Registration Dossier

Diss Factsheets

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension) has been performed following the ECHA decision (CCH-D-2114382075-49-01/F). Due to a delay in the performance of the study, the final study report is not yet available and therefore only initial requested information can be presented. The robust study summary and endpoint summary will be updated when the final study report will become available. This is expected to be in August 2022. Argumentation of the delay is provided by the testing facility and attached in the “Attached justification” field. This is in agreement with approach outlined the after enquiry with ECHA (INC000000319505)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
2018
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
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

Constituent 1
Chemical structure
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 19 Nov 2021

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 / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 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)

Effect levels (P0)

Dose descriptor:
NOAEL
Sex:
male/female
Remarks on result:
other: will be derived after all information is available

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Sex:
male/female
Remarks on result:
other: will be derived after all information is available

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Interim results EOGRTS ZBEC


 


Mortality – F1-generation (post weaning)


The following mortalities were considered not to be the result of treatment with the test item.


 


Male No. 375 (300 mg/kg/day, Cohort 1B), which was dosed for 34 days, was found dead in its cage. There were no clinical signs or adverse changes in body weight and food consumption prior to its death. At necropsy, enlarged thyroids, esophagus perforation and in its thoracic cavity a gelatin-like, watery turbid substance (which is a similar consistency as the formulation) were noted. Based on these findings, this death was considered to be the result of an oral gavage error.


 


Female No. 559 (control group, Cohort 1A), which was dosed for 35 days, was found dead in its cage. Labored respiration, gasping and piloerection were noted prior to its death with no adverse changes in body weight and food consumption on the days prior to its death. Female No. 632 (300 mg/kg/day, Cohort 1A), which was dosed for 35 days, was euthanized in extremis after consultation with the veterinarian as it exhibited hunched posture, labored respiration and piloerection. For both Female Nos. 559 and 632 a gelatin-like, watery turbid substance was noted in their thoracic cavity at necropsy. Therefore, although no perforation was noted during necropsy, based on the gelatin-like, watery turbid substance in their thoracic cavity, these deaths were considered the result of an oral gavage error.


 


One male (No. 321, 100 mg/kg/day, Cohort 1C), which was dosed for 18 days, was inadvertently caught between the shelter and the lid of the cage resulting in its untimely death.


 


One female (No. 645, 300 mg/kg/day, Cohort 1B), which was dosed for 32 days, was inadvertently caught between the cage and the lid of the cage resulting in its untimely death.


 


Clinical Signs – F1-generation (post weaning)


No toxicologically relevant signs were noted thus far.


 


Incidental findings that were noted included hunched posture, wounds, scabs, alopecia, bent or broken tail apex, piloerection, salivation, exophthalmos and rales. 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. At the incidence observed, these were considered signs of no toxicological relevance.


 


Body Weights – F1-generation (post weaning)


Body weights and body weight gains were considered similar to the control for all treated animals up to 1000 mg/kg/day.


 


Food Consumption – F1-generation (post weaning)


Food consumption was considered similar to the control for all treated animals up to 1000 mg/kg/day until Week 8 of treatment. From Week 8 until 11 of treatment, absolute and relative food consumption was increased in males at 1000 mg/kg/day.


 


Reproductive/Developmental data – post-weaning


Vaginal patency: not affected by treatment with the test item up to 1000 mg/kg/day.


Balanopreputial Separation: not affected by treatment with the test item up to 1000 mg/kg/day.


Estrous Cycle Determination (cohort 1A): not affected by treatment with the test item up to 1000 mg/kg/day.


Time to onset of first estrous (cohort 1A): not affected by treatment with the test item up to 1000 mg/kg/day.


Sperm analysis (cohort 1A): Sperm motility, concentration and morphology parameters were considered unaffected by treatment with the test item. The slightly higher number of spermatozoa with a detached head at 1000 mg/kg/day, was considered unrelated to treatment in absence of a dose related trend.


 


Hematology and coagulation – Cohort 1A


In males, a higher white blood cell count (WBC; 1.22x of control) was observed which was mainly cuased by a higher lymphocyte count (1.23x of control) at 1000 mg/kg/day.


In females, mean corpuscular volume (MCV) and mean corpuscular hemoglobin level (MCH) were slightly decreased at 1000 mg/kg/day.


 


Clinical chemistry – Cohort 1A


In males, the following changes were noted at 1000 mg/kg/day:


-increase in urea concentration of 1.17x of control


-Increase in cholesterol of 1.22x of control


 


Urinalysis – Cohort 1A


In females, a slightly lower specific gravity (0.99x of control) was observed at 1000 mg/kg/day


 


Splenic lymphocyte subpopulation analysis - Cohort 1A


Th/Tc was slightly increased at 100 mg/kg/day.


 


Thyroid hormones - Cohort Surplus and PND 4 pups


No statistically significant changes were noted


 


Macroscopic examination – F1-generation (post weaning)


Cohort 1A (PND 89-95): 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 1B (PND ≥97): 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 (After VO/BPS positive): 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): no alterations that were considered to have arisen as a result of treatment with the test item.


 


Organ weights – F1-generation (post weaning)


Cohort 1A (PND 89-95): In males, relative liver weights were increased with 5% at 1000 mg/kg/day. Any other changes were considered not related to treatment with the test item due to the minimal magnitude of change and/or absence of a dose-related trend.


Cohort 1B (PND ≥97): In males, relative prostate gland weights were increased with 18 and 13% at 300 and 1000 mg/kg/day, respectively.


Cohort surplus (PND 22): considered not affected by treatment with the test item up to 1000 mg/kg/day.


 

Applicant's summary and conclusion