Zinc bis(dibutyldithiocarbamate)

Toxicity to reproduction

Administrative data

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

Reliability:

1 (reliable without restriction)

Justification for type of information:

Information on type of information: The extended one-generation reproductivity- with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B and 3) has been performed following the ECHA decision (CCH-D-2114375518-38-01/F). Due to a delay in the performance of the study, the final study report is not yet available and therefore only initial requested information can be presented. The robust study summary and endpoint summary will be updated when the final study report will become available. This is expected to be in August 2022. Argumentation of the delay is provided by the testing facility and attached in the “Attached justification” field. This is in agreement with approach outlined the after enquiry with ECHA (INC000000319505).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD guidance document on the current implementation of internal triggers in test guideline 443 for an extended one-generation reproductive toxicity test, in the United States and Canada, No. 117

Version / remarks:

2011

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Test guideline 4

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2014

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

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

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

- Basis for dose level selection: The dose levels were selected based on the results of a 90-day study (data on file at Sponsor site), teratology study (Test Facility Study No. 20150794) and simplified reproductive toxicity study (reproduction/developmental toxicity screening test, Test Facility Study No. 20150803). In all three studies Wistar Han rats received the test item by daily oral gavage with corn oil as vehicle. In the 90-day study, rats received 0, 10, 50 and 250 mg/kg/day test item. Treatment-related effects noted in the high-dose group included, clinical observations (i.e. salivation), reduction in male body weights, increased thrombocytes (females), decreased total protein and/or albumin concentrations and A/G ratio (both sexes), increased inorganic phosphate concentration (both sexes), increased urinary volume (males) and ketones (females), increased inorganic phosphate concentration (both sexes), increased relative weights of liver (24% and 28% in males and females, respectively), kidneys (27% and 13% in males and females, respectively), the spleen (24% in females only), adrenals (19% in males only) and decreased thymus weight (18% and 29% in males and females, respectively). Increased relative weights of the liver (both sexes) and kidneys (males) were also noted at 50 mg/kg. No treatment-related macro- or microscopic findings were noted up to 250 mg/kg/day. In the teratology study, rats received 0, 25, 75 and 250 mg/kg/day test item. The body weight gain corrected for gravid uterus of the dams was reduced in a dose related trend from females at 25 mg/kg/day onwards; the body weight gain was statistically significantly lower at 75 and 250 mg/kg/day when compared with concurrent control (6.3 and 2.9% vs. 14.0%, respectively). At 250 mg/kg/day food consumption was reduced throughout treatment and at 75 mg/kg a lower food consumption was noted during the first three days of treatment. Furthermore, mean combined fetal body weight at 250 mg/kg/day was decreased by approximately 8%. The remaining maternal and developmental parameters were unaffected by treatment up to 250 mg/kg/day. Based on the body weight effects during the teratology study and the organ weight changes in the 90-day study at 250 mg/kg/day, this dose level was considered inappropriate for the simplified reproductive study in which gestation and lactation periods would be included for female rats. Therefore, rats were dosed at dose levels of 0, 30, 62.5 and 125 mg/kg/day during the simplified reproductive study. No test item-related mortality was observed, and clinical signs were limited to slight salivation after dosing. Body weight and food consumption of F0-males was considered unaffected by treatment. For F0-females, mean body weight and relative food consumption were 8 and 25% lower towards the end of the gestation period, but no relevant differences were observed during the lactation period. At necropsy, no macroscopic abnormalities were noted. Liver weights were increased starting 62.5 mg/kg/day and kidney weights were increased at 125 mg/kg/day in males only. Mating, fertility and precoital time were considered unaffected by treatment. No toxicologically relevant effects on duration of gestation and pup mortality, clinical signs and macroscopy were observed. Number of dead and living pups at first litter check, viability index and sex ratio were unaffected by treatment. At 125 mg/kg/day, pup body weights were decreased from Day 1. Combined male and female pup body weights on Day 13 were 18% lower than control at 125 mg/kg/day. As the mean body weight (gain) of treated F1-pups was similar between all groups on PND 28, even for the pups selected from litters that were overall lighter on PND 13, this decrease was considered not to be a limiting factor for selection of 125 mg/kg/day as high dose. Based on the abovementioned results and in consultation with the sponsor, the following dose levels were selected: 0, 10, 35 and 125 mg/kg/day. The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

-Exclusion of extension of Cohort 1B

- 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 by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants. The total number of animals to be used in this study is considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Test material

Test material information

Constituent 1

Reference substance name:

Zinc bis(dibutyldithiocarbamate)

EC Number:

205-232-8

EC Name:

Zinc bis(dibutyldithiocarbamate)

Cas Number:

136-23-2

Molecular formula:

C18H36N2S4Zn

IUPAC Name:

zinc bis(dibutyldithiocarbamate)

Details on test material:

solid
Mw 474.2g/mol
purity: 98.9

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
-Females nulliparous and non-pregnant: yes
-Age/weight at dosing: F0-animals: approximately 6 weeks/ F0-males: 140 to 230 g; F0-females: 110 to 160 g.
-Housing: On arrival, prior to mating and during the post-weaning period, animals were grouped housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Makrolon type IV; height 18 cm or type 2000P; 61x43.5x21.5 cm depending on body weight). During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (type III; height 18 cm). During the post-mating phase, males were housed in Makrolon type IV or type 2000P cages (61x43.5x21.5 cm) with a maximum of 5 males/cage. During the lactation phase, females were housed in Makrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination (unscheduled deaths, spares, and pups of Cohort 2B) or until weaning on PND 21 (Cohorts 1A, 1B, 2A, 3). During locomotor activity monitoring, F1- Cohort 2A animals were housed individually in a Hi-temp polycarbonate cage (dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water for a maximum of 2 hours. Cages containing sterilized wooden fibers as bedding material equipped with water bottles. The cages contained appropriate bedding and were equipped with water bottles. Animals were separated during designated procedures/activities.
-Diet: SM R/M-Z pellets, Ad libitum, except during designated procedures. During motor activity measurements, F1- Cohort 2A animals did not have access to food for a maximum of 2 hours.
-Water: Municipal tap water, provided via water bottles ad libitum. During motor activity measurements, F1- Cohort 2A animals did not have access to water for a maximum of 2 hours.
-Acclimatisation period: At least 5 days

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

IN-LIFE DATES: from 21 Jul 2021 to 25 Feb 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

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


VEHICLE
- Amount of vehicle: 4 mL/kg
- Concentration in vehicle: 2.5, 8.75, 31.25 mg/mL
- The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube.

Details on mating procedure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and concentration were determined based on Zinc content since there is no better, feasible analytical method available for this type of molecule. Dose formulation samples were collected for analysis in week 1, 9, 17 and 25 of treatment.

-Sample for analysis: all Groups (concentration analysis) and Group 2 and 4 (homogeneity analysis).
-Sample volume: 2 x approximately 500 mg
-Acceptance criteria: For concentration, the criteria for acceptability were mean sample concentration results within or equal to ± 10% for solutions or ±15% for suspensions of target concentration. For homogeneity, the criteria for acceptability were relative standard deviation (RSD) of concentrations of ≤ 10% for each group.

Stability analysis of the test item in the vehicle was not determined since the available analytical method is only capable of measuring the zinc content in the test substance and, therefore, unable to measure the test item itself. Additional development of an analytical method for the test item was tried, 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 preparation were performed with approved procedures and documented in detail. This GLP exception was therefore considered as being minor with no impact on the outcomes and the integrity and the achievement of the objective of the study.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week.
F0-males were treated for a minimum of 11 weeks, including 10 weeks prior to mating (with the objective of covering at least one spermatogenic cycle) and during the mating period, up to and including the day before scheduled necropsy.
F0-females were treated for a minimum of 16 weeks, including 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females were not dosed during littering.
Prior to weaning, pups were not treated directly but could potentially be exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B, 2A and 3 were dosed up to and including the day before scheduled necropsy. The F1-animals of Cohort 2B and Spares (not assigned to one of the cohorts) were not dosed.
The dose volume for each animal were based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The first day of dosing were designated as Day 1 (exception: alternate animals used for replacement after Day 1 assumed the day of the animal being replaced). The dosing formulations were stirred continuously during dose administration. A dose control system (DCS) were used as additional check to verify the dosing procedure according to Standard Operating Procedures

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 2

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 3

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

F0 Animals: 25
F1 Animals:
-Cohort 1A: 20
-Cohort 1B: 20
-Cohort 2A: 10
-Cohort 2B: 10
-Cohort 3: 10
-Positive control: 10

Control animals:

yes, concurrent vehicle

Details on study design:

The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on the results of a 90-day study (data on file at Sponsor site), teratology study (Test Facility Study No. 20150794) and simplified reproductive toxicity study (reproduction/developmental toxicity screening test, Test Facility Study No. 20150803). In all three studies Wistar Han rats received Zinc bis(dibutyldithiocarbamate) (ZDBC) by daily oral gavage with corn oil as vehicle.
In the 90-day study, rats received 0, 10, 50 and 250 mg/kg/day test item. Treatment-related effects noted in the high-dose group included, clinical observations (i.e. salivation), reduction in male body weights, increased thrombocytes (females), decreased total protein and/or albumin concentrations and A/G ratio (both sexes), increased inorganic phosphate concentration (both sexes), increased urinary volume (males) and ketones (females), increased inorganic phosphate concentration (both sexes), increased relative weights of liver (24% and 28% in males and females, respectively), kidneys (27% and 13% in males and females, respectively), the spleen (24% in females only), adrenals (19% in males only) and decreased thymus weight (18% and 29% in males and females, respectively). Increased relative weights of the liver (both sexes) and kidneys (males) were also noted at 50 mg/kg. No treatment-related macro- or microscopic findings were noted up to 250 mg/kg/day.
In the teratology study, rats received 0, 25, 75 and 250 mg/kg/day test item. The body weight gain corrected for gravid uterus of the dams was reduced in a dose related trend from females at 25 mg/kg/day onwards; the body weight gain was statistically significantly lower at 75 and 250 mg/kg/day when compared with concurrent control (6.3 and 2.9% vs. 14.0%, respectively). At 250 mg/kg/day food consumption was reduced throughout treatment and at 75 mg/kg a lower food consumption was noted during the first three days of treatment. Furthermore, mean combined fetal body weight at 250 mg/kg/day was decreased by approximately 8%. The remaining maternal and developmental parameters were unaffected by treatment up to 250 mg/kg/day.
Based on the body weight effects during the teratology study and the organ weight changes in the 90-day study at 250 mg/kg/day, this dose level was considered inappropriate for the simplified reproductive study in which gestation and lactation periods would be included for female rats.
Therefore, rats were dosed at dose levels of 0, 30, 62.5 and 125 mg/kg/day during the simplified reproductive study. No test item-related mortality was observed, and clinical signs were limited to slight salivation after dosing. Body weight and food consumption of F0-males was considered unaffected by treatment. For F0-females, mean body weight and relative food consumption were 8 and 25% lower towards the end of the gestation period, but no relevant differences were observed during the lactation period. At necropsy, no macroscopic abnormalities were noted. Liver weights were increased starting 62.5 mg/kg/day and kidney weights were increased at 125 mg/kg/day in males only. Mating, fertility and precoital time were considered unaffected by treatment. No toxicologically relevant effects on duration of gestation and pup mortality, clinical signs and macroscopy were observed. Number of dead and living pups at first litter check, viability index and sex ratio were unaffected by treatment. At 125 mg/kg/day, pup body weights were decreased from Day 1.
Combined male and female pup body weights on Day 13 were 18% lower than control at 125 mg/kg/day. As the mean body weight (gain) of treated F1-pups was similar between all groups on PND 28, even for the pups selected from litters that were overall lighter on PND 13, this decrease was considered not to be a limiting factor for selection of 25 mg/kg/day as high dose.
Based on the above mentioned results and in consultation with the sponsor, the following dose
levels were selected: 0, 10, 35 and 125 mg/kg/day.
The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Positive control:

Yes: Group 5.

2.0 mg/mL cyclophosphamide were prepared once and filled out in daily portions for all treatment days under sterile conditions. Formulations were stored under conditions set to maintain 4°C (refrigerator).
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. Formulations was gently inverted prior to dosing. The dose volume for each animal was based on body weight measurement on Day 1 of cyclophosphamide treatment.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency was at least once daily. Animals were observed within their cage unless necessary for identification or confirmation of possible findings.
- Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

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

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

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

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

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

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

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with hematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.
One epididymis (right) was removed, placed in labeled bags, and kept in the freezer at ≤ 15°C. After thawing, the right epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples. In the case of any abnormalities in the right epididymis, the right side organ(s were fixed in modified Davidson's solution, and the left side organ was used for evaluation of sperm numbers. If abnormalities were found in both epididymides, both these organs were fixed in modified Davidson's solution and no evaluation of sperm numbers was performed.

Litter observations:

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

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

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

F1-Generation from Weaning (PND 21) onward (Cohort 1A, 1B, 2A and 3):
- Mortality/Moribundity Checks: At least twice daily throughout the study, animals were observed for general health/mortality and moribundity. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
- Clinical Observations: Clinical observations were performed at least twice daily, up to the day prior to necropsy. These observations were at least conducted prior to dosing and after dosing. Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade were predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was be scored.
- Arena Observations: Animals were observed for specific clinical signs in a standard arena once on the first Day 8 of weaning and thereafter at weekly intervals during the treatment period
- Body Weights: Animals were weekly weighed. This started on a specific date on which all pups were at least at PND 21. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation.
- Food Consumption: Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy.
- Water Consumption: Water consumption was monitored by visual inspection of the water bottles on regular basis throughout the study.
- Vaginal Patency: Vaginal patency (vaginal opening) was monitored daily for all females from PND 25 onwards until vaginal patency was present, by visual inspection of the vaginal area.
- Balanopreputial Separation: Balanopreputial separation (prepuce opening) was monitored daily for all males from PND 35 onwards until balanopreputial separation was present, by visual inspection of the genital area. Body weight was recorded on the day of acquisition of balanopreputial separation.

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

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

Clinical Pathology -F1-animals, PND 4 pups
On PND 4 at culling, blood was collected from two surplus pups per litter by decapitation, between 7.00 and 10.30 a.m. in the necropsy room, and samples were pooled per litter.
Pooled serum of culled PND 4 pups was used for measurement of T4 only.

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

Cohort specific investigations: 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 was 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), average maximum response amplitude (MaxN) and average latency to achieving the maximum response amplitude (Tmax) 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 (see Table 7)
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: Cohort 3 and positive control animals
- Immunization: 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 0.5 mL of the 4*10^8 SRBC/mL in sterile PBS formulation.
This immunization was performed 2 to 4 hours after treatment of the positive control and Cohort 3 animals with cyclophosphamide. The SRBC formulation was dosed between 30 minutes and 3 hours after removal from ice. Prior to dosing of each animal, the SRBC formulation was gently inverted. The animals were restrained during the injection procedure (without sedation) and injected using a disposable needle and butterfly needle.
- 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 in an ultra-low freezer set to maintain -80°C until analysis. The antibody response to the immunization with SRBC was determined by measuring the anti-SRBC IgM levels in serum using a validated ELISA method.

Cohort specific investigations: Cohort Cohort 2B
On PND 21-22, 1.0 mL of blood was collected from all Cohort 2B animals (10/sex/group), if possible. Blood was drawn, between 8.00 and 12.00 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.

Postmortem examinations (parental animals):

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

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

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

Postmortem examinations (offspring):

SACRIFICE
- Unscheduled Deaths until weaning:
Recognizable fetuses of females that die spontaneously or are euthanized in extremis were examined externally and sexed (both externally and internally, if possible). Live fetuses were euthanized by decapitation. Pups that are sacrificed in extremis, younger than 7 days, were euthanized by decapitation. Pups sacrificed in extremis on or after PND 7 were euthanized by an intraperitoneal injection of sodium pentobarbital.
Pups found dead during the weekend were fixed in identified containers containing 70% ethanol if not being subjected to necropsy on the same day.
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology. For pups found dead from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally, if possible). Descriptions of all abnormalities were recorded. Abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Culled Pups (PND 4): On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation. Sex was determined both externally and internally (if possible). Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.

GROSS NECROPSY
For all animals, necropsy procedures was performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. Tissues were preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution) unless otherwise indicated. Additional tissue samples may be collected to elucidate abnormal findings. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. Organ weight as a percent of body weight (using the terminal body weight) were calculated.

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

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

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

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

Cohort specific terminal procedures - Cohort 1B
- Scheduled Deaths - Cohort 1B: Scheduled necropsy of Cohort 1B were conducted on ≥ PND 105. Cohort 1B animals were not deprived of food overnight before necropsy. These animals had a terminal body weight recorded and were deeply anesthetized using isoflurane and subsequently exsanguinated (See Table 3).

Cohort specific terminal procedures - Cohort 2A and 2B
Scheduled necropsy of Cohort 2A were conducted on PND 76-90. Scheduled necropsy of Cohort 2B were 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 x2H2O, 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 were measured for all animals selected for neuropathology. Subsequently, the brain was fixed in 10% buffered formalin together with selected PNS tissues. Representative samples of the tissues identified in the Tissue Collection and Preservation (see Table 4) was collected.

Cohort specific terminal procedures - Cohort 3 and Positive Control Animals
Scheduled necropsy of Cohort 3 were 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. Descriptions of all macroscopic abnormalities were recorded. In
case of macroscopic abnormalities, gross lesions was preserved in the most appropriate
fixative together with the identification marks (see Table 5).

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.
Numerical data collected on scheduled occasions was analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation was reported whenever possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate. Inferential statistics was performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
Pairwise comparisons were made between the following groups: Group 2 vs. Group 1; Group 3 vs Group 1; Group 4 vs group 1.
Statistical analysis was not performed by the Testing Facility on T-cell dependent antibody response (TDAR) data due to limitations incurred as a result of the inter-animal variable nature of the antibody response and semi-quantitative nature of the assay.

-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 was applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were 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 (at least 3 groups) was compared using an overall Kruskal-Wallis. Whenever, the overall test is significant, the Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
-Incidence:
An overall Fisher’s exact test was used to conduct pairwise group comparisons of interest

Reproductive indices:

See Table 6

Offspring viability indices:

See Table 6

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks on result:

other: Will be derived after all information is available.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

not specified

Results: F1 generation

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks on result:

other: Will be derived after all information is available.

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

INTERIM RESULTS

Analysis of dose preparations

Week 1: Formulations were prepared accurately and homogenously.

Week 9: Formulations were prepared accurately and homogenously.

Week 17: Formulations were prepared accurately and homogenously.

Week 25: Formulations of Groups 3 and 4 were prepared accurately and homogenously. The formulation of Group 2 was below specification (77% vs 85% for suspensions), even after samples were re-analysed. An out of specification procedure was initiated for further investigation, but so far no explanation was found. As the NOAEL is expected to be at a higher dose, this lower accuracy of Group 2 is not expected to have an impact on the study outcome.

 

Mortality – F0

No mortality occurred that was considered to be related to treatment with the test item.

One Group 3 male (No. 71) was found dead on Day 46 of treatment. This animal was found stuck between the food hopper and shelter, and therefore its death was not attributed to treatment with the test item.

One Group 1 female (No. 118) had a total litter loss on Day 1 of lactation. This animal was subsequently sent for necropsy. At necropsy, one dead male fetus was found in the uterus.

 

Clinical signs – F0

No clinical signs of toxicity were noted during the observation period.

Salivation was intermittently recorded for most/all animals at 125 mg/kg/day between Weeks 2-4.

 

Body weights – F0

At 125 mg/kg/day, lower body weight gain was recorded for females from Week 3 of treatment while mean body weights of these females was similar to control means during this period. During post-coitum, variations in mean body weight and weight gain did not show clear relationship to the dose. Body weight of these females was again lower during the Lactation period, but these lower mean body weights appear to partially recover during this period along with higher weight gain. Males at this dose also showed a lower mean body weight from Week 5 of treatment onwards, with slightly lower weight gain (not statistically significant).

The statistically significantly lower body weights (and occasional lower mean weight gain) recorded for females at 10 mg/kg/day from Week 2 of treatment onwards, occurred in the absence of a dose-related trend, and was therefore considered unrelated to treatment with the test item.

 

Food consumption – F0

At 125 mg/kg/day, a statistically significantly higher relative food consumption was noted between Weeks 8 and 10 of treatment for males, and in Week 2 of treatment for females. During post-coitum, absolute and relative food consumption was only lower over post-coitum Days 7-11, but since food consumption was similar to control levels during other post-coitum days, this was considered unrelated to treatment with the test item. During Lactation Days 1-4, a reduced absolute and relative mean food consumption was noted for females at 125 mg/kg/day which partially recovered thereafter.

 

Note: As males did not mate on the same date, food consumption during the mating period has to be corrected for the actual number of males present in their home cage each day.

 

Haematology – F0

Males: considered not affected by treatment

Females: At 125 mg/kg/day, a higher mean reticulocyte count and increased mean red blood cell distribution width were noted. Other changes were considered not to be related to treatment with the test item as these occurred in the absence of a dose-related trend and were minor.

 

Clinical biochemistry – F0

Males: At 125 mg/kg/day, higher mean ALT, AST and inorganic phosphate values and lower albumin values were recorded. Other (statistically significant) changes were considered not to be related to treatment with the test item as these occurred in the absence of a dose-related trend and were minor.

Females: Higher mean ALT, AST and NA values were recorded at 125 mg/kg/day and lower mean potassium values were recorded from 35 mg/kg/day onwards. Other (statistically significant) changes were considered not to be related to treatment with the test item as these occurred in the absence of a dose-related trend and were minor.

 

Thyroid hormones  – F0

Males: Considered not affected by treatment. Mean TSH values of treated males were lower than concurrent control, but this was attributed to a relatively high control mean caused by a single animal with a high value (No. 6, 1.620 mU/L).

Females: Mean TSH values of females treated at 10 and 35 mg/kg/day were lower than concurrent control (0.60 and 0.77x, respectively), but this was attributed to a relatively high control mean caused by a single animal with a high value (No. 114, 1.380 mU/L). Without this animal the control mean would be 0.2667 mU/L. This also means that the mean TSH value of the females at 125 mg/kg/day may actually be considered increased (1.28x of control).  

 

Urinalysis – F0

Males: At 35 and 125 mg/kg/day, a higher mean urinary pH was recorded (a clear dose-related trend was absent)  recorded. Also mean urinary volume was higher at 125 mg/kg/day (with high standard deviation). Other (statistically significant) changes were considered not to be related to treatment with the test item as these occurred in the absence of a dose-related trend and were minor.

Females: At 125 mg/kg/day, a higher mean urinary pH was recorded. Other (statistically significant) changes were considered not to be related to treatment with the test item as these occurred in the absence of a dose-related trend and were minor.

 

Macroscopic examination – F0

Males: considered not affected by treatment

Females: considered not affected by treatment

 

Organ weights – F0

Males: higher absolute and relative liver, kidney and adrenal weights at 35 and 125 mg/kg/day (not always statistically significant for absolute weights; the lower terminal body weight at 125 mg/kg/day may have substantiated the relative increase at 125 mg/kg/day). A clear dose-related trend was absent.

Females: higher absolute and relative liver, kidney and adrenal weights, and lower ovary weights at 125 mg/kg/day (not statistically significant for absolute kidney weights). Note that the high standard deviation for control uterus weights required further clarification.

Other (statistically significant) changes in absolute or relative organ weights were considered not to represent an effect of treatment with the test item as these occurred in the absence of a dose-related trend and/or were attributed to variations in terminal body weight.

 

Reproduction data

One female in each dose group (including control) did not show evidence of mating. Of the mated females, two control females, and two females at 10 mg/kg/day were found to be not pregnant and were sent for necropsy between Day 26-27 Post Coitum. One control female (No. 118) had a total litter loss on Day 1 of lactation.

• Estrous cycle – F0: Not affected by treatment with the test item; all females had a regular estrous cycle of 4 to 5 days.


• Precoital time: At 125 mg/kg/day, three females at had a precoital time of 13 or 14 days, due to which the mean precoital time at this dose level was higher than the control mean (3.6 days vs 2.8 days). Although this longer precoital time was only recorded for a few animals, iIt could not be excluded that this represented a test item-related effect, also taking into account historical control data (a total of 6 out of 303 historical control animals had a precoital time of 13 or 14 days).


• Most females showed evidence of mating within 5 days after commencement of the pairing period. One control female had a precoital time of 12 days and This was considered not to represent an effect of the test item.


• Number of implantation sites: Considered not affected by treatment.


• Sperm analysis: Sperm parameters were considered not affected by treatment. The statistically significantly higher epididymal sperm count at 125 mg/kg/day occurred in the absence of a clear dose-related trend and the opposite effect would be considered a toxicological concern rather than the recorded apparent minor increase.

 

Developmental data  - pre-weaning

• Gestation duration: considered unaffected by treatment with the test item.


• Number of dead and living pups at first litter check: considered unaffected by treatment with the test item.


• Postnatal loss (live birth/viability/weaning index): Considered not affected by treatment with the test item.


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


• Anogenital distance:  At 35 and 125 mg/kg/day, slightly lower absolute anogenital distances were recorded for both male and female pups. A clear dose-related trend was absent. Anogenital distance corrected for body weight did not show any relevant differences. The changes in absolute distances were therefore considered secondary to the lower pup weights.


• Areolae/nipple retention: Not affected by treatment with the test item.


• Early postnatal pup development (mortality, clinical signs, body weight and macroscopy): Mean body weight of both male and female pups at 125 mg/kg/day was reduced compared to control from Postnatal Day 1 onwards (0.86x of control on PND 21 for both sexes combined). At 10 and 35 mg/kg/day, lower mean pup body weights were recorded from Postnatal Day 7 onwards (statistically significant on most occasions). Since these variations occurred in absence of a dose-related trend and were not consistently seen with continuing treatment, they were considered not to represent an effect of the test item. No clinical signs of toxicity were noted in the pups during the observation period thus far.

 

Mortality – F1 – post weaning

No test item-related mortality occurred.

One Group 2 female (No. 627, Cohort 1B) was sacrificed in extremis. It was accidentally trapped between the lid of the cage and the cage and therefore its death was not attributed to treatment with the test item.

 

Clinical signs – F1– post weaning

No clinical signs of toxicity were noted during the observation period.

Salivation was recorded for most/all animals at 125 mg/kg/day between from Week 3 of dosing onwards.