Registration Dossier

Endpoint:

extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Oct 2020 to 23 Apr 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

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

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

- Basis for dose level selection: The dose levels in this study were selected based on a Dose Range Finder Study with oral administration by gavage of the test item for a minimum of 28 days to Wistar Han rats. In the Dose Range Finder Study, animals were dosed 10, 50 and 125 mg/kg/day.
In the Parental generation, two females at 125 mg/kg/day were sacrificed in extremis on Lactation Days 13 and 6, respectively. Both female lost approximately 20% of their body weight within a few days and presented with clinical signs of toxicity. Based on the similarities between the circumstances surrounding the deaths of both females at 125 mg/kg/day and in the absence of histopathological evaluation, a relationship to treatment with the test item could not be excluded. As such, these deaths at 125 mg/kg/day were considered test-item related.
Females during the post-coitum and lactation phase showed lower mean body weight (gain) at 50 and 125 mg/kg/day, compared to concurrent controls (8-13%). A similar trend on mean body weights was observed at 10 mg/kg/day (4%), although not statistically significant. This was without apparent effect on food consumption during this phase. Given the magnitude of the effect at 50 and 125 mg/kg/day, the test item-related effects on body weights in females at 50 and 125 mg/kg/day were considered adverse. In 50 and 125 mg/kg/day group males and in 125 mg/kg/day group females, test item-related higher mean liver weights were noted. Based on the size of the effect, only the increased mean liver weights at 125 mg/kg/day in males and females (>20%) were considered adverse.

In the F1 generation, at 50 and 125 mg/kg/day, mean pup body weights were decreased from Day 1 onwards (not always statistically significant). Combined male and female pup body weights on Day 13 were respectively 14% and 18% lower compared to concurrent controls. Based on the magnitude of the response, decreased body weights of pups at 50 and 125 mg/kg/day were considered adverse.

Based on test-item related mortality at 125 mg/kg/day, adverse effects on body weights in females at 50 and 125 mg/kg/day and increased liver weights in males and females at 125 mg/kg/day, a parental NOAEL was set at 10 mg/kg/day.
Based on lack of adverse effects on reproduction, the reproduction NOAEL was set of at least 125 mg/kg/day.
Based on test-item-related adverse effects on body weights of pups at 50 and 125 mg/kg/day, the Developmental NOAEL was set at 10 mg/kg/day

Based on the abovementioned results (observed parental toxicity, unaffected mating and fertility up to the high dose level, sufficient litters available for evaluation at the high dose, observed effects on pup body weights at the mid and high dose level), taking into account the 10 week dosing period prior to start mating and in consultation with the sponsor, the following dose levels were selected: 10, 30 an 100 mg/kg/day.

-Exclusion of extension of Cohort 1B

- Inclusion of developmental neurotoxicity Cohorts 2A and 2B

- Inclusion 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 and for neurotoxicity and immunotoxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental/neurological/immunological 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, neurological and immunological 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.

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 6 - 7 wks
- Weight at study initiation: (P) Males:155 and 206 g ; Females: 101 and 145 g
- Housing: On arrival, prior to mating and during the post-weaning period, animals were group 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).
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 their home cage (Makrolon plastic cages, type IV; height 18 cm or type 2000P; 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 or weaning (on PND 21).
During locomotor activity monitoring, F1- Cohort 2A animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water for a maximum of 2 hours.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
- Diet: ad libitum except during designated procedures. During motor activity measurements, F1- Cohort 2A animals will not have access to food for a maximum of 2 hours.
- Water: freely available to each animal via water bottles. During motor activity measurements, F1- Cohort 2A animals will not have access to water for a maximum of 2 hours.
- Acclimation period: The F0-Animals and positive control animals were allowed to acclimate to the Test Facility toxicology accommodation for respectively 12 and 8 or 9 days (depending on the initiation of dosing for the two subsets of positive control animals) before the commencement of dosing.

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

IN-LIFE DATES: From: 12 Oct 2020 To: 23 Apr 2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation: The dosing formulations was prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations (w/w) were homogenized, by stirring the formulations for at least 15 minutes (prior to visual inspection), to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations was kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle will be continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

VEHICLE
- Amount of vehicle: 4 mL/kg
- The dose volume for each animal was based on the most recent body weight measurement. The dose formulations were stirred continuously during dosing. 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
- After successful mating each pregnant female was caged in Makrolon plastic cages (type III, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for Analysis: On three occasions (Week 1 of treatment - Day 3; Week 11 of treatment - Day 79; Week 22 of treatment - Day 156) all Groups were collected for concentration analysis and Group 2 and Group 4 were collected for homogenicity

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 was a coefficient of variation (CV) of concentrations of ≤ 10% for each group.

Stability analysis of the test item in the vehicle could not be determined, since the available analytical method was only capable of measuring the zinc content in the test substance and, therefore, unable to measure the test item itself (as demonstrated during development of the analytical procedure of test substance with a similar structure, Zinc bis(dibenzyldithiocarbamate (ZBEC; Test Facility Study No. 506837)). Additional development of an analytical method for ZBEC was tried in Test Facility Study No. 20247984, however, due to high complexity in chemical behavior of the molecule it was technically not possible to develop a UPLC-UV method on the intact molecule. Derivatization approach appeared to be not robust enough. Therefore, it has been decided to develop an analytical method based only on quantification of zinc ion.

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 preparations 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.
This study was conducted in accordance with the procedures described herein. All deviations authorized/acknowledged by the Study Director are documented in the Study Records. The report represents an accurate and complete record of the results obtained.
There were no deviations from the above regulations that affected the overall integrity of the study or the interpretation of the study results and conclusions.

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 and post-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, 2A and 3 were dosed up to and including the day before scheduled necropsy. The F1-animals of Cohort 2B, Cohort Surplus and Spares (not assigned to one of the cohorts) were not dosed. Duration of treatment for Cohort 1A was 9-10 weeks; Cohort 1B 10-11 weeks; Cohort 2A 7-8 weeks and Cohort 3 4-5 weeks.

Frequency of treatment:

Once daily

No. of animals per sex per dose:

F0: 25
F1:
Cohort 1A and 1B: 20
Cohort 2A, 2B, 3: 10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose levels in this study were selected to be 0, 10, 30 and 100 mg/kg/day based on a Dose Range Finder Study with oral administration by gavage of the test item for a minimum of 28 days to Wistar Han rats. In the Dose Range Finder Study, animals were dosed 10, 50 and 125 mg/kg/day (see justification for study design)

Positive control:

yes; Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany and used as positive control animals for the TDAR assay. At initiation of dosing, these animals were 7 weeks old and weighed between 212 and 235 g (males) and between 122 and 147 g (females)
2.0 mg/mL cyclophosphamide formulation was administered via intraperitoneal injection using a disposable needle and syringe once daily on five consecutive days prior to necropsy (i.e. starting between PND 48-54), approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. The dose volume for each animal was based on body weight measurement on the first day of cyclophosphamide treatment.

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.

DETAILED 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. These observations were conducted after dosing.

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. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE
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, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

WATER CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.
As a test item-related effect on water consumption was suspected in treatment Week 5, semi-quantitative measurement of water intake was introduced at discretion of the Study Director for all males and females of Group 1 and 4 between Days 31-47 of treatment, by reading approximate water consumption from the marks on the water bottles. From Day 48 onward, subjective appraisal was maintained during the study, as in consultation with the sponsor, no quantitative investigation was introduced.

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 (F0-Animals) were collected into tubes without anticoagulant. Blood samples were processed for serum. 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. Any remaining sample will be discarded at finalization.
- 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 for all F0-females 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):

Parameters examined in F0, Cohort 1A male parental generations:
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

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. The number of live and dead pups was determined on PND 1 and daily thereafter. 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. For animals of Cohort 2B and Surplus, a terminal weight was recorded on the day of scheduled necropsy.
- 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, 2A and 3):
- Mortality/Moribundity Checks: Throughout the study, animals were observed for general health/mortality and moribundity at leat 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.
- 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. For animals of Cohorts 1A, 1B, and 2A, a terminal weight was recorded on the day of scheduled necropsy.
- 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.

Cohort specific investigations F1-Generation from Weaning (PND 21) onward (Cohort 1A):
- Stage of Estrus 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. In addition, estrous stages were determined by examining the cytology of vaginal lavage sample, taken on the day of scheduled necropsy.

Cohort specific investigations F1-Generation from Weaning (PND 21) onward (Cohort 2A):
- Acoustic startle response: Acoustic startle response (habituation) was assessed using the StartleMonitor System (Kinder Scientific, Poway, USA). This was performed once in a sound- attenuated room between PND 23-25. To the extent possible, treatment groups were balanced across devices and the time of testing will be counterbalanced across dose group and sex. The animals were tested in sets of up to 3. The test sessions consisted of a five-minute acclimation period with a 65 ± 5-dB broadband background white noise. The startle stimulus for each trial was a 115 ± 5-dB mixed frequency noise burst stimulus (approximately 20 milliseconds in duration). Responses were recorded during the first 250 milliseconds following onset of the startle stimulus for each trial. The test session consisted of 50 trials with an eight- second intertrial interval. Average response amplitude (AveN) were analyzed in five blocks of 10 trials each
- Functional Observation Battery: The Functional Observation Battery (FOB) tests was conducted once between PND 63-75 in the order of sequence indicated below and may be divided between several days. The detailed functional observations and locomotor activity was conducted in separate room(s) specially equipped for these purposes.
1.Detailed functional observations: consist of a number of tests conducted in- and out-side the home cage
2. Rectal temperature: measured immediately after the detailed functional observations
3.Locomotor activity: tested using the Kinder Scientific Motor Monitor System. Recording period was one hour under normal laboratory light conditions.
4.Hearing ability. Score 0 = normal/present, score 1 = abnormal/absent
5.Pupillary reflex (both eyes).
6.Fore- and hindlimb grip strength: recorded per animal as the mean of three measurements, using a grip strength meter (Series M4-10, Mark-10 Corporation).
7.Landing (hind) foot splay:recorded per animal as the mean of three measurements

Cohort specific investigations F1-Generation from Weaning (PND 21) onward (Cohort 3):
- Immunization with KLH: All F1-Animals of Cohort 3 and all positive control animals were immunized once, 5 days before scheduled necropsy (i.e. once between PND 48-54) via intravenous injection into the tail vein (approximately 1 mL/min) with 1.0 mL of the 300 µg/mL KLH formulation.
This immunization was performed 2 to 4 hours after treatment of the positive control animals with cyclophosphamide. The animals were restrained during the injection procedure and injected using a disposable needle and butterfly needle. After injection, the injection site was marked with indelible ink. On the day of KLH injection, clinical sign observations were performed after this injection for animals of Cohort 3 and the positive control animals.
- Positive control animals were checked for: mortality/moribundity, clinical observations, body weights, food consumption and water consumption.
- T-Cell Dependent Antibody Response (TDAR) Assay: Blood of all Cohort 3 animals (10 animals/sex/group) and positive control animals (10 animals/sex), except for animals which were sacrificed in extremis or found dead, were collected on pre-immunization and 5 days after immunization (PND 53-59). Animals were not deprived of food prior to sampling. Samples were collected, between 7.00 and 10.30 a.m., from the jugular vein in the animal facility. Blood samples at a target volume of 0.5 mL were collected into tubes without anticoagulant. Blood samples were allowed to clot for at least 30 minutes and centrifuged within 2 hours after collection. Within 1 hour after centrifugation, serum of these samples were divided into 2 aliquots and subsequently stored in labeled polypropylene tubes at ≤-75°C until shipment.

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. Pooled serum of culled PND 4 pups was used for measurement of T4 only.

Clinical Pathology - F1- animals of Cohort 2B
On PND 21-22, blood was collected from all Cohort 2B animals (10/sex/group), if possible. Blood was drawn, between 8.00 and 11.30 a.m., from the retro-orbital sinus under anaesthesia using isoflurane as part of the necropsy procedure. Serum of Cohort 2B animals was used for measurement of both T4 and TSH.

Clinical Pathology – Cohort 1A
Blood of 10 selected animals/sex/group of Cohort 1A animals was collected on the day of scheduled necropsy for
- 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 (ALAT), Creatinine, Aspartate aminotransferase (ASAT),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) were collected into tubes without anticoagulant. Blood samples were processed for serum. Serum of Cohort 1A animals was used for measurement of both T4 and TSH.
-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
Animals surviving until scheduled euthanasia were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of approximately 24 hours before necropsy. Water was available.

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 4 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios were calculated. Representative samples of the tissues identified in Table 5 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.

Postmortem examinations (offspring):

SACRIFICE
- Unscheduled Deaths: 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 external abnormalities were recorded. External 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.
- 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.

GROSS NECROPSY
See Table 2 for details

HISTOPATHOLOGY / ORGAN WEIGTHS
The same tissues indicated in Table 4 (Tissue Collection and Preservation for the F0- Generation) were prepared for microscopic examination and weighed, respectively, for Cohort 1A animals and unscheduled deaths.
For Chohort 2A and 2B animals investigations, see Table 5

Cohort specific terminal procedures - 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. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
- For all 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.
- In addition to the general procedures, HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared for the Cohort 1A animals of Group 1 and 4 for quantitative evaluation of follicles (primordial and small growing follicles counted together), as well as corpora lutea.

Cohort specific terminal procedures - Cohort 1B
- Scheduled necropsy of Cohort 1B was conducted on ≥ PND 97. Cohort 1B animals were not deprived of food overnight before necropsy. These animals were weighed and deeply anesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Cohort specific terminal procedures - Cohort 2A and 2B
- Scheduled necropsy of Cohort 2A was conducted on PND 76-90. Scheduled necropsy of Cohort 2B was conducted on PND 21-22. The animals were not deprived of food overnight before necropsy. Terminal body weight was recorded. The animals were first anesthetized using isoflurane and subsequently sacrificed by whole body (in situ) perfusion using heparinized saline (0.9% NaCl) followed by a 4% paraformaldehyde solution (adjusted to pH 7.4; HCl, KCl, NaH2PO4 x H2O, Na2HPO4 x 2H2O, paraformaldehyde and NaOH, aqua dest.). All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. After perfusion, the cranium was removed, exposing the brain. The skull including the brain was placed in 10% buffered formalin and allowed to fix for at least 7 days prior to removal from the skull. The fixed brains were removed and weighed, and the length and maximum width of the brain was measured for all animals selected for neuropathology. Subsequently, the brain was fixed in 10% buffered formalin together with selected PNS tissues.
- In addition to the general procedures, the entire brain from all groups was processed to the block stage up front at the same time to avoid effects of fixation duration on morphometry. Sections of the brains of all Cohort 2A and 2B animals (all groups) were also stained for myelin and cell bodies using Luxol Fast Blue and Cresyl Violet. For morphometric analysis, 3 consecutive sections were taken from neocortical, hippocampal and cerebellar areas to ensure homologous sections are obtained. Morphometric (quantitative) analyses of CNS tissues was performed for Cohort 2A and 2B animals of Groups 1 and 4. As test item-related effects were suspected, this was extended to animals in the intermediate dose groups (Groups 2 and 3). Analyses included measurements from neocortical, hippocampal, and cerebellar areas selected.

Cohort specific terminal procedures - Cohort 3
- Scheduled necropsy of Cohort 3 was conducted on PND 53-59. Positive control animals were euthanized on the same date(s). These animals were not deprived of food overnight before necropsy. The animals were deeply anesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Cohort specific terminal procedures - Cohort Surplus
- Scheduled necropsy of Cohort Surplus was conducted on PND 22-24. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. The animals were deeply anesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

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). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means will applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, will be used in order to select the best fitting statistical model
Non-Parametric:
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). The startle data set and the follicle count data set (at least 3 groups) will be compared using an overall Kruskal-Wallis. Whenever, the overall test is significant, the Wilcoxon Rank-Sum test will be applied to compare the treated groups to the control group.
Incidence:
An overall Fisher’s exact test will be used to compare all groups. The above pairwise comparisons will be 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

Reproductive indices:

See Table 3

Offspring viability indices:

Seee Table 3

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Note: Clinical signs that (only) occurred in animals that were found dead or were sacrificed in extremis are described under Mortality – F0-generation.

Reflux after dosing was noted in individual animals at 30 mg/kg/day (Nos. 61 and 172) and 100 mg/kg/day (No. 94) on a single day during the treatment period. This clinical sign was also noted in two animals at 100 mg/kg/day that were sacrificed in extremis.
In addition, the following clinical signs were noted: rales in a single male at 30 and 100 mg/kg/day. Piloerection in individual animals of the control, 10, 30 and 100 mg/kg/day group (females only in the control and 10 mg/kg/day group ) and hunched posture in a single female at 100 mg/kg/day. As these clinical signs were noted in most of the animals that died prematurely, as well as in several animals that survived until scheduled necropsy, a relationship to treatment could not be excluded.
Salivation was observed after dosing among animals of the 10, 30 and 100 mg/kg/day groups (both sexes). Both, a higher incidence as well as an earlier onset in relation to the treatment period was noted with increasing dose. Considering the nature and minor to moderate severity of the effect and its time of occurrence (i.e. after dosing) it was regarded to be a physiological response rather than a sign of systemic toxicity.
No treatment-related clinical signs were noted during weekly arena observations.
Other findings that were noted (pre- and/or post-dose) included skin scabs, wounds, alopecia, a broken tail apex, exophthalmos, yellow discoloration of the genital region, and chromodacryorrhoea. These clinical signs were noted incidentally, 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/or did not show any apparent dose-related trend. At the incidence observed, they were considered not to be signs of toxicological relevance.

Mortality:

mortality observed, treatment-related

Description (incidence):

A total of twelve F0-animals (Table 1) were found dead or were sacrificed in extremis during the treatment period. This concerned 2/50, 2/50 and 8/50 animals of the control, 30 and 100 mg/kg/day groups, respectively, whereas all 50 animals of the low dose group (10 mg/kg/day) survived until scheduled necropsy. Full histopathological examination was performed for the animals at 100 mg/kg/day including microscopic evaluation of the nasopharynx and nasal cavity.

Based on the findings in the nasal cavity/ nasopharynx, in combination with the clinical signs (including rales and gasping) and/or severe body weight loss noted for most of these animals., all (except Animal No. 188) decedents were considered to be reflux-related and not directly related to treatment with the test item. For Female No. 188 (100 mg/kg/day), no definite cause of moribundity could be determined. As this was a single death which, given the timing (Day 1 of lactation), might be related to the pregnancy, it was considered not toxicologically relevant.
One female of the control group (No. 116) was euthanized on Day 2 of lactation, as it had a total litter loss. In addition, Female No. 112 (control group) was sacrificed in extremis because of difficulties with delivery. For details see table below and Section 9.4.7. At the single incidence observed and as both females were control animals, these deaths were not test item-related.
Female No. 163 at 30 mg/kg/day was found dead on Day 36 of treatment. Based on the combination of macroscopic and microscopic findings (watery clear fatty fluid in the pericardium of the heart and watery-clear fluid in the thoracic cavity, together with the microscopic finding of a granulocytic inflammatory cell infiltrate in the pleura of the lungs), this spontaneous death was considered to likely be gavage procedure-related and not test item-related. Moreover, female No. 160 (30 mg/kg/day) was sacrificed at initiation of mating at discretion of the Study Director, as it had no vaginal opening and as such could not be used for mating. Its death was not test-item related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg/day, body weight gain of males was lower compared to controls from Day 15 of treatment onwards. This resulted in lower body weights, reaching statistical significance from Day 36 onwards (up to 7.5% lower at the end of the treatment period). A similar trend was observed at 30 mg/kg/day, although changes compared to controls were very slight (up to 2.5% lower for body weight at the end of the treatment period; statistical significance was not achieved).
Noteworthy, body weights in females of the control group were considered somewhat higher compared to all treated groups throughout the treatment period, causing statistically significant changes in body weight (gain) at several occasions. Any statistically significant changes in weight (gain) of these animals during the pre-mating and mating period occurred in the absence of a dose-related trend and were considered to be unrelated to treatment.
At 100 mg/kg/day, female body weight and body weight gain were lower compared to controls during the post-coitum phase, reaching statistical significance on several occasions (up to 9% lower for body weights at Day 20 post-coitum phase). A similar trend was observed for body weight during post-coitum at 30 mg/kg/day (incidentally reaching statistical significance throughout the post-coitum phase). Body weight gain at 30 mg/kg/day was considered lower on Day 20 post-coitum only (not statistically significant).
As a result, body weights at the start of the Lactation phase were lower at 100 mg/kg/day. Due to the observed increased body weight gain at 100 mg/kg/day during the Lactation phase, body weights at 100 mg/kg/day showed apparent recovery to normal values by Day 21 of Lactation.
Body weight and body weight gain at 10 mg/kg/day during the post-coitum and lactation phase were considered unaffected by treatment with the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg/day, relative food consumption was somewhat decreased in males during the first week of treatment (4% compared to concurrent controls in males), with apparent recovery from the second week of treatment onwards. Subsequently, relative food consumption was increased compared to concurrent controls from Day 15 onwards, reaching statistical significance on multiple occasions.
In females treated at 100 mg/kg/day, absolute and relative food consumption during post-coitum and lactation were lower compared to concurrent controls (13% and 4% lower for relative food consumption on respectively Day 20 post-coitum and Day 21 of lactation; occasionally reaching statistical significance).
At 10 and 30 mg/kg/day (both sexes), food consumption before or after correction for body weight was considered not affected by treatment with the test item. Further statistically significant differences in absolute and/or relative food consumption for males and females at were considered not to represent a change of toxicological significance since no trend was apparent regarding dose and duration of treatment.

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

effects observed, treatment-related

Description (incidence and severity):

Increased water consumption at 100 mg/kg/day (both sexes) was noted during visual inspection over the first weeks of treatment. At discretion of the Study Director, water consumption was monitored semi-quantitatively for all animals in the control and high dose group between Days 31 and 47 of treatment by reading approximate water consumption of the water bottles. These observations confirmed the findings during visual inspection that water consumption at 100 mg/kg/day was increased. In consultation with the Sponsor, no further semi-quantitative measures of water consumption were performed during the study, as it was considered that sufficient information was obtained from these data to perform toxicological evaluation for this endpoint.
Water consumption at 10 and 30 mg/kg/day (monitored by visual inspection) was considered unaffected by treatment with the test-item.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following changes distinguished males treated at 100 mg/kg/day from control animals (the differences were statistically significant unless indicated otherwise; relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
- Increased mean white blood cell count (WBC; 1.39x of control)
- Increased mean neutrophil count (NEUT; 1.63x of control)
- Increased mean lymphocyte count (LYMPH; 1.33x of control)
- Increased mean monocyte count (MONO; 1.33x of control; not statistically significant)
- Increased mean eosinophil count (EOS; 1.11x of control; not statistically significant)
- Increased mean basophil count (BASO; 1.89x of control; not statistically significant)
- Increased mean large unstained cell count (LUC; 1.55x of control)
Hematology parameters in males at 10 and 30 mg/kg/day and females up to 100 mg/kg/day were considered not to have been affected by treatment with the test item.
The statistically significantly lower mean corpuscular hemoglobin concentration at 10 mg/kg/day (HGB; 0.98x of control) was considered not to be of toxicological relevance given the small magnitude of the change and in the absence of a dose-related trend.

Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.
The trend towards a lower Activated Prothrombin Time (APTT) in males and females at 100 mg/kg/day (not statistically significant), was considered not toxicologically relevant, due to the general overlap and variability of individual values and the direction of the change.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following changes distinguished animals treated from control animals (the differences were statistically significant unless indicated otherwise; relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
- Decreased alanine aminotransferase in females at 10, 30 and 100 mg/kg/day (ALT; 0.94x, 0.83x , and 0.79x of control, respectively; not statistically significant at 10 and 30 mg/kg/day)
- Decreased total protein in males at 100 mg/kg/day, and in females at 10, 30 and 100 mg/kg/day (TPROT; 0.95x of control in males, and in females 0.96x of control at all dose levels; not statistically significant in females at 10 mg/kg/day)
- Decreased albumin in males at 100 mg/kg/day and females at 10, 30 and 100 mg/kg/day (ALB; 0.94x of control in males, and in females 0.95, 0.94 and 0.92x of control, respectively)
- Decreased glucose in males at 100 mg/kg/day (GLUC; 0.84x of control)
- Increased cholesterol in females at 100 mg/kg/day (CHOL; 1.43x of control)
- Decreased inorganic phosphate in females at 100 mg/kg/day (PHOS; 0.80x of control)
The statistically significant lower inorganic phosphate in males at 30 mg/kg/day (0.84x of control), decreased creatinine in males at 100 mg/kg/day and females at 10 and 100 mg/kg/day (CREAT; 0.92x of control in males and in females 0.88 and 0.86x of control, respectively; not statistically significant in males at 100 mg/kg/day), increased chloride in females at 30 and 100 mg/kg/day (CL; 1.02x of control at both dose levels; not statistically significant at 30 mg/kg/day) and the increased glucose in females at 30 mg/kg/day (1.19x of control) were considered not to be of toxicological relevance in the absence of a dose-related response.

Endocrine findings:

no effects observed

Description (incidence and severity):

Serum levels of TSH and total T4 in F0-males and F0-females were considered not to be affected by treatment with the test item.
The increase in TSH in males at 100 mg/kg/day (1.43x of control, not statistically significant) was due to the high individual value of Animal No. 86 (0.879 mU/L) and was considered not toxicologically relevant. After excluding this value, mean TSH at 100 mg/kg/day was 0.1652 mU/L, which is comparable to the control group (i.e. 0.1653 mU/L).

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In males, a dose dependent increase in urine volume was noted, reaching statistical significance at 100 mg/kg/day (up to 2.05x of control).
Also in females, at 100 mg/kg/day, a higher urine volume was observed, but this change was less pronounced than in high dose males (no statistical significance was achieved). Noteworthy, a high variability in urine volume was noted in females at 100 mg/kg/day which was caused by an unusual high individual value of Female No. 197 (> 60mL urine overnight). However, after excluding this value from the group mean, urine volume at 100 mg/kg/day was still somewhat increased compared to controls (18.1 mL at 100 mg/kg/day vs 15.7 mL in the control group, i.e. 1.15x of control).
Moreover, the following changes in urine parameters distinguished treated animals from control animals:
- Clarity of the urine was scored as cloudy in 3/10 males and 4/10 females at 100 mg/kg/day (vs. 0/10 in concurrent controls, both sexes).
- Increased incidence of blood in urine and red blood cells in sediment samples in males at 100 mg/kg/day (BLD; score 3+ in 3/10 vs. negative for blood in all concurrent controls and RBC; score up to 3+ in 3/10 vs. 1+ in 1/10 concurrent controls).
- Increased number of leukocyte esterase positive urine samples in males at 100 mg/kg/day (LEUK EST; 6/10 vs. 0/10 in concurrent controls).
- Increased number of ketone positive urine samples in females at 100 mg/kg/day (KET; 6/10 with score 1+ vs. negative for ketones in all concurrent controls).
- Increased unclassified crystals in sediment of females at 100 mg/kg (UNCLASS CRYS; score 3+ in 7/10 females vs. 1/10 in concurrent controls).
- Increased urinary pH in 2/10 males at 100 mg/kg/day (8.0 vs. maximally 7.0 in concurrent controls).
The statistically significant slight decrease in specific gravity at 30 and 100 mg/kg/day (0.99x of control at both dose levels) was considered not toxicologically relevant due to the small magnitude of the effect and in the absence of a dose related trend.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the adrenal gland of the 30 and 100 mg/kg/day group males and are summarized in Table 3.
In the adrenal glands of males starting at 30 mg/kg/day, an increased incidence and severity of vacuolation of the zona glomerulosa was present (up to slight degree). This microscopic finding likely correlated with the increased adrenal gland weight.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings 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.

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. Estrous stage/length could not be determined for Female No. 106 of the control group and Female Nos. 189 and 190 at 100 mg/kg/day (all with normal litters). An irregular cycle was noted for Female No. 193 at 100 mg/kg/day (with normal litter). Given their incidental nature, absence of a dose-related incidence and/or absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Sperm motility and morphology were considered unaffected by treatment up to 100 mg/kg/day.
A statistically significantly lower percentage of progressive sperm was recorded for males at 10 and 30 mg/kg/day. This finding was considered not to be related to treatment with the test item, since no similar change was observed in males of the 100 mg/kg/day group.
Sperm count in the epididymis was statistically significantly higher at 100 mg/kg/day (1.20x of concurrent control). Given the direction of the change, this finding was considered not toxicologically relevant.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating Index: Female mating index (number of mated females as percentage of the number of paired females) and male mating index (number of mated males as percentage of the number of paired males) was unaffected by treatment with the test item. Both male and female mating indices were 96, 100, 100, 100% for the control, 10, 30 and 100 mg/kg/day groups, respectively. Except for Female No. 109 (control group), all females showed evidence of mating.

Precoital time was unaffected by treatment with the test item. All females showed evidence of mating within 5 days.

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

Female fertility index (number of pregnant females as percentage of the number of mated females) and male fertility index (number of pregnant females as percentage of the number of mated males) was considered not to be affected by treatment with the test item. Both male and female fertility indices were 100, 96, 91, 100% for the control, 10, 30 and 100 mg/kg/day groups, respectively.
One female at 10 mg/kg/day and two females at 30 mg/kg/day were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment.
Noteworthy, Male No. 92 and Female No. 192 are excluded from calculation of the fertility indices since for Female No. 192 at 100 mg/kg/day, pregnancy status could not be determined as she was sacrificed on Day 5 of post-coitum, at which stage implantation of the conceptus has not been completed.

Couples that did not succeed in producing healthy offspring:
In the control group, there was 1/25 couple with total litter loss and 1/25 couple that did not mate. In the 10 mg/kg/day group, there was one female that was not pregnant after mating. In the 30 mg/kg/day group, there were two females that were not pregnant after mating. In the 100 mg/kg/day group, there was one female sacrificed on lactation Day 1 due to maternal toxicity, and there was another female sacrificed on Day 5 post-coitum.
No abnormalities were seen in the reproductive organs or mammary glands in the control group, or animals at 10 and 100 mg/kg/day that could explain reproductive performance. For the couples at 30 mg/kg/day, the lack of pregnancy was due to the bilateral testes and epididymides findings. These were not considered test item-related based on the absence of these bilateral findings in high dose males (100 mg/kg/day).
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present (except for the two males (Nos. 58 and 61) at 30 mg/kg/day with bilateral testes and epididymides findings).

Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation was unaffected by treatment with the test item. Except for one female in the control group, all pregnant females had live offspring. The gestation indices were 96, 100, 100 and 100% for the control, 10, 30 and 100 mg/kg/day groups, respectively.
No signs of difficult or prolonged parturition were noted among the pregnant females that were considered test item-related. Female No. 112 (control group) was sacrificed on Day 0 of the Lactation phase because of complications during parturition. 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 (total number of offspring born as percentage of total number of uterine implantation sites) was considered not affected by treatment with the test item. Post-implantation survival indices were 93, 92, 91 and 91% for the control, 10, 30 and 100 mg/kg/day groups, respectively.
Litter size was considered not affected by treatment with the test item. Live litter sizes were 9.9, 10.6, 10.5 and 9.8 living pups/litter for the control, 10, 30 and 100 mg/kg/day groups, respectively.
Sex ratio was considered not to be affected by treatment with the test item.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During Lactation:
No clinical signs occurred among pups that were considered to be related to treatment with the test item.
Abnormal posture of the left hindleg was noted for one pup of Female No. 125 (control group) that was sacrificed in extremis on PND 4. In addition, lethargy and abnormal posture of the left hindleg was noted for one pup of Female No. 143 (10 mg/kg/day) that was found missing on PND 2. Moreover, abnormal posture of the right hindleg was noted for one pup of Female No. 173 (30 mg/kg/day) that was sacrificed in extremis on PND 4. Abnormal posture of the foreleg was noted for one pup of Female No. 188 that was sacrificed in extremis on PND1.
For one pup of Female No. 116 (control group) and two pups of Female No. 150 (10 mg/kg/day) who were all missing on PND 2, for five pups of Female No. 188 (100 mg/kg/day) that were sacrificed in extremis on Day 1 of lactation, as well as for all live pups of Female No. 127 on PND 1 (all survived) and one pup of Female No. 150 between PND 5-7 (pup survived), less or no milk in the stomach and/or cold to touch and lethargy (Litter No. 188 only) was noted at first litter check.
Lethargy, wound on the abdomen and/or no milk was noted for two pups of Female No. 174 (30 mg/kg/day) that were sacrificed in extremis on PND 1.
One pup of Female No. 176 (100 mg/kg/day) was noted with agenesis of both eyes. At the isolated incidence, this finding was considered congenital rather than related to treatment with the test item.
The nature and incidence of other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

After PND21
Reflux after dosing was noted in individual animals of the control group (Female No. 528), 10 mg/kg/day group (Male No. 622), 30 mg/kg/day group (Male No. 374) and 100 mg/kg/day group (Male No. 481) on a single day during the treatment period. This clinical sign was also noted in several treated F1-animals of all dose levels that were found dead or were sacrificed in extremis.
Further test item-related clinical signs observed for surviving animals included rales, gasping, hunched posture and piloerection, and were seen across the dose groups as described below:
• At 10 mg/kg/day, rales were observed incidentally prior to and/or after dosing (both sexes), mostly on single days.
• At 30 mg/kg/day, rales, hunched posture and piloerection were observed incidentally prior to and/or after dosing (both sexes) mostly during the first three weeks of treatment, with a maximum of seven consecutive days.
• At 100 mg/kg/day, piloerection was observed for the majority of the animals prior to and/or after dosing (both sexes) during the first two weeks of treatment, with a maximum of ten consecutive days. In addition, rales, gasping, and/or hunched posture were observed incidentally prior to and/or after dosing (males only) mostly during the first two weeks of treatment.
Salivation was observed after dosing with the test item in males starting at 10 mg/kg/day and in females at 30 and 100 mg/kg/day from Weeks 1-2 onwards. The incidence increased with dose. In females at 10 mg/kg/day, salivation was noted sporadically only and at a similar incidence as for control females. Considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing), it was regarded to be a physiological response rather than a sign of systemic toxicity and therefore not toxicologically relevant.
Other findings that were noted (pre- and/or post-dose) included skin scabs, wounds, alopecia, broken upper incisors, a missing or broken tail apex or tail, a smaller size of the lower right hind leg with concurrent hypotonia, and a smaller or larger size of the eye and/or an eye affected by chromodacryorrhea, exophthalmos or enophthalmos. In addition, rales, gasping, squeaking, hunched posture and piloerection were noted in a single positive control animal (Female No. 826). These clinical signs were noted incidentally, 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, were limited to (positive) control animals and/or did not show any apparent dose-related trend. They were therefore considered not to be signs of toxicological relevance.
No clinical signs were noted during weekly arena observations.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

During lactation
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment with the test item. The live birth indices were 95, 96, 99 and 98% for the control, 10, 30 and 100 mg/kg/day groups, respectively. A total of 29 pups were found dead at first litter check: In the control group twelve pups (Litter Nos. 102, 103, 112 [eight pups, 113 and 120). At 10 mg/kg/day, ten pups (Litter Nos. 127, 142 and 150 [seven pups, also see below]). At 30 mg/kg/day, two pups (Litter Nos. 172 and 174) and at 100 mg/kg/day, five pups (Litter Nos. 188 and 196). Noteworthy, Female No. 150 (10 mg/kg/day) was noted with seven dead pups at first litter check (also see Section 9.4.6). Out of the remaining four pups, three were noted with less milk in their stomach at first litter check (note: these three pups were found missing on PND 2 (also see Section 9.4.7)) and only one pup remained until scheduled necropsy. 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 considered not to be affected by treatment with the test item. Viability indices were 97, 98, 98 and 95% for the control, 10, 30 and 100 mg/kg/day groups, respectively. Seven pups (Litter Nos. 112, 113, 116 and 125) of the control group, and five (Litter Nos. 129, 143 and 150), five (Litter Nos. 154, 162, 173 and 174) and ten pups (Litter Nos. 188, 196 and 200) at 10, 30 and 100 mg/kg/day were found missing on PND 2 or 3 or were sacrificed in extremis on PND 1 or 4. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these 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. Female No. 112 (control group) was sacrificed in extremis together with the remainder of its litter (four pups) on Day 0 of the lactation phase (the day of delivery) . Female No. 116 (control group) had total litter loss (one pup missing) on Day 2 of lactation. Female No. 188 at 100 mg/kg/day was sacrificed in extremis together with its litter (five remaining live pups) on Day 1 of lactation. Of the eight pups in its litter, three pups were dead at first litter check, the remaining five live pups were small, cold to the touch, and lethargic. Only three pups had a small amount of milk in their stomach, the remaining two pups had no milk.
Weaning index (the number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling)) was considered not to be affected by treatment with the test item. The weaning indices were 100, 100, 100 and 95% for the control, 10, 30 and 100 mg/kg/day groups, respectively. The observed lower weaning index at 100 mg/kg/day was due to mortality in a single litter (Litter No. 200). From this litter, one pup was found dead on PND 5. In addition, the remaining seven pups from this litter were sacrificed on PND 6, together with their dam as per protocol, as Female No. 200 was sacrificed in extremis. No toxicological relevance was attributed to the single dead pup since the mortality incidence remained within the range considered normal for pups of this age.

After PND21
A total of 36 F1-animals were found dead or were sacrificed in extremis during the treatment period , most of which during the first week after weaning (first week of treatment of F1-animals). This concerned 3, 8 and 25 animals of the 10, 30 and 100 mg/kg/day groups, respectively, whereas all F1-animals of the control group survived until scheduled necropsy.
Histopathological examination, including microscopic evaluation of the nasopharynx and nasal cavity, was required for Cohort 1A animals only that were not replaced (i.e. Female Nos. 777 and 778 at 100 mg/kg/day).
For female No. 777 main macroscopic findings were lungs not collapsed, large and small intestines that were distended with gas and a spleen that was reduced in size. There were no correlating microscopic findings. There was a marked neutrophilic inflammation and massive ulceration in the nasopharynx and marked neutrophilic inflammation, marked epithelial necrosis, marked ulceration and marked mucopurulent exudate in the nasal cavities. For this animal the microscopic findings in the nasal cavity and nasopharynx were considered to be the main causes of moribundity.
For female No. 778 main macroscopic findings were large and small intestines (with exception of the jejunum) that were distended with gas. There were no correlating microscopic findings. There was a moderate neutrophilic inflammation, marked ulceration and moderate mucopurulent exudate in the nasopharynx and moderate neutrophilic inflammation, moderate epithelial necrosis, moderate ulceration with minimal fusion of the turbinates, moderate mucopurulent exudate and slight squamous metaplasia in the nasal cavities. For this animal the microscopic findings in the nasal cavity and nasopharynx were considered to be the main causes of moribundity.
The macroscopic findings and microscopic nasopharynx/nasal cavity findings for both Cohort 1A premature Cohort 1A-decedents were similar to those seen in the F0 premature F0-decedents and were also considered to be reflux-related and hence not directly related to treatment with the test item.
The deaths of Animal Nos. 320A, 640A (10 mg/kg/day), 367A, 408A, 719 (30 mg/kg/day), 465A, 473A, 508A, 783, 788, 791, and 808A (100 mg/kg/day) were mainly considered related to the challenges of the gavage procedure with this particular test item in small animals, based on the necropsy findings (trachea or esophagus perforation and/or fluid in the thoracic cavity). However, as there were no premature decedents in the control group, a dose-relationship for premature decedents was apparent, and based on the similarities in the clinical appearance of these – and other remaining – early deaths in Groups 2-4 and the premature deaths in the F0-generation, the involvement of another cause could not be excluded (i.e. reflux/regurgitation).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During Lactation:
Body weights of pups were lower at 100 mg/kg/day from PND 1 onwards (5 % for both male, female and combined pup weights ), reaching statistical significance from PND 4 onwards.
At 100 mg/kg/day, male, female and combined pup weights were up to respectively 14, 13 and 14% lower compared to concurrent controls at PND 21.
Body weights up to 30 mg/kg/day were considered unaffected by treatment with the test item.

After PND21
Test item-related changes in body weight were observed starting at 10 mg/kg/day.
At start treatment on PND 21 mean body weight at 30 and 100 mg/kg/day was already respectively 4 and 14% lower in both sexes (not statistically significant in males at 30 mg/kg/day). Moreover, mean body weight gain of F1-animals at 30 and 100 mg/kg/day was lower than controls from Week 1 of treatment onwards (males) or between Weeks 1 to 3 (females), which together with the lower start weights resulted in a lower mean body weight of these animals throughout the treatment period (not always statistically significant for either parameter). Mean terminal body weights of Cohort 1A and 1B males were 10 and 5%, and 17 and 12% lower than control at 30 and 100 mg/kg/day, respectively (not statistically significant for Cohort 1B males at 30 mg/kg/day). Mean terminal body weights of Cohort 1A and 1B females were 7 and 8%, and 12 and 12% lower than control at 30 and 100 mg/kg/day, respectively, despite the apparent higher body weight gain in females at 100 mg/kg/day from Week 7 of treatment onwards (not statistically significant).
At 10 mg/kg/day, mean body weight and body weight gain were only slightly lower than controls during the first weeks of treatment. Mean values were considered comparable to concurrent controls from Week 4 (females) or 6 (males) of treatment onwards. The initially lower body weight gains resulted in only minor (< 5%; not reaching statistical significance) differences in mean terminal body weight of these animals when compared to concurrent controls. As such, this effect was considered not toxicologically relevant.
Note: “Day 1” of treatment is the day that the first animals were weaned. Body weight of animals that were weaned during the subsequent 7 days were all entered under that day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean absolute food consumption was slightly lower at 100 mg/kg/day between Weeks 1-8 of treatment in males, and in females between Weeks 1-6 of treatment (not always statistically significant). In addition, food consumption corrected for body weight was in general slightly increased in males and females throughout the treatment period (4-16% and 5-14% higher than controls in males and females, respectively; not always statistically significant).
At 30 mg/kg/day, slightly lower mean absolute food consumption was noted in males. The changes were attributed to the lower body weight of these males as no relevant differences were noted in the food consumption corrected for body weight. As such, this finding at 30 mg/kg/day was considered not toxicologically relevant.
At 30 mg/kg/day (females) and 10 mg/kg/day (both sexes), food consumption before or after correction for body weight was considered not affected by treatment with the test item.

Note: Due to the replacement of several F1-animals at 30 and 100 mg/kg, mostly during the first week of weaning, (relative) food consumption during the first week of treatment should be interpreted with caution.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A:
The mean lymphocyte (LYMPH), neutrophil (NEUT) and monocyte (MONO) counts were increased when compared with concurrent control for F1-animals of both sexes treated at 100 mg/kg/day, resulting in increased mean total white blood cell (WBC) counts. Additionally, an increase in mean large unstained cells (LUC) count was observed at 100 mg/kg/day for both sexes.
Except for the mean neutrophil count, the same trend in abovementioned white blood cell parameters was observed for males treated at 30 mg/kg/day.
The fold changes for these parameters when compared with control are presented in Table 5.
In addition, the mean corpuscular hemoglobin concentration (MCHC) was slightly increased for females at 100 mg/kg/day (1.04x of control). For 5/10 females at 100 mg/kg/day, individual values were just above the range of the concurrent controls. Given the small magnitude of the effect and as correlating parameters were all unaffected, the change in MCHC was considered not test item-related. Any other changes in hematology parameters (regardless of achieving statistical significance) were considered to be unrelated to administration of the test item due to the minimal magnitude of the change, variation in direction of change, absence of a dose-related response and/or absence of biological relevance.
At 100 mg/kg/day, activated prothrombin time (APTT) was shorter compared to controls (0.84x of controls) in males. This was only partly attributed to Animal No. 449, with a remarkable low APTT value, as the sample possibly contained a clot. When excluding this value, APTT values at 100 mg/kg/day remained shorter compared to controls (0.87x of controls). However, as individual values (except for Animal No. 449) remained with the concurrent control range, this finding was considered not toxicologically relevant.
Clotting parameters in females were considered unaffected by treatment with the test item.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

On PND 4 and 21-22 – F1-Pups:
Serum T4 levels in male and female pups, culled on PND 4 were considered not to be affected by treatment with the test item.
At 100 mg/kg/day, serum TSH levels in male and female pups of Cohort 2B on PND 21-22 were increased (1.24x and 1.28x of controls, respectively). All values remained within the range of the historical controls (Historical control data for TSH (µIU/mL) in Cohort 2B animals (period 2017-2021): Male rats mean = 0.107, P5-P95 = 0.051 – 0.261 (n=30); Female rats mean = 0.081, P5-P95 = 0.030 – 0.180 (n=30)).
Serum T4 levels in male and female pups of Cohort 2B on PND 21-22 were considered not to be affected by treatment with the test item.

Cohort 1A:
The following changes distinguished treated Cohort 1A animals from control animals (the differences were statistically significant unless indicated otherwise; relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
- Mean concentration of albumin was decreased for males starting at 30 mg/kg/day (0.95 and 0.93x of control at 30 and 100 mg/kg/day, respectively) and for females at 100 mg/kg/day (0.94x of control).
- As a result, mean concentration of total protein was decreased for males starting at 30 mg/kg/day (0.96 and 0.93x of control at 30 and 100 mg/kg/day, respectively; not statistically significant at 30 mg/kg/day) and for females at 100 mg/kg/day (0.96x of control).
- Mean concentration of total bilirubin was increased for males at 100 mg/kg/day (1.24x of control).
- Mean concentration of urea was increased for males at 100 mg/kg/day (1.30x of control).
- Mean concentration of potassium was decreased for males at 100 mg/kg/day (0.94x of control).
Given the magnitude and/or dose response relationship, these changes were considered to be test item related.
Any other changes in clinical biochemistry parameters (regardless of achieving statistical significance) were considered to be unrelated to administration of the test item due to the minimal magnitude of the change, variation in direction of change, absence of a dose-related response and/or absence of biological relevance.
Thyroid hormone analyses:
Serum levels of T4 and TSH in Cohort 1A males and females were considered unaffected by treatment with the test item up to 100 mg/kg/day.
The increased mean TSH concentration in males treated at 30 and 100 mg/kg/day (1.62 and 1.65x of control; not statistically significant) was attributed to the relatively high value in a single animal of both groups (Nos. 368 and 457). After excluding these values, mean TSH at 30 and 100 mg/kg/day was respectively 0.1906 and 0.1937 mU/L (vs. 0.1489 mU/L in concurrent controls). As remaining individual values generally remained within concurrent control range, no relationship with the test item was indicated.
The increased mean TSH concentration in females treated at 10 and 100 mg/kg/day (1.60 and 1.53x of control; not statistically significant) and decreased mean TSH concentration in females treated at 30 mg/kg/day (0.78x of control; not statistically significant) was considered unrelated to treatment with the test item in absence of a dose-related response.

Urinalysis findings:

not specified

Description (incidence and severity):

The following changes in urine parameters distinguished animals treated at 100 mg/kg/day from control animals:
- Clarity of the urine was scored as turbid in 2/10 males (vs. 0/10 in concurrent controls).
- Dark yellow discoloration of urine in 3/10 males (vs. 0/10 in concurrent controls).
- Increased incidence and/or severity of blood in urine of 6/10 males and 1/10 females (up to score 4+ in males and +2 in females vs. negative for blood in controls, both sexes).
- Increased incidence and severity of ketone in urine of 4/10 females (up to score 2+ vs. 1+ in 1/10 concurrent controls).
- Increased incidence of white blood cells in sediment samples of 4/10 males (vs. 1/10 in concurrent controls).
- Increased incidence and severity of the presence of red blood cells in sediment samples of 4/10 males (score up to 2+ vs. 1+ in 2/10 concurrent controls).
- Increased urinary pH in 2/10 males (8.5 vs. maximally 7.5 in concurrent controls).

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

BALONOPREPUTIAL SEPARATION AND VAGINAL OPENING - F1 generation
At 100 mg/kg/day, the age at attainment of balonopreputial separation (BPS) in males was increased (41.6 days vs. 40.6 in controls). A similar trend was observed at 30 mg/kg/day (41. days vs. 40.6 in controls), although statistical significance was not achieved.
In females at 10, 30 and 100 mg/kg/day, the age at attainment of vaginal opening (VO) was dose-dependently increased compared to controls (respectively 31.6, 32.5 and 33.8 days vs. 31.0 in controls).
Body weight at attainment of VO or BPS was similar (females at 10, 30 and 100 mg/kg/day) or reduced (males at 30 and 100 mg/kg/day) compared to controls, indicating that the observed delay in sexual maturation for both males and females was due to the test item-related growth retardation observed at these dose levels.
In addition, the age at first estrus was increased at 100 mg/kg/day (38.4 days vs. 34.3 in controls). This was considered related to the increased age at attainment of vaginal opening at 100 mg/kg/day and as such regarded secondary to the observed test-item related growth retardation.
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 Female No. 763 (100 mg/kg/day) for which it took slightly longer (9 days). At the isolated incidence, no toxicological relevance was attached to this finding.

ESTROUS CYCLE - Cohort 1A
Length and regularity of the estrous cycle were not affected by treatment with the test item.
At both 30 and 100 mg/kg/day, a single female was noted with an irregular cycle (Nos. 694 and 769, respectively). Based on the single incidence and in the absence of a dose-related trend, this finding was considered not test item-related.
At 30 mg/kg/day, one female (No. 693) was noted with an extended estrus (4 consecutive days of estrous). At the single incidence and in the absence of a dose-related trend, this finding was considered unrelated to treatment with the test item.
For three control females (Nos. 522, 529 and 540), two females at 10 mg/kg/day (Nos. 603 and 619) and one female at 100 mg/kg/day (No. 780), 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. In the absence of a dose-related trend, this finding was considered unrelated to treatment with the test item.

SPERM ANALYSIS - Cohort 1A
Sperm motility and concentration were unaffected up to 100 mg/kg/day.
At 30 and 100 mg/kg/day, the mean number of cells with detached head was increased (5 vs. 3 in controls). In the absence of a dose-related response at the individual level, this finding was considered unrelated to treatment with the test item.
All other sperm morphology parameters were considered unaffected by treatment with the test item.
The statistically significant lower number of cells with coiled tail at 10 mg/kg/day and higher number of cells with abnormal neck at 30 mg/kg/day were considered not related to treatment with the test item in the absence of a dose-related trend.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance (normalized for body weight) was increased in male and female pups at 100 mg/kg/day. Compared to concurrent controls, corrected anogenital distance was 5 and 12% higher in males and females, respectively.
A similar trend was observed in males at 30 and 100 mg/kg/day and females at 100 mg/kg/day for absolute anogenital distance (3, 3 and 10% increased compared to concurrent controls in respectively males and females), although values did not reach statistical significance.
Noteworthy, for female pup 9 of Litter No. 102 (control group) and pup female 2 of Litter No. 135 (10 mg/kg/day), higher AGD values were noted compared to other female pups (respectively 2.20 mm and 2.89 mm vs. a mean AGD of 0.92 mm in control females). No clear explanation was found for these higher values and at necropsy, the sex of these females was confirmed.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 100 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at 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 was a higher liver weight in males and females at 30 and 100 mg/kg/day (relative to body weight only).
There was a significant higher adrenal gland weight in males starting at 30 mg/kg/day (relative to body weight only).
There was a lower pituitary gland weight in females starting at 30 mg/kg/day (absolute and/or relative to body weight).
The (significant) differences in brain weight in males at 30 and 100 mg/kg/day were in opposite directions for absolute and relative organ weight and were considered secondary to a test item-related effect on terminal body weight. In addition, the significant differences in seminal vesicles starting at 10 mg/kg/day, pituitary gland, kidney, heart, prostate gland, spleen, testes and epididymides starting at 30 mg/kg/day and thymus at 100 mg/kg/day in males were also considered to be secondary to test item-related effect on terminal body weight. For females, the absolute brain weight was significantly lower at 30 and 100 mg/kg/day, and this was considered secondary to a test item-related effect on terminal body weight. In addition, the significant organ weight differences in thymus starting at 10 mg/kg/day, kidney starting at 30 mg/kg/day and the thyroid gland at 100 mg/kg/day in females were also considered to be secondary to test item-related effect on terminal body weight. For females, the absolute brain weight was significantly lower at 30 and 100 mg/kg/day and this was considered secondary to a test item-related effect on terminal body weight. In addition, the significant differences in thymus starting at 10 mg/kg/day, kidney starting at 30 mg/kg/day and the thyroid gland and ovaries in females at 100 mg/kg/day were also considered to be secondary to test item-related effect on terminal body weight. As such, these changes were considered not to be a sign of direct toxicity.

Cohort 1B (≥ PND 97)
Due to high mortality observed (mostly) during the first week of treatment of F1-animals, some of the Cohort 1B animals were used to replace early decedents from Cohorts 1A, 2A, 2B and 3. As a result, the number of Cohort 1B animals at 100 mg/kg/day was lower at the end of treatment (in particular in females).
There was a lower pituitary gland weight in females starting at 30 mg/kg/day (absolute and/or relative to body weight, not statistically significant for absolute).
The statistically significant changes in absolute epididymides and seminal vesicles weights in males at 100 mg/kg/day were in opposite directions for absolute and relative organ weight and were considered secondary to a test item-related effect on terminal body weight. In addition, the significant differences in relative weights of adrenal (females only) and testes at 30 and/or 100 mg/kg/day were also considered to be secondary to test item-related effect on terminal body weight. As such, these changes were considered not to be a sign of direct toxicity.

Cohort Surplus (PND 22-24)
Note: Due to insufficient number of female pups at 100 mg/kg/day to fill the different cohorts, there were no Cohort Surplus females at 100 mg/kg/day.
Organ weights were considered affected by treatment with the test item at 100 mg/kg/day.
In males, thymus and spleen weights (absolute and relative to body weight) were considered decreased by treatment with the test item at 100 mg/kg/day, although statistical significance was only achieved for absolute thymus weight.
In females, thymus weights (absolute and relative to body weight) were significantly increased at 30 mg/kg/day (i.e. the highest dose tested in females of Cohort Surplus).

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups sacrificed at the end of the lactation period (including spare pups) that were considered to be related to treatment with the test item.

Cohort 1A (PND 89-95):
There were no test item-related gross observations.
All of the recorded macroscopic findings (including the reddish/dark red discoloration observed in the mesenteric lymph node of 6/10 high dose males of Cohort 1A) were considered to be within the range of background gross observations encountered in rats of this age and strain.
Cohort 1B (≥ PND 97):
There were no test item-related macroscopic observations.
All of the recorded macroscopic findings in Cohort 1B animals were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in 3/20, 1/20 and 3/18 and 1/9 females of respectively the control, 10, 30 and 100 mg/kg/day group, is related to the stage in the estrous cycle and is a normal finding.
Cohort 2A (PND 76-90):
There were no test item-related macroscopic observations.
All of the recorded macroscopic findings in Cohort 2A animals were within the range of background gross observations encountered in rats of this age and strain.
Cohort 3 (PND 53-59):
At 100 mg/kg/day, a smaller thymus was noted in 3/10 males and 4/10 females (vs. 0/10 in controls).
Other findings that were noted among control and/or treated animals of Cohort 3 were considered to be of no toxicological significance, since they remained within the range of biological variation for rats of this age and strain.
Cohort 2B (PND 21-22):
There were no test item-related macroscopic observations.
Noteworthy, Male No. 412 (30 mg/kg/day) was noted with gray discoloration of the lungs and liver. As this was an incidental finding and unrelated to dose, it was considered not toxicologically relevant.
Any other findings that were noted among control and/or treated animals of Cohort 2B were considered to be of no toxicological significance, since they remained within the range of biological variation for rats of this age and strain.
Cohort Surplus (PND 22-24):
There were no test item-related macroscopic observations.
At the individual level, Male Pup No. 1 of Litter 176 of Cohort Surplus (100 mg/kg/day) was noted with agenesis of both eyes. Based on the single incidence of this finding and the absence of other eye-related findings in other pups at 100 mg/kg/day, this finding was considered not toxicologically relevant.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

HISTOPATHOLOGY - F1
Test item-related microscopic findings were noted in the adrenal glands of the 100 mg/kg/day group males and are summarized in Table 6. In the adrenal glands of males at 100 mg/kg/day, an increased incidence and severity of vacuolation of the zona fasciculata was present (up to a slight degree). This microscopic finding likely correlated with the increased adrenal gland weight.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings 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.

SPLENIC LYMPHOCYTE SUBPOPULATION - Cohort 1A
Splenic lymphocyte subpopulations were considered unaffected by treatment with the test item.

TESTIS STAGING - Cohort 1A
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes 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 - Cohort 1A
There were no test item-related effects on the ovarian follicle and corpora lutea counts in F1-females (Cohort 1A) at 100 mg/kg/day when compared to control group females. Any variation between group mean counts represented biological variability and was not statistically significant.

BRAIN INVESTIGATIONS - Cohort 2A and 2B
There were no test item-related findings in the H&E or Luxol Fast Blue/Cresyl Violet stained sections of brain (Cohorts 2A and 2B) and peripheral nerves (Cohort 2A) in the high dose group males or females.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

FUNCTIONAL TESTS - Cohort 2A
1. Acoustic Startle Response
For females at 100 mg/kg/day, a lower mean average and maximum response amplitude in the acoustic startle response (down to 0.67x and 0.58x of control) was recorded in all 5 blocks of trials (not statistically significant for maximum response amplitude in time block 4).
A similar trend was observed for means of average and maximum response amplitude in females at 30 mg/kg/day (down to 0.80x and 0.71x of control, respectively) and in males at 100 mg/kg/day (down to 0.77x and 0.71 x of control, respectively), although statistical significance was not reached (except for maximum response amplitude in block 5 in males at 100 mg/kg/day).
In addition, at 100 mg/kg/day there was a trend towards a longer mean latency time to achieving the maximum response amplitude in block 1 (1.05x and 1.09x of control in males and females, respectively) and block 2 (males only: 1.16x of control). However, differences were relatively slight (reaching no statistical significance) with great overlap in individual values, and recovery towards control values was observed in the remaining blocks of trials. Therefore, no toxicological relevance was attached to this finding.
At 10 mg/kg/day (males and females) and 30 mg/kg/day (males), acoustic startle response (both as overall mean and per block) was considered not affected by treatment.
Detailed clinical observations revealed no symptoms that were considered to be related to treatment with the test item.

2. Detailed clinical observations
All clinical signs were within the normal range of behavioral findings for this type of study. Noteworthy, increased activity was noted in a single animal at 100 mg/kg/day (Female No. 802). As this was an isolated finding, it was considered unrelated to treatment with the test item.

3. Rectal temperature
Rectal temperature was considered not to be affected by treatment with the test item.
The slight increase in rectal temperature at 30 mg/kg/day (1.01x of control for both sexes), reaching statistical significance, was considered not test item-related in the absence of a dose-related trend.

4. Motor activity test
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
The statistically significant increase in ambulations in males at 30 mg/kg/day was considered not toxicologically relevant in the absence of a dose-related trend.

5. Functional observations
No test item-related or toxicologically relevant changes in functional observation parameters (hearing ability, pupillary reflex, foot splay and grip strength) were noted.
Noteworthy, although not statistically significant, a trend towards a lower grip strength of the fore legs was observed in males at 100 mg/kg/day, with 6/10 animals showing values below the concurrent control range. As values remained within the range of the historical controls, this finding was considered not toxicologically relevant (Historical control data for Grip Strength Foreleg (gram) in F1-animals (period 2017-2021): Male rats mean = 1084, P5-P95 = 798 – 1474 (n=90)).
A statistically significantly higher mean grip strength hind leg value was recorded for males at 10 mg/kg/day. In absence of a dose-related response, this variation was considered not to represent an effect of the test item.

NEUROPATHOLOGY AND MORPHOMETRY – Cohorts 2A and 2B
1. Fixed Brain Weights
There were brain weight findings as depicted in Table 7 and Table 8.
Significant differences in fixed brain weights (absolute and/or relative to body weights) were noted in the 100 mg/kg/day groups males and females. These brain weight differences were attributed to the lower terminal body weight and considered not a direct effect of the test item. A significant difference in brain weight (relative to body weight) was noted in the 100 mg/kg/day groups females. This brain weight difference was attributed to the lower terminal body weight and considered not a direct effect of the test item.

2. Brain Dimensions
There were brain dimension changes as depicted in Table 9 and Table 10
There was a significant decrease in brain width in males treated at 30 and 100 mg/kg/day. These small width differences were considered secondary to the lower terminal body weight and the resulting lower brain weight. There were no other (significant) changes in brain width or length.
There was a significant decrease in brain length in males treated at 100 mg/kg/day. This small length difference was considered secondary to the lower terminal body weight and the resulting lower brain weight. There were no other (significant) changes in brain width or length.

3. Brain histopathology
There were no test item-related findings in the H&E or Luxol Fast Blue/Cresyl Violet stained sections of brain (Cohorts 2A and 2B) and peripheral nerves (Cohort 2A) in the high dose group males or females.

4. Brain morphometry
Morphometric analysis of the brain at PND 21-22 (Cohort 2B) revealed statistically significantly lower cerebellum height in males at 100 mg/kg/day, but this was no longer significant after evaluation of all dose groups. In females, statistically significantly lower caudate putamen width was noted at 100 mg/kg/day.
At PND 76-90 (Cohort 2A), statistically significantly lower caudate putamen width was noted in males at 30 and 100 mg/kg/day as well as lower corpus callosum and hippocampus thickness at 100 mg/kg/day.
In females (Cohort 2A), statistically significantly lower corpus callosum thickness was noted at 10, 30 and 100 mg/kg/day and lower caudate putamen width was noted at 30 mg/kg/day. Initially, significantly lower caudate putamen width was noted at 100 mg/kg/day, but this was no longer significant after evaluation of all dose groups.
There were no other statistically significant differences.

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

T-CELL DEPENDENT ANTIBODY RESPONSE (TDAR) EVALUATION - Cohort 3 and positive control animals
Daily administration of Zinc bis(diethyldithiocarbamate) by oral gavage to Wistar rats (10, 30 and 100 mg/kg/day) did not result in any test item-related effects on TDAR results.
As expected, all animals were negative for the presence of anti-KLH IgM antibodies before the administration of KLH. In addition, immunosuppression was observed in the Cyclophosphamide positive control males and females (Group 5), with decreases of at least 89% in the group mean when compared to the vehicle control group, which reached statistical significance in females.
Overall, there were no test item-related effects on the anti KLH IgM antibody levels in dosed groups when compared to the vehicle control group. There were some differences in group mean when compared to the vehicle control group (-50% to 78%). However, these differences were mostly due to high or low responders within the vehicle or dosed groups, impacting the group mean. When taking into account the overall distribution of the anti-KLH IgM titers within each group, there were no clear trends and most animals remained within the overall male and female vehicle control group range.

Dose Formulation Analyses
Accuracy: The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). A small response at m/z 66 (single charged zinc isotope of quantification of the test item) was detected in all analyzed Group 1 formulations (i.e., for use in Weeks 1, 11 and 22). The responses were higher than the responses obtained in the analytical blanks (4% HNO3 in H2O). The maximum contribution to Group 2 samples calculated was 0.12%. This response was considered negligible and regarded to have no impact on the outcome of the study. The small response in the Group 1 samples might be explained by signal interference due to formation of polyatomic ions during sample analysis. Polyatomic ions are formed in the high-temperature plasma, either due to incomplete atomization or from recombination reactions during the extraction of ions into the mass spectrometer. These ions may be derived from the sample matrix, reagents used for sample preparation, plasma gases (argon) or entrained atmospheric gases Ref 3. Corn oil is a complex mixture of various components; therefore it is reasonable to assume that some components of corn oil can create polyatomic ions which can interfere with detection of single charged zinc (e.g. sulfur present in corn oil can be converted into sulfur dioxide cation which one isotope form has a mass of 63.961. This isotope can interfere with single charged zinc isotope used for quantification which has an atomic mass of 63.929).
Homogeneity: The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Table 1:Mortality Incidence F₀-animals

Group	Animal No.	Sex	Day of Death	Day of Treatment	Death Status	Relevant Observations prior to death	Relevant Necropsy Findings	Relevant Histopathological Findings
Control	112	F	14 Jan 2021 (pre-dose)	95 (Day 0 lactation)	Sacrificed in extremis, complications during parturition[1] Of note: sacrificed together with the remainder of its litter, as per protocol (also see Section 9.4.7).	Hunched posture, piloerection, pale appearance	Pale discolouration of the liver and thyroid gland (both sides)	n.a.
Control	116	F	18 Jan 2021 (post-dose)	99 (Day 2 lactation)	Other, total litter loss.	-	-	n.a.
30 mg/kg/day	160	F	21 Dec 2020 (pre-dose)	71	Other, not mated (no vaginal opening)	-	Vaginal entrance not open, small vagina, fluid in uterus and cervix, pelvic dilatation of kidneys.	n.a.
30 mg/kg/day	163	F	16 Nov 2020 (pre-dose)	36	Spontaneous death	-	Beginning autolysis, watery-clear fatty fluid in the pericardium, many dark red foci on the thymus and watery clear fluid in the thoracic cavity.	A granulocytic inflammatory cell infiltrate in the pleura of the lungs.
100 mg/kg/day	80	M	27 Dec 2020 (pre-dose)	77	Sacrificed in extremis	Hunched posture, piloerection, ptosis, rales, gasping, diarrhea and red staining on the snout. Severe body weight loss (17% in <7 days), based on additional measurements, see Table 2.27 Appendix 2. Of note: three days prior to sacrifice, reflux after dosing was noted.	Intestinal tract distended with gas, many reddish foci on the thymus, dark red discolouration of the mesenteric lymph nodes.	Moderate decreased lymphocyte cellularity of the thymus (likely due to the poor condition of the animal). In addition, there was a slight neutrophilic inflammation, marked ulceration and marked mucopurulent exudate in the nasopharynx and slight ulceration of the turbinates, moderate mucopurulent exudate and minimal respiratory/olfactory epithelium metaplasia and minimal epithelial degeneration in the nasal cavities. For this animal the microscopic findings in the nasal cavity and nasopharynx were considered to be the main causes of moribundity.
	88	M	16 Nov 2020 (pre-dose)	36	Sacrificed in extremis	Rales and/or labored respiration, piloerection, hunched posture. Severe body weight loss (20% in 7 days)	Intestinal tract distended with gas, many reddish foci on the thymus and a small thymus.	Slight decreased lymphocyte cellularity of the thymus (likely due to the poor condition of the animal). In addition, there was a moderate neutrophilic inflammation, slight ulceration, slight squamous cell metaplasia and moderate mucopurulent exudate in the nasopharynx and slight ulceration with slight fusion of the turbinates and moderate mucopurulent exudate in the nasal cavities. For this animal the microscopic findings in the nasal cavity and nasopharynx were considered to be the main causes of moribundity.
	95	M	09 Nov 2020 (pre-dose)	29	Sacrificed in extremis	Severe body weight loss (15% in 7 days)	-	Slight decreased lymphocyte cellularity of the thymus (likely due to the poor condition of the animal). In addition, there was a slight neutrophilic inflammation and slight squamous cell metaplasia in the nasopharynx and minimal ulceration with slight fusion and slight fibrosis of the turbinates, slight mucopurulent exudate, slight squamous cell hyperplasia and slight basal cell hyperplasia in the nasal cavities. For this animal the microscopic findings in the nasal cavity and nasopharynx were considered to be the main causes of moribundity.
	177	F	22 Oct 2020 (post-dose)	11	Sacrificed in extremis	Hunched posture, piloerection, ptosis, some type of seizure and squeaking when touched[2].	Thickening of the glandular mucosa of the stomach and yellowish gelatinous content of the jejunum.	Moderate meningitis of the brain and minimal meningitis of the spinal cord (cervical and thoracic segments). In addition, there was a minimal neutrophilic inflammation and slight squamous cell metaplasia in the nasopharynx and moderate ulceration with slight fusion of the turbinates and slight mucopurulent exudate and minimal squamous cell metaplasia and minimal basal cell hyperplasia in the nasal cavities. For this animal the meningitis in the brain and spinal cord and the nasal cavity and nasopharynx findings were considered to be the main causes of moribundity.
	180	F	21 Dec 2020 (pre-dose)	71	Sacrificed in extremis	Hunched posture, piloerection, ptosis, rales, gasping, labored breathing, red staining on the nose and swollen abdomen. Severe body weight loss (20% in 7 days).	Intestinal tract distended with gas, small spleen, dark red discolouration of the mesenteric lymph nodes, thickened limiting ridge of the stomach and lungs not collapsed.	Slight decreased lymphocyte cellularity of the thymus and spleen (likely due to the poor condition of the animal). In addition, there was a moderate neutrophilic inflammation, massive ulceration and moderate mucopurulent exudate in the nasopharynx and moderate neutrophilic inflammation, marked ulceration with moderate fusion of the turbinates and marked mucopurulent exudate in the nasal cavities. For this animal the microscopic findings in the nasal cavity and nasopharynx were considered to be the main causes of moribundity.
	188	F	13 Jan 2021 (post-dose)	94 (Day 1 lactation)	Sacrificed in extremis, based on clinical appearance of dam and litter Of note: sacrificed together with its litter, as per protocol	Piloerection, hunched posture and lean appearance[3]. Low body weight gain during post-coitum (0.64x of Group 4 mean on Day 20 post-coitum).	Small spleen and thymus.	Moderate decreased lymphocyte cellularity of the thymus (likely due to the poor condition of the animal). There were no nasopharynx or nasal cavity findings. No definite cause of moribundity could be determined.
	192	F	28 Dec 2020 (pre-dose)	78 (Day 5 post-coitum)	Sacrificed in extremis	Hunched posture, piloerection, rales, labored respiration and gasping. Severe body weight loss (15% in <7 days), based on additional measurements	Intestinal tract distended with gas.	Moderate decreased lymphocyte cellularity of the thymus (likely due to the poor condition of the animal). In addition, there was a slight neutrophilic inflammation, marked ulceration and marked mucopurulent exudate in the nasopharynx and marked neutrophilic inflammation, moderate ulceration with moderate fusion of the turbinates, marked mucopurulent exudate and slight squamous cell metaplasia in the nasal cavities. For this animal the microscopic findings in the nasal cavity and nasopharynx were considered to be the main causes of moribundity.
	200	F	21 Jan 2021 (pre-dose)	102 (PND 6)	Sacrificed in extremis Of note: sacrificed together with its litter, as per protocol	Piloerection, hunched posture, lean appearance, hypothermia, rales and gasping. Severe body weight loss (24% in <7 days), based on additional measurements. Of note: two days prior to sacrifice, reflux after dosing was noted.	Intestinal tract distended with gas, irregular surface of the glandular mucosa of the stomach, lungs not collapsed and small thymus.	Moderate decreased lymphocyte cellularity of the thymus (likely due to the poor condition of the animal). In addition, there was a slight neutrophilic inflammation, marked ulceration and marked mucopurulent exudate in the nasopharynx and marked ulceration of the turbinates, marked mucopurulent exudate, slight squamous cell metaplasia, slight respiratory/olfactory epithelium metaplasia and slight basal cell hyperplasia in the nasal cavities. For this animal the microscopic findings in the nasal cavity and nasopharynx were considered to be the main causes of moribundity.

- No (relevant) findings.

n.a. Not applicable.

[1] Observation taken from Study Daybook.

[2] Observation taken from Study Daybook.

[3] Observations taken from study communications in raw data.

Table 2: Mean Percent Organ Weight Differences from Control Groups - F₀-Generation

	Males			Females
Dose Level (mg/kg/day):	10	30	100	10	30	100

BODY WEIGHT
Absolute	-1	-4	-10**	-5	-7**	-7**

LIVER
Absolute	4	2	8*	-6	-1	6
Relative to body weight	5*	6**	20**	-1	7	14**

ADRENAL GLANDS
Absolute	6	6	4	-	-	-
Relative to body weight	8	8*	15**	-	-	-

SEMINAL VESICLES
Absolute	-14**	-15**	-20**	NA	NA	NA
Relative to body weight	-14*	-12*	-12*	NA	NA	NA

PITUITARY GLAND
Absolute	-	-	-	-6	-13**	-16**
Relative to body weight	-	-	-	-2	-8	-10*

*: P<0.05, **: P<0.01, -: no significant differences, NA: not applicable

Table 3: Summary Test Item-Related Microscopic Findings – Males F₀-Generation

	Males
Dose level (mg/kg/day):	0	10	30	100

ADRENAL GLAND ^a	25	25	25	25
Vacuolation zona glomerulosa
Minimal	-	-	6	13
Slight	-	-	1	5

^a = Number of tissues examined from each group.

Table 4: Mortality Incidence F₁-Animals

Group	Animal No.	Cohort	Sex	Day of Death	Day of Treatment^a	Death Status	Relevant Observations prior to Death	Relevant Necropsy Findings	Relevant Histopathological Findings^b
10 mg/kg/day	320A*	1B	M	06 Feb 2021 (PND 21)	1 (post-dose)	Spontaneous death	-	Lungs not collapsed, fluid in thoracic cavity.	n.a.
	330A*	2A	M	09 Feb 2021 (PND 25)	5 (post-dose)	Spontaneous death	-	Lungs not collapsed.	n.a.
	640A*	1B	F	08 Feb 2021 (PND 24)	4 (post-dose)	Spontaneous death	Of note: reflux after dosing was noted on the day of its death.	Fluid in thoracic cavity, trachea perforation.	n.a.
30 mg/kg/day	367A^$	1A	M	06 Feb 2021 (PND 24)	4 (post-dose)	Spontaneous death	-	Lungs not collapsed, irregular surface of the glandular mucosa of the stomach, stomach distended with gas and watery-clear fluid in thoracic cavity.	n.a.
	368A*	1A	M	04 Feb 2021 (PND 22)	2 (pre-dose)	Spontaneous death	-	Lungs not collapsed.	n.a.
	376A^$	1A	M	26 Feb 2021 (PND 41)	21 (pre-dose)	Sacrificed in extremis	Rales and gasping. Of note: reflux after dosing was noted two days prior to its death.	Diaphragmatic hernia of the right median lobe of the liver and perforation(s) in the diaphragm (the latter was considered congenital and not related to the gavage procedure).	n.a.
	398	1B	M	06 Feb 2021 (PND 22)	2 (post-dose)	Sacrificed in extremis	Rales, gasping and piloerection.	-	n.a.
	408A^$	2A	M	09 Feb 2021 (PND 25)	5 (post-dose)	Sacrificed in extremis	Hunched posture, flat posture, rales, gasping, piloerection and pale appearance.	Perforations of the esophagus, stomach and duodenum distended with gas.	n.a.
	421A*	3	M	05 Feb 2021 (PND 23)	3 (pre-dose)	Spontaneous death	Hunched posture, piloerection, rales and gasping	Few dark red foci on the thymus.	n.a.
	707	1B	F	09 Feb 2021 (PND 27)	7 (post-dose)	Sacrificed in extremis	Rales (moderate), gasping, flat posture, piloerection, salivation and pale appearance. Of note: reflux after dosing was noted on the day of its death.	-	n.a.
	719	1B	F	15 Mar 2021 (PND 59)	39 (pre-dose)	Spontaneous death	-	Lungs not collapsed, many reddish foci on the glandular mucosa of the stomach, many dark red foci on the thymus, and watery-clear fluid in the thoracic cavity.	n.a.
100 mg/kg/day	446A*	1A	M	04 Feb 2021 (PND 22)	2 (pre-dose)	Spontaneous death	-	Lungs not collapsed.	n.a.
	451A*	1A	M	07 Feb 2021 (PND 24)	4 (pre-dose)	Spontaneous death	Piloerection on the day prior to its death.	- (no macro data available due to cannibalism)	n.a.
	454A^$	1A	M	07 Feb 2021 (PND 23)	3 (pre-dose)	Spontaneous death	-	Lung not collapsed.	n.a.
	456A^$	1A	M	07 Feb 2021 (PND 23)	3 (post-dose)	Sacrificed in extremis	Rales, gasping and piloerection.	-	n.a.
	458A^$	1A	M	09 Feb 2021 (PND 24)	4 (post-dose)	Sacrificed in extremis	Hunched posture, rales, gasping and piloerection.	Stomach, duodenum, jejunum, ileum, caecum and colon distended with gas.	n.a.
	465A*	1B	M	04 Feb 2021 (PND 22)	2 (pre-dose)	Spontaneous death	-	Watery-clear fluid in thoracic cavity.	n.a.
	472	1B	M	08 Feb 2021 (PND 25)	5 (post-dose)	Sacrificed in extremis	Hunched posture, rales, gasping, piloerection and pale appearance.	Stomach, duodenum, jejunum, ileum, caecum and colon distended with gas.	n.a.
	473A*	1B	M	06 Feb 2021 (PND 22)	2 (pre-dose)	Spontaneous death	Hunched posture, gasping, piloerection and ptosis.	Watery-clear fluid in thoracic cavity.	n.a.
	502A*	3	M	06 Feb 2021 (PND 23)	3 (post-dose)	Spontaneous death	Flat posture, gasping and piloerection.	Beginning autolysis and lungs failure to collapse.	n.a.
	502N^$	3	M	09 Feb 2021 (PND 24)	3 (pre-dose) Of note: Inadvertently, dosing for this animal was commenced on PND 22 (see Appendix 15)	Spontaneous death	Flat posture, gasping and piloerection.	Lungs not collapsed.	n.a.
	508A^$	3	M	07 Feb 2021 (PND 22)	2 (post-dose)	Sacrificed in extremis	Rales, gasping, piloerection and ptosis.	Watery-clear fluid in thoracic cavity.	n.a.
	766A^$	1A	F	07 Feb 2021 (PND 25)	5 (pre-dose)	Spontaneous death	Piloerection.	Lungs not collapsed.	n.a.
	772A^$	1A	F	08 Feb 2021 (PND 25)	5 (pre-dose)	Spontaneous death	Piloerection.	-	n.a.
	777	1A	F	16 Apr 2021 (PND 91)	70 (pre-dose)	Sacrificed in extremis	Hunched posture, rales (moderate), piloerection and dehydration (slight) in combination with severe body weight loss (11% in one day). Of note: reflux after dosing was noted on the day prior to its death.	Lung not collapsed, duodenum, jejunum, ileum, caecum and colon distended with gas and spleen reduced in size.	There were no correlating microscopic findings. There was a marked neutrophilic inflammation and massive ulceration in the nasopharynx and marked neutrophilic inflammation, marked epithelial necrosis, marked ulceration and marked mucopurulent exudate in the nasal cavities. For this animal the microscopic findings in the nasal cavity and nasopharynx were considered to be the main causes of moribundity.
	778A^$	1A	F	08 Feb 2021 (PND 23)	3 (post-dose)	Sacrificed in extremis	Rales, gasping and piloerection.	Stomach, duodenum, jejunum, ileum, caecum and colon distended with gas.	n.a.
	778N	1A	F	16 Apr 2021 (PND 91)	70 (pre-dose)	Sacrificed in extremis	Hunched posture, rales (moderate), swollen abdomen, piloerection and dehydration (moderate) in combination with severe body weight loss (16% in three day). Of note: reflux after dosing was noted two days prior to its death.	Duodenum, ileum, caecum and colon distended with gas.	There were no correlating microscopic findings. There was a moderate neutrophilic inflammation, marked ulceration and moderate mucopurulent exudate in the nasopharynx and moderate neutrophilic inflammation, moderate epithelial necrosis, moderate ulceration with minimal fusion of the turbinates, moderate mucopurulent exudate and slight squamous metaplasia in the nasal cavities. For this animal the microscopic findings in the nasal cavity and nasopharynx were considered to be the main causes of moribundity.
	783	1B	F	24 Mar 2021 (PND 51)	30 (post-dose)	Spontaneous death	-	Clear oil-like fluid, dark-red watery-cloudy fluid and hemorrhagic/clotted blood in thoracic cavity.	n.a.
	786A*	1B	F	04 Feb 2021 (PND 22)	2 (pre-dose)	Spontaneous death	-	Lungs not collapsed.	n.a.
	788	1B	F	07 Feb 2021 (PND 24)	4 (pre-dose)	Spontaneous death	Piloerection.	Watery-clear fluid in thoracic cavity.	n.a.
	790	1B	F	26 Feb 2021 (PND 42)	22 (pre-dose) Note: Not dosed on the day preceding its death as this was considered not possible because of its health status.	Sacrificed in extremis	Hunched posture, rales, gasping, swollen abdomen (moderate), piloerection and pale appearance. Of note: reflux after dosing was noted on the day of its death.	Lungs not collapsed, duodenum, jejunum, ileum, caecum and colon distended with gas.	n.a.
	791	1B	F	07 Feb 2021 (PND 24)	4 (pre-dose)	Sacrificed in extremis	Hunched posture, rales, gasping, piloerection, pale appearance and ptosis.	Watery-clear fluid in thoracic cavity.	n.a.
	792	1B	F	04 Feb 2021 (PND 21)	1 (post-dose)	Spontaneous death	Hunched posture, rales, piloerection and ptosis (both eyes).	Lungs not collapsed.	n.a.
	800	1B	F	07 Feb 2021 (PND 22)	2 (pre-dose)	Spontaneous death	-	Lungs not collapsed.	n.a.
	804A^$	2A	F	07 Feb 2021 (PND 25)	5 (pre-dose)	Spontaneous death	Piloerection.	-	n.a.
	808A^$	2A	F	09 Feb 2021 (PND 26)	6 (pre-dose)	Sacrificed in extremis	Hunched posture, rales, gasping, piloerection and pale appearance.	Perforation in the esophagus, ileum and caecum distended with gas.	n.a.
^aTime point of death compared to dosing (i.e. pre-dose or post-dose) was based on information taken from the study daybook. ^b No histopathological examination was conducted on unscheduled deaths of Cohort 1B, 2A, 2B and 3 animals, as sufficient histopathological information was obtained from Cohort 1A. Also, no histopathological examination is conducted by default for (surviving) animals of these cohorts. ^* Animals were replaced with a spare animal. ^$ Animals were replaced with an animal weaned previously in a different cohort. A = Old animal number. These animal numbers were introduced when these animals were replaced by another animal. N = New animal number. These animal numbers were introduced for the animals used to replace an animal that was found dead or was sacrificed in extremis. - = No (relevant) findings. n.a. Not applicable.

Table 5: Summary Test Item-Related Hematology Changes (fold change tCtrl) – F₁-Cohort 1A

	Males			Females
Dose Level (mg/kg/day):	10	30	100	10	30	100
No. of Animals Analyzed	10	10	10	10	10	10

WBC (10⁹/L)	1.03x	1.08x	1.32x**	0.77x	0.93x	1.27x
NEUT (10⁹/L)	1.06x	0.81x	1.29x	0.80x	1.03x	1.42x
LYMPH (10⁹/L)	1.03x	1.15x	1.33x**	0.76x	0.92x	1.24x
MONO (10⁹/L)	0.99x	1.10x	1.47x	1.09x	1.05x	1.89x^##
LUC (10⁹/L)	0.88x	1.21x	2.00x^#	0.83x	0.83x	1.96x
^#/ ^## Kruskal-Wallis & Dunn: ^# = p ≤ 0.05, ^## = p ≤ 0.01 * / ** Anova & Dunnett: * = p ≤ 0.05, ** = p ≤ 0.01

Table 6: Summary Test Item-Related Microscopic Findings – Males F₁-Generation Cohort 1A

	Males
Dose level (mg/kg/day):	0	10	30	100

ADRENAL GLAND ^a	20	20	20	20
Vacuolation zona glomerulosa
Minimal	1	-	-	9
Slight	-	-	-	3

^a = Number of tissues examined from each group.

Table 7: Mean Percent Brain Weight Differences from Control Groups - F₁-Generation Cohort 2A

	Males			Females
Dose level (mg/kg/day):	10	30	100	10	30	100

BODY WEIGHT
Absolute	2	-5	-17**	-2	-7	-13**

FIXED BRAIN
Absolute	0	-4	-10**	1	-1	-7**
Relative to body weight	-3	0	8*	2	5	7

*: P<0.05, **: P<0.01

Table 8: Mean Percent Brain Weight Differences from Control Groups - F₁-Generation Cohort 2B

	Males			Females
Dose level (mg/kg/day):	10	30	100	10	30	100

BODY WEIGHT
Absolute	-2	-6	-14**	-2	-2	-16**

FIXED BRAIN
Absolute	-3	-4	-7	1	1	-4
Relative to body weight	-2	2	9	2	4	14*

*: P<0.05, **: P<0.01

Table 9: Summary Brain Dimensions - F₁-Generation Cohort 2A

	Males				Females
Dose level (mg/kg/day):	0	10	30	100	0	10	30	100

BRAIN DIMENSION ^a	10	10	10	10	10	10	10	10

Brain Length (mm)	23.96	24.07	23.57	23.75	23.06	23.26	23.47	23.16
Brain Width (mm)	16.17	16.23	15.43**	15.24**	15.41	15.62	15.65	15.40

^a = Number of tissues examined from each group.

** Dunnett-test based on pooled variance significant at 1% (**) level

Table 10: Summary Brain Dimensions - F₁-Generation Cohort 2B

	Males				Females
Dose level (mg/kg/day):	0	10	30	100	0	10	30	100

BRAIN DIMENSION ^a	10	10	10	10	10	10	10	10

Brain Length (mm)	21.26	20.51	20.57	20.10*	20.51	20.41	20.32	20.50
Brain Width (mm)	15.63	15.37	15.37	15.30	15.38	15.44	15.44	14.86

^a = Number of tissues examined from each group.

* Dunnett-test based on pooled variance significant at 5% (*) level.

Table 11:Mean Percent Organ Weight Differences from Control Groups - F₁-Generation Cohort 1A

	Males			Females
Dose level (mg/kg/day):	10	30	100	10	30	100

BODY WEIGHT
Absolute	-3	-10**	-17**	-3	-7**	-12**

LIVER
Absolute	4	-3	-3	0	0	7
Relative to body weight	6	8*	18**	3	8**	22**

ADRENAL GLANDS
Absolute	7	0	-7	-	-	-
Relative to body weight	13	13*	13*	-	-	-

PITUITARY GLAND
Absolute	-	-	-	-9	-16**	-23**
Relative to body weight	-	-	-	-6	-9	-13*

*: P<0.05, **: P<0.01, -: no significant differences, NA: not applicable

Table 12: Mean Percent Organ Weight Differences from Control Groups - F₁-Generation Cohort 1B

	Males			Females
Dose level (mg/kg/day):	10	30	100	10	30	100

BODY WEIGHT
Absolute		-5	-12**		-8**	-12**

PITUITARY GLAND
Absolute	-	-12*	-18**	-	-15*	-25**
Relative to body weight	-	-4	-4	-	-8	-13

*: P<0.05, **: P<0.01, -: no significant differences, NA: not applicable

Table 13: Mean Percent Organ Weight Differences from Control Groups – F₁-Generation Cohort Surplus

			Males			Females
Dose Level (mg/kg/day)			10	30	100	10	30	100

BODY WEIGHT
		Absolute	2	-2	-11*	4	4	-
THYMUS
	Absolute		-9	2	-22**	10	20*	-
	Relative to body weight		-11	4	-14	8	15*	-

SPLEEN
	Absolute		4	5	-30	11	16	-
	Relative to body weight		3	9	-22	8	10

*: P<0.05, -: no Cohort Surplus females were available in this study

Table 14 Developmental data

	GROUP 1 CONTROL	GROUP 2	GROUP 3	GROUP 4
		10 MG/KG	30 MG/KG	100 MG/KG

LITTERS TOTAL	24	24	21	22

DURATION OF GESTATION
MEAN (+)	21.5	21.4	21.3	21.5
ST.DEV.	0.7	0.7	0.5	0.6
N	24	24	21	22

DEAD PUPS AT FIRST LITTER CHECK
LITTERS AFFECTED (#)	5	3	2	2
TOTAL	12	10	2	5
MEAN (+)	0.5	0.4	0.1	0.2
ST.DEV.	1.6	1.5	0.3	0.8
N	24	24	21	22

LIVING PUPS AT FIRST LITTER CHECK
% OF MALES / FEMALES (#)	46 / 54	54 / 46	48 / 52	55 / 45
TOTAL	238	254	220	215
MEAN (+)	9.9	10.6	10.5	9.8
ST.DEV.	3.9	3	2.6	2.6
N	24	24	21	22

POSTNATAL LOSS
% OF LIVING PUPS	2.9	2	2.3	4.7
LITTERS AFFECTED (#)	4	3	4	3
TOTAL (#)	7	5	5	10
MEAN (+)	0.3	0.2	0.2	0.5
ST.DEV.	0.9	0.7	0.5	1.3
N	24	24	21	22

CULLED PUPS
TOTAL	67	70	53	42

LIVING PUPS DAY 4 P.P.
TOTAL	164	179	162	163
MEAN (+)	6.8	7.5	7.7	7.4
ST.DEV.	2.7	1.8	0.8	1.8
N	24	24	21	22

BREEDING LOSS DAYS 5 - 21 P.P.	0	0	0	4.9
% OF LIVING PUPS AT DAY 4 P.P.
LITTERS AFFECTED (#)	0	0	0	1
TOTAL (#)	0	0	0	8 ##
MEAN (+)	0	0	0	0.4
ST.DEV.	0	0	0	1.7
N	24	24	21	22

LIVING PUPS DAY 21 P.P.
% OF MALES / FEMALES (#)	48 / 52	50 / 50	50 / 50	54 / 46
TOTAL	164	178	162	155
MEAN (+)	6.8	7.4	7.7	7
ST.DEV.	2.7	1.8	0.8	2.4
N	24	24	21	22

+/++ Steel-test significant at 5% (+) or 1% (++) level
# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Conclusions:

Based on the results of this Extended One-Generation Reproductive Toxicity Study (including Cohorts 1, 2 and 3), the following No Observed Adverse Effect Levels (NOAELs) of Zinc bis(diethyldithiocarbamate) were established:
30 mg/kg/day for F0 local toxicity, based on reflux-related findings;
at least 100 mg/kg/day for both systemic and reproduction toxicity (F0);
30 mg/kg/day, based on the lower pup body weights, for developmental toxicity of the F1 generation until weaning at PND 21;
30 mg/kg/day, based on the observed growth retardation at 100 mg/kg/day, for developmental toxicity of the F1 generation post-weaning at PND 21;
at least 100 mg/kg/day for both developmental neurotoxicity and immunotoxicity of the F1 generation.

Executive summary:

In this GLP compliant Extended One Generation Reproductive Toxicity Test, performed according to the OECD TG 443, with the inclusion of Cohorts 1A and 1B (without extension to F2), 2A, 2B and 3, the objective of the study was to provide an evaluation of the pre- and postnatal effects of Zinc bis(diethyldithiocarbamate) 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 Zinc bis(diethyldithiocarbamate) 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.

Furthermore, the information obtained from the developmental neurotoxicity and developmental immunotoxicity assessments characterized potential effects in those systems.

The dose levels in this study were selected to be 0, 10, 30, 100 mg/kg/day, based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with oral exposure of Zinc bis(diethyldithiocarbamate) in rats (Test Facility Study No. 20238263). Higher doses could not be tested as these were not tolerated in the dose range finder study (Test Facility Study No. 20238263), based on observed test-item related mortality at 125 mg/kg/day, adverse effects on body weights in females during the post-coitum and lactation phase at 50 and 125 mg/kg/day and increased liver weights in males and females at 125 mg/kg/day.

Chemical analyses of formulations were conducted at three occasions during the study to assess accuracy and homogeneity.

For the F0-generation, the following parameters and endpoints 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, functional observations including acoustic startle response, immunotoxicity assessments using TDAR assay, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations, neurohistopathological examinations and morphometric analysis.

In addition, the following reproduction/developmental parameters were determined: 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).

Formulation analyses confirmed that formulations of test item in Corn oil were prepared accurately and homogenously.

Parental results

Local parental toxicity was observed at 100 mg/kg/day. No systemic toxicity was noted up to 100 mg/kg/day.
In total 8/50 F0-animals at 100 mg/kg/day did not survive until scheduled necropsy. In 7/8 of these premature decedents, most frequently observed findings prior to death and at necropsy included hunched posture, piloerection, rales, gasping, labored respiration, reflux after dosing, (mostly severe) body weight loss, and/or intestines distended with gas. Full histopathological examination was performed for these high dose animals, including microscopic evaluation of the nasopharynx and nasal cavity. Macroscopic and microscopic findings for premature decedents did not indicate a misgavage as possible cause for moribundity/death in the 100 mg/kg/day group. Main microscopic findings in these high dose animals consisted of slightly to moderately decreased lymphocyte cellularity of the thymus (likely due to the poor condition of the animals), with correlating gross finding of thymus reduced in size in a single female. In addition, abnormalities in the nasopharynx (minimal to moderate neutrophilic inflammation, slight squamous cell metaplasia, slight to massive ulceration and/or moderate to marked mucopurulent exudate), and nasal cavity (slight to marked mucopurulent exudate, minimal to slight squamous cell metaplasia, minimal to slight basal cell hyperplasia, moderate mucopurulent exudate and/or minimal epithelial degeneration), including turbinates as distinct substructure (moderate to marked neutrophilic inflammation, minimal to marked ulceration with slight to moderate fusion and/or slight fibrosis) were observed.
Furthermore, in a single female at 100 mg/kg/day, on top of these findings in the nasopharynx/nasal cavity, moderate meningitis of the brain and minimal meningitis of the spinal cord (cervical and thoracic segments) were noted. Meningitis of the brain and spinal cord is a very uncommon finding in rats and the etiology of this finding is unclear. The porte d’entrée of the meningitis might be the nasopharynx and sinus in the tympanic bullae and as such it may have spread from the nasal cavity to the cranial cavity along the trigeminal nerves. Gavage-related reflux and retrograde aspiration of gastric contents is described for rodents as a cause of unexpected changes in nasal cavity and nasal sinuses (similar to those findings recorded for premature decedents in this study) and is associated with mortalityRef 4,5,6. Gastro-esophageal reflux may be triggered or facilitated by additional factors in combination with gavage dosing. A high dosing volume, delayed gastric emptying and/or a high viscosity and irritating properties of the test item can play a roleRef 4,6. In the present study however, a clear cause for the observed reflux could not be determined. When stomach content ends up in nasal cavities, conditions are generated for ubiquitary bacteria. This was most likely the case in the current study causing the inflammatory changes in the brain, spinal cord, nasopharynx and nasal cavity of all but one of the premature decedents. Extensive meningeal inflammatory responses after reflux and retrograde aspiration of gastric contents has not been described, but there are publications where an inflammation of the inner/middle ear with progression to the meninges in the cranial cavity are describedRef 7,8. The meningitis in this study is considered to be caused by reflux/regurgitation of the stomach contents with stepwise inflammatory changes in nasopharynx and nasal cavity and progression along the Gasserian ganglion and the trigeminal nerves to the meninges in the cranial cavity and meninges of the spinal cord. This is further substantiated by the degenerative, regenerative and inflammatory lesions that are present in the nasopharynx/nasal cavity of all but one of the premature decedents. A relation of the meningitis to systemic exposure to the test item is highly unlikely, as there were no comparable findings in the meninges of the brain or spinal cord in any of the surviving treated animals.
Based on the abovementioned findings, these premature decedents at 100 mg/kg/day were considered to be reflux-related. Since these reflux-related findings might be interpreted as local irritating effects rather than systemic effects, they are regarded as not relevant for systemic effect level determination and not taken into account for establishing the systemic NOAEL.
In addition, one female at 100 mg/kg/day was sacrificed in extremis on Day 1 of lactation based on the clinical appearance of both the dam (hunched posture, piloerection, lean appearance and reduced body weight gain during the post-coitum) and its litter. There were no findings in nasal cavity and no definite cause of moribundity could be determined. As this was a single death which, given the timing (Day 1 of lactation), might be related to the pregnancy, it was considered not toxicologically relevant.
In animals that survived until scheduled necropsy, reflux after dosing was noted in two and one animal at 30 and 100 mg/kg/day, respectively, on a single day during the treatment period. In addition, rales, piloerection, and hunched posture were observed incidentally in individual animals, mostly at 30 and 100 mg/kg/day. As these clinical signs were also noted in most of the animals that died prematurely, a relationship to treatment is very likely. These clinical signs were likely also reflux-related and considered a local, rather than a systemic response. As such, they are regarded as not relevant for systemic effect level determination and not taken into account for establishing the systemic NOAEL.
Other test item-related findings consisted of reduced body weight gain (30 and 100 mg/kg/day) and food consumption (100 mg/kg/day) in both sexes. At 100 mg/kg/day, body weight (gain) of males was lower compared to controls from Day 15 of treatment onwards, resulting in slightly lower terminal body weights at the high dose level. In females at 100 mg/kg/day, body weight and body weight gain were lower compared to controls during the post-coitum phase. As a result, body weights at the start of the Lactation phase were lower at 100 mg/kg/day, with apparent recovery to normal values by the end of the lactation period. In males at 30 mg/kg/day, a similar trend on body weight (gain) was observed as at 100 mg/kg/day, although statistical significance was not achieved, and changes compared to controls were minimal by the end of the treatment period. Moreover, a trend towards lower body weight (gain) during post-coitum was also observed in females at 30 mg/kg/day, albeit less pronounced. Based on the slight magnitude of the change (< 10%) in males and the apparent recovery to normal values in females by the end of the treatment period, these findings on body weight were considered not adverse.
In males at 100 mg/kg/day, relative food consumption was initially somewhat decreased during the first week of treatment. It should be noted that in these males there was a general trend towards higher food intake (relative to body weight) compared to the concurrent control group from Day 15 of treatment onwards. The delayed growth of high dose males in the presence of higher food intake is indicative for a lower food efficiency, i.e. more food has to be ingested to reach the same growth in the animal’s mass. In females at 100 mg/kg/day, absolute and relative food consumption were lower compared to concurrent controls during post-coitum and lactation.
Hematology changes in males at 100 mg/kg/day consisted of increased (differential) white blood cell counts (white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, and large unstained cell count [LUC]). LUCs are either larger monocytes or lymphocytes that cannot be classified, and given the increase in lymphocytes and monocytes observed in these high dose animals, the increased LUC count was considered test item-related as well. In the absence of corroborative findings at the organ level, these changes in white blood cell parameters were considered not adverse.
The following changes in clinical chemistry parameters were noted that were considered test item-related: decreased alanine aminotransferase in females at 10, 30 and 100 mg/kg/day, slightly decreased total protein in males at 100 mg/kg/day and in females starting at 10 mg/kg/day, slightly decreased albumin in males at 100 mg/kg/day and females starting at 10 mg/kg/day, decreased glucose in males at 100 mg/kg/day, and decreased inorganic phosphate in females at 100 mg/kg/day. Moreover, increased cholesterol was noted in females at 100 mg/kg/day. In the absence of corroborative findings at the organ level, these changes were considered not adverse.
There were several changes in urine parameters in males and/or females, mostly at 100 mg/kg/day that were considered test item-related: increased incidence of cloudy urine (both sexes) and increased incidence and severity of blood in urine and presence of red blood cells in sediment (males only), increased number of leukocyte esterase positive urine samples (males only), increased ketone in urine (females only), increased unclassified crystals in sediment (females only), and increased urinary pH in individual animals (males only). Moreover, an increase in urine volume collected for urinalysis was noted in males (dose dependent, from 10 mg/kg/day onwards) and females (100 mg/kg/day). At 100 mg/kg/day this corresponded to the observed increased water consumption during the treatment period (not determined for Group 2 and 3). In the absence of correlating findings at the organ level, these findings were considered not adverse.
At the organ level, higher adrenal gland weights were noted in males at 30 and 100 mg/kg/day, which correlated microscopically to the increased incidence and severity of vacuolation of the zona glomerulosa noted in males at these dose levels. However, in the absence of any degenerative finding and in view of the low severities recorded (minimal to slight), this finding in the adrenal gland was considered not adverse.
Additional changes in organ weights included higher liver weights in males starting at 10 mg/kg/day and females at 100 mg/kg/day, lower seminal vesicle weight in males starting at 10 mg/kg/day, and lower pituitary gland weights in females starting at 30 mg/kg/day, but these were without microscopic or macroscopic correlate and considered not adverse.
No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. coagulation parameters, TSH and total T4 thyroid hormone levels, and macroscopic examination).
Reproductive results
No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg/day).
No test item-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, sperm analysis, spermatogenic profiling, and histopathological examination of reproductive organs).
Developmental results
Developmental toxicity was observed at 100 mg/kg/day.
At 100 mg/kg/day, body weights of male and female pups were lower from PND 1 onwards (5% at PND 1 and up to 13-14% at PND 21). Based on the magnitude of the change and as the effects were persistent throughout the pre-weaning period, this finding was considered adverse.
In addition, anogenital distance (normalized for body weight) was increased in male and female pups at 100 mg/kg/day. As absolute anogenital distance was unaffected by treatment with the test-item, the change in normalized anogenital distance was considered secondary to the observed lower body weights of pups at PND 1 and was therefore considered not a direct effect of the test item.
At 100 mg/kg/day, serum TSH levels in male and female pups of Cohort 2B on PND 21-22 were increased. As all values remained within the historical control range, this change was considered not adverse.
No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, post-implantation survival, live-birth, viability and lactation indices, duration of gestation, parturition, litter size, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination.
F1-Generation results
In the F1-generation, toxicity was observed at 100 mg/kg/day.
A total of 36 F1-animals were found dead or were sacrificed in extremis during the treatment period, most of which during the first week of treatment. This concerned 3, 8 and 25 animals of the 10, 30 and 100 mg/kg/day groups, respectively, whereas all F1-animals of the control group survived until scheduled necropsy. Most frequently observed findings prior to death for these early decedents included rales, gasping, piloerection, hunched posture, pale appearance, ptosis and reflux after dosing, and/or (severe) body weight loss. Moreover, most frequently observed macroscopic findings included lungs not collapsed, and/or intestines distended with gas. As most of the premature decedents from the F1-generation were replaced, no histopathological evaluation was performed for these animals. For the two Cohort 1A animals that were not replaced, histopathological evaluation showed abnormalities in the nasopharynx (moderate to marked neutrophilic inflammation, marked to massive ulceration, and/or moderate mucopurulent exudate), and nasal cavity (moderate to marked neutrophilic inflammation, moderate to marked epithelial necrosis, moderate to marked ulceration [with minimal fusion of the turbinates in one animal], moderate to marked mucopurulent exudate and/or slight squamous metaplasia) that were considered the main causes of moribundity in these two animals. Moreover, the available in-life and necropsy data for the premature F1-decedents are comparable to those seen previously in the premature decedents in the F0-generation which deaths were considered reflux-related (see Parental results). Based on these similarities, it is very likely that at least 24 out of the 36 F1-animals that died preterm in the F1-generation were reflux-related.
For the remaining 12 F1-animals that died preterm, the cause of death was considered mainly related to the challenges of the gavage procedure with this particular test item, especially in relatively small animals , based on the necropsy findings (trachea or esophagus perforation and/or fluid in the thoracic cavity). However, as there were no preterm deaths in the control group and a dose-response relationship was observed in the number of premature decedents, the involvement of another cause could not be readily excluded (i.e. reflux/regurgitation).
Based on the abovementioned findings, in combination with the findings observed in F0-animals, the preterm deaths were considered to be either related to the gavage procedure with this particular test item in relatively small animals and as such not directly related to treatment with the test item, or reflux-related. Since reflux-related findings might be interpreted as local effects rather than systemic effects, they are regarded as not relevant for systemic effect level determination and not taken into account for establishing the NOAEL.
In animals that survived until scheduled necropsy, test-item related clinical observations were noted across the dose groups, with increasing incidence and severity with increasing dose levels. These findings consisted of reflux after dosing in individual animals (including animals of the control group) on a single day during the treatment period, as well as rales, gasping, hunched posture and piloerection, noted mostly during the first two/three weeks of treatment. These clinical signs were likely also reflux-related and considered a local, rather than a systemic response. As such, they are regarded as not relevant for systemic effect level determination and not taken into account for establishing the NOAEL.
Test item-related changes in body weight were observed starting at 30 mg/kg/day. At 30 and 100 mg/kg/day, mean body weight gain of F1-animals was lower than controls from Week 1 of treatment onwards (males) or between Weeks 1 to 3 (females), which together with the lower weights at start of treatment (PND 21) resulted in a lower mean body weight of these animals throughout the treatment period, despite the apparent higher body weight gain in females at 100 mg/kg/day from Week 7 of treatment onwards. At 30 mg/kg/day, the changes were considered not adverse given the small size of the effect. At 100 mg/kg/day however, based on the magnitude of the effect (>10%) and since no recovery occurred during the treatment period, the effects on body weight were considered adverse.
At 100 mg/kg/day, mean absolute food consumption was slightly lower between Weeks 1-8 and Weeks 1-6 of treatment in males and females, respectively. It should however be noted that food consumption corrected for body weight was in general slightly increased in males and females throughout the treatment period. This is indicative for a lower food efficiency, i.e. more food has to be ingested to reach the same growth in the animal’s mass. This effect was less pronounced in females, which had a higher body weight gain compared to concurrent controls from Week 7 of treatment onwards.
Sexual maturation was slightly delayed. In males, the age at attainment of balonopreputial separation (BPS) was increased at 100 mg/kg/day. A similar trend was observed at 30 mg/kg/day, albeit less pronounced. In females, the age at attainment of vaginal opening (VO) was dose-dependently increased at 10, 30 and 100 mg/kg/day. However, body weight at attainment of VO or BPS was similar or reduced compared to controls, indicating that the observed delay in sexual maturation for both males and females was due to the test item-related growth retardation observed at these dose levels. Moreover, the age at first estrus was increased in females at 100 mg/kg/day. This was considered related to the increased age at attainment of VO and as such regarded secondary to the observed adverse test-item related growth retardation.
Changes in hematology parameters were observed at 30 (males only) and 100 mg/kg/day (both sexes). Comparable to F0-males, in F1-animals of both sexes treated at 100 mg/kg/day absolute and differential white blood cell counts (neutrophils, lymphocytes, monocytes, and large unstained cells) were increased at 100 mg/kg/day. Except for the mean neutrophil count, the same trend in abovementioned white blood cell parameters was observed for males at 30 mg/kg/day. In the absence of any corroborative findings at the organ level, these changes were considered not adverse.
Changes in clinical biochemistry parameters in males and/or females of Cohort 1A that were observed at 30 and/or 100 mg/kg/day included decreased concentrations of albumin and consequently total protein, increased total bilirubin and urea, and decreased potassium. As these changes occurred in the absence of correlating findings at the organ level, they were considered not adverse.
Furthermore, there were several changes in urine parameters at 100 mg/kg/day, that were considered test item-related: increased incidences of males with turbid and/or dark-yellow discolored urine, increased incidence and/or severity of blood in urine (both sexes), increased presence of ketones in urine (females only), increased presence of white blood cells in sediment and increased incidence and severity of the presence of red blood cells in sediment (males only), and increased urinary pH in individual animals (males only). These were largely the same changes as seen previously in the F0-generation. However, also for the F1-generation no correlating findings at the organ level were noted. Therefore, these findings were considered not adverse.
Morphological findings were noted in the adrenal glands of F1-animals of Cohort 1A. Changes in the F1-generation were the same as seen previously in the F0-generation: an increased incidence and severity of vacuolation of the zona fasciculata to a slight degree in males at 100 mg/kg/day. This microscopic finding likely correlated with the increased adrenal gland weight. However, in the absence of any degenerative findings and in view of the low severities recorded (minimal to slight), this finding was considered not adverse.
Remaining test item-related organ weight changes in males and/or females of Cohort 1A and/or 1B at 30 and/or 100 mg/kg/day were largely comparable as seen previously in the F0-generation and consisted of higher liver weight and lower pituitary gland weight (females only). In addition, in males of Cohort Surplus, thymus and spleen weights were decreased at 100 mg/kg/day. In Cohort Surplus females, thymus weights were increased at 30 mg/kg/day (Note: there were no Surplus females at 100 mg/kg/day). In the absence of any macroscopic or microscopic correlate and/or as changes were no longer present after prolonged treatment with the test item (in case of Cohort Surplus) these changes were considered to be not adverse.
At necropsy, a smaller thymus was noted at 100 mg/kg/day in Cohort 3 (both sexes). No histopathological evaluation of the thymus was performed for Cohort 3 animals. However, in the absence of corroborative macroscopic and/or microscopic changes in the thymus in animals from other cohorts that were treated for a much longer period, this finding was considered not toxicologically relevant.
Histopathologically, no test-item related effects were noted at stage-dependent qualitative evaluation of spermatogenesis, on ovarian follicle counts or morphology of reproductive organs in both sexes.
No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. TSH and T4 thyroid hormone levels, coagulation parameters, length and regularity of the estrous cycle, sperm analysis, and histopathological examination of reproductive organs, incl. spermatogenic profiling and ovarian follicle count).
F1-Generation (Post-Weaning) – Developmental Neurotoxicity
Cohort 2A females at 100 mg/kg/day showed a lower mean average and maximum response amplitude during the acoustic startle response test performed between PND 23-25. A similar trend was observed in females at 30 mg/kg/day and in males at 100 mg/kg/day, albeit less pronounced. These findings were considered secondary to the observed adverse test item-related lower body weights and/or food consumption at the mid and high dose levels and as such regarded not a direct effect of treatment with the test item.
In addition, similar as for the F0-animals, in the F1-animals in Cohort 1A there was a decrease in absolute fixed brain weight in males 100 mg/kg/day and an increase in mean relative to body weight brain weight in males at 30 and 100 mg/kg/day (mean terminal body weights were also lower). The same phenomenon of decreased absolute and increased relative (fixed) brain weight also appeared to be present in males and/or females of F1 Cohorts 2A (PND 76-90) and 2B (PND 21-22).
Although absolute brain weight is generally considered to remain constant in rodents experiencing body weight loss, studies have shown that such brain weight ‘sparing’ is not seen when body weight loss occurs during the period of brain development, as being applicable to the current F1-generationRef 9,10,11. Overall, in the absence of any other indicators of CNS toxicity, such as macroscopic or histopathological changes, the brain weight changes were considered to be related to a test item-related effect on final body weight and as such not adverse.
The differences in brain width in Cohort 2A males and brain length in Cohort 2B males were considered secondary to the lower terminal body weight and the resulting lower brain weight, as these differences were not consistent over time (i.e. there was a lower brain length in the younger animals with a normal brain width, but a lower brain width and a normal brain length in the older animals).
Morphometric analysis of the brain on PND 21-22 (Cohort 2B) revealed lower cerebellum height in males at 100 mg/kg/day (no longer significant after evaluation of all groups) and lower caudate putamen width in females at 100 mg/kg/day. Moreover, on PND 76-90 (Cohort 2A) lower caudate putamen width was noted in males and females starting at 30 mg/kg/day (for females no longer significant at 100 mg/kg/day after evaluation of all groups), as well as a lower corpus callosum thickness in males at 100 mg/kg/day and in females starting at 10 mg/kg/day and a lower hippocampus thickness in males at 100 mg/kg/day.
For both sexes there was no clear correlate between the significant differences in morphometry values between the PND 21-22 and the PND 76-90 animals. Overall, all these effects could be attributed to a test item-related effect on terminal body weight and the resulting lower brain weight, or a spurious higher control group value (corpus callosum thickness in Cohort 2A females on PND 76-90).
There were no microscopic abnormalities observed and no neurological signs noted in these animals after treatment with the test item up to 100 mg/kg/day and morphometric analysis revealed no indicators for neurotoxicity.
Taken together, there was no evidence of Zinc bis(diethyldithiocarbamate) being a developmental neurotoxicant.
No further test item-related changes were observed in the remaining developmental neurotoxicity endpoints tested: brain histopathology (Cohorts 2A and 2B), and functional observation tests (Cohort 2A; PND 63-75), including detailed clinical observations, rectal temperature, motor activity test, hearing ability, pupillary reflex, foot splay and grip strength.
F1-Generation (Post-Weaning) – Developmental Immunotoxicity
No test item-related changes in developmental immunotoxicity endpoints (i.e. TDAR assay, splenic lymphocyte subpopulations, lymphoid histopathology and organ weights) were observed in Cohort 1A animals.
Higher doses could not be tested as these were not tolerated in the dose range finder study (Test Facility Study No. 20238263), based on observed test-item related mortality at 125 mg/kg/day, adverse effects on body weights in females during the post-coitum and lactation phase at 50 and 125 mg/kg/day and increased liver weights in males and females at 125 mg/kg/day.
In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1, 2 and 3), the following No Observed Adverse Effect Levels (NOAELs) of Zinc bis(diethyldithiocarbamate) were established:
Local toxicity (F0): 30 mg/kg/day
Systemic toxicity (F0): at least 100 mg/kg/day
Reproduction NOAEL (F0): at least 100 mg/kg/day
Developmental NOAEL (F1 until weaning at PND 21): 30 mg/kg/day (based on the lower pup body weights at 100 mg/kg/day from PND 1 onwards)
Developmental NOAEL (F1 post-weaning): 30 mg/kg/day15 (based on the observed growth retardation at 100 mg/kg/day)
Developmental neurotoxicity (F1): at least 100 mg/kg/day

Note: In the present study, several premature decedents were observed in the F₀-generation at 100 mg/kg/day and F₁-generation at 10, 30 and 100 mg/kg/day. In the F₀-generation, all except for one, premature decedents at 100 mg/kg/day were considered to be reflux-related based on the observed clinical, macroscopic and microscopic findings (including degenerative/regenerative and inflammatory lesions in the nasopharynx/nasal cavity and secondary meningitis) and not directly related to treatment with the test item.
In the F₁-generation, the combination of the observed clinical and macroscopic findings together with the findings observed in F₀-animals, suggested that the premature deaths were either related to the gavage administration procedure in very young and small animals (i.e. not directly related to treatment with the test item) or reflux-related.
Since these reflux-related findings might be interpreted as local effects rather than systemic effects, they are regarded as not relevant for systemic effect level determination and not taken into account for establishing the NOAEL.

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