10^9/L) [G]	NEUT (10^9/L) [G]	LYMPH (10^9/L) [G]	MONO (10^9/L) [G]	EOS (10^9/L) [G1]	BASO (10^9/L) [G1]	LUC (10^9/L) [G]	RBC (10^12/L) [G]	RETIC (10^9/L) [G]	RDWG (%) [G]	HGB (g/L) [G]	HCT (L/L) [G]	MCV (fL) [G]	MCH (pg) [G1]	MCHC (g/L) [G]	PLT (10^9/L) [G]
0 mg/kg/day Group 1	Mean	6.362	1.011	5.104	0.117	0.074	0.014	0.039	8.584	221.26	11.32	155.8	0.4639	54.06	18.17	336.0	693.7
	SD	1.427	0.455	1.190	0.033	0.036	0.008	0.015	0.333	24.32	0.35	3.0	0.0119	1.44	0.79	7.2	148.0
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
100 mg/kg/day Group 2	Mean	5.405	0.943	4.243	0.102	0.076	0.008	0.031	8.566	200.80	11.20	156.7	0.4693	54.77	18.29	334.1	729.8
	SD	0.868	0.233	0.750	0.029	0.026	0.006	0.013	0.303	27.79	0.43	4.5	0.0162	0.94	0.37	6.6	91.0
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	tCtrl	0.85	0.93	0.83	0.87	1.03	0.57	0.79	1.00	0.91	0.99	1.01	1.01	1.01	1.01	0.99	1.05
300 mg/kg/day Group 3	Mean	6.837	1.072	5.494	0.129	0.089	0.012	0.044	8.660	204.34	11.63	155.6	0.4721	54.52	17.96	329.5	713.7
	SD	1.103	0.258	0.910	0.044	0.029	0.004	0.016	0.278	16.75	0.45	4.7	0.0137	1.36	0.57	6.2	57.2
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	tCtrl	1.07	1.06	1.08	1.10	1.20	0.86	1.13	1.01	0.92	1.03	1.00	1.02	1.01	0.99	0.98	1.03
1000 mg/kg/day Group 4	Mean	7.791 *	1.182	6.289 *	0.147	0.097	0.015	0.056	8.501	227.31	11.64	153.3	0.4628	54.50	18.06	331.5	718.0
	SD	1.555	0.440	1.123	0.049	0.048	0.011	0.028	0.468	23.52	0.31	5.9	0.0198	1.27	0.47	6.6	69.9
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	tCtrl	1.22	1.17	1.23	1.26	1.31	1.07	1.44	0.99	1.03	1.03	0.98	1.00	1.01	0.99	0.99	1.04

[G] - Anova & Dunnett: * = p ≤ 0.05
[G1] - Kruskal-Wallis & Dunn

 

Table 11: Summary of Hematology values (females, F1 Generation, Cohort 1A)

Sex: Female		Reporting Hematology
		WBC (10^9/L) [G]	NEUT (10^9/L) [G]	LYMPH (10^9/L) [G1]	MONO (10^9/L) [G]	EOS (10^9/L) [G]	BASO (10^9/L) [G]	LUC (10^9/L) [G]	RBC (10^12/L) [G]	RETIC (10^9/L) [G]	RDWG (%) [G1]	HGB (g/L) [G]	HCT (L/L) [G]	MCV (fL) [G]	MCH (pg) [G]	MCHC (g/L) [G]	PLT (10^9/L) [G]
0 mg/kg/day Group 1	Mean	4.359	0.577	3.592	0.092	0.062	0.009	0.030	7.857	200.39	10.64	148.8	0.4375	55.74	18.96	340.3	708.5
	SD	1.042	0.172	0.938	0.040	0.015	0.007	0.016	0.681	28.97	0.28	11.4	0.0357	1.23	0.75	8.6	88.7
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
100 mg/kg/day Group 2	Mean	3.599	0.450	3.005	0.061	0.046	0.006	0.028	8.044	191.76	10.35 *	149.4	0.4443	55.24	18.56	336.0	733.6
	SD	0.875	0.146	0.768	0.017	0.016	0.005	0.015	0.358	34.81	0.14	5.3	0.0209	0.79	0.50	9.1	100.1
	N	8	8	8	8	8	8	8	8	8	8	8	8	8	8	8	8
	tCtrl	0.83	0.78	0.84	0.67	0.75	0.69	0.92	1.02	0.96	0.97	1.00	1.02	0.99	0.98	0.99	1.04
300 mg/kg/day Group 3	Mean	3.931	0.596	3.162	0.084	0.058	0.007	0.026	7.983	188.13	10.62	146.3	0.4350	54.48	18.34	336.8	743.4
	SD	1.471	0.320	1.133	0.047	0.028	0.005	0.016	0.564	30.48	0.35	9.3	0.0339	1.53	0.45	8.0	71.8
	N	9	9	9	9	9	9	9	9	9	9	9	9	9	9	9	9
	tCtrl	0.90	1.03	0.88	0.92	0.93	0.74	0.85	1.02	0.94	1.00	0.98	0.99	0.98	0.97	0.99	1.05
1000 mg/kg/day Group 4	Mean	4.306	0.582	3.561	0.077	0.052	0.004	0.028	8.016	208.71	10.83	145.8	0.4349	54.27 *	18.19 *	335.2	725.7
	SD	1.777	0.238	1.515	0.030	0.015	0.007	0.022	0.251	33.80	0.40	4.0	0.0127	0.92	0.41	5.4	69.9
	N	9	9	9	9	9	9	9	9	9	9	9	9	9	9	9	9
	tCtrl	0.99	1.01	0.99	0.83	0.84	0.49	0.93	1.02	1.04	1.02	0.98	0.99	0.97	0.96	0.99	1.02

[G] - Anova & Dunnett: * = p ≤ 0.05
[G1] - Kruskal-Wallis & Dunn

 

Table 12: Summary of of Urea and Cholesterol Clinical Chemistry Values (males, F1 Generation, Cohort 1A)

Sex: Male		UREA (mmol/L) [G]	CHOL (mmol/L) [G]
0 mg/kg/day Group 1	Mean	3.71	1.562
	SD	0.33	0.284
	N	10	10
100 mg/kg/day Group 2	Mean	3.71	1.731
	SD	0.57	0.215
	N	10	10
	tCtrl	1.00	1.11
300 mg/kg/day Group 3	Mean	3.98	1.677
	SD	0.49	0.249
	N	10	10
	tCtrl	1.07	1.07
1000 mg/kg/day Group 4	Mean	4.34 *	1.908 *
	SD	0.48	0.292
	N	10	10
	tCtrl	1.17	1.22

[G] - Anova & Dunnett

 

Applicant's summary and conclusion

Conclusions:

Based on the results of this Extended One Generation Reproductive Toxicity Study (including Cohort 1), No Observed Adverse Effect Levels (NOAELs) of 1000 mg/kg/day of the test item were established for F0 General Toxicity, F0 Reproductive Toxicity and F1, pre and post weaning Developmental Toxicity.

Executive summary:

This OECD TG 443, performed under GLP conditions,  included Cohort 1. The objective of this study was to provide an evaluation of the pre- and postnatal effects of the test item on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of the test item on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, and lactation. The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on the results of a preliminary reproductive toxicity study (simplified reproduction/developmental toxicity screening test) with oral exposure of the test item in rats. Chemical analyses of formulations were conducted at four occasions during the study to assess accuracy and homogeneity and confirmed that formulations of test item in Corn oil were prepared accurately and homogenously. For the F0 generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. For the F1 generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus, estrous cycle, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined for the F0‑generation: estrous cycle, mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones). In total five F0 animals (two F1 males, one at 100 and 300 mg/kg/day each, and three F1 females, one at 100 and two at 300 mg/kg/day) did not survive until scheduled necropsy. All these mortalities were considered to be procedure-related and not caused by the test item.

Regarding the F0 Generation results, at 300 and 1000 mg/kg/day, total T4 was decreased in F0 males which was considered non-adverse. At 1000 mg/kg/day, a higher mean white blood cell count with concurrent higher mean lymphocyte count and an increase in mean prothrombin time were noted in F0 males. A higher mean white blood cell count with concurrent higher mean neutrophil count were noted in F0-females. These findings were considered non-adverse. No test item-related changes were noted in any of the other parameters investigated in this study for the F0 generation (i.e., mortality/moribundity, clinical signs, body weight, food consumption, urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations).

Regarding F1 Generation, pre-weaning developmental results, at 1000 mg/kg/day, higher mean TSH levels were recorded in male and female pups of Cohort Surplus on PND 22 which was considered non-adverse. No test item-related changes were noted in reproduction and any of the other developmental parameters investigated in the study for the F0 or F1 generation (i.e., estrous cycle, mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones)).

In the post-weaning F1 Generation, at 300 and 1000 mg/kg/day, higher mean TSH levels were observed in females of Cohort 1A which was considered non-adverse. At 300 and 1000 mg/kg/day, a higher number of spermatozoa with a detached head and abnormal neck was recorded for F1-males which was considered non-adverse. At 1000 mg/kg/day, a higher mean white blood cell count with concurrent higher mean lymphocyte count, a higher mean cholesterol concentration and higher mean urea concentration were observed in F1-males. These findings were considered non-adverse. No test item-related changes were noted in any of the other parameters investigated in this study for the F1 generation (i.e., mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus, estrous cycle, urinalysis, gross necropsy findings and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations).

In conclusion, based on the results of this Extended One Generation Reproductive Toxicity Study (including Cohort 1), No Observed Adverse Effect Levels (NOAELs) of 1000 mg/kg/day of the test item were established for F0 General Toxicity, F0 Reproductive Toxicity and F1, pre and post weaning Developmental Toxicity.