Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-05-2019 to 24-04-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes (incl. certificate)
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: Dose levels of 30, 100 and 300 mg/kg/day were selected in conjunction with the Sponsor based on the result of a preliminary juvenile toxicity study in Han Wistar rats, whereby the rats were dosed from Day 21 to 35 of age with 0, 30, 100 and 300 mg/kg/day (Envigo Study No. XS89HN) and on the results of a sub-chronic 13 week toxicity study (OECD 408), in which Wistar Han rats received OTNE at daily dose levels of 0, 30, 120 and 500 mg/kg/day. In the preliminary juvenile toxicity study, the only effect of treatment was at 300 mg/kg/day. Slightly increased liver weights recorded in males and females receiving 300 mg/kg/day was however, considered not to be adverse. In the sub-chronic 13 week toxicity study (OECD 408), the body weight adjusted weight of the liver was increased in males and females receiving 120 or 500 mg/kg/day (12-15% and 50%≤ respectively). Microscopic changes in the liver were characterized by centrilobular hepatocellular hypertrophy, the incidence of which was 3/10, 6/10 and 9/10 when receiving 30, 120 or 500 mg/kg/day respectively and hepatocellular vacuolation, the incidence of which was 8/10 and 9/10 when receiving 120 or 500 mg/kg/day respectively. Body weight adjusted weight of the kidneys was increased in males receiving 120 or 500 mg/kg/day (13% and 36% respectively) and in females receiving 500 mg/kg/day only (13%). Microscopic changes in the kidneys were noted for males only, receiving 30, 120 or 500 mg/kg/day and consisted of an accumulation of alpha2-urinary microglobulins. The severity of these findings was generally highest for males that received 500 mg/kg/day and lowest for males receiving 120 mg/kg/day. Body weight adjusted weight of the spleen for males receiving 500 mg/kg/day was increased (37%) and microscopic changes were characterized by increased extramedullary erythropoiesis in 8/10 males receiving 500 mg/kg/day and 3/10 for females receiving 120 mg/kg/day and 5/10 for females receiving 500 mg/kg/day. Based on especially the effects in liver weight up to 50% indicating overload of the metabolic capacity. Based on especially the effects in liver, indicating metabolic overload and set NOAEL of 120 mg/kg bw in the 90-day study, a high dose of 300 mg/kg/day was considered suitable for this study with low and intermediate dose levels of 30 and 100 mg/kg/day providing approximately 3-fold dose increments.
- Exclusion of extension of Cohort 1B: The conditions to include the extension of Cohort 1B are currently not met.
- Termination time for F2: not applicable, as cohort 1B will not be extended
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B: The available in vivo dataset for OTNE does not indicate specific concerns on neurotoxicity after repeated exposure (for details please refer to in Chapter 7.9.1 of this dossier wich contains an evaluation of neurotoxicity findings in the available 28-day and 90-day repeated dose toxicity studies for OTNE). The structural analogue AETT suggested by ECHA does not give a cause for concern due to the difference of metabolic pathways of OTNE and AETT, as the neurotoxic metabolite of AETT, a gamma-diketone, is not generated during the metabolisation of OTNE (Cammer, 1980).
- Exclusion of developmental immunotoxicity Cohort 3: No triggers described in Column 2 of Section 8.7.3., Annex X and further elaborated in ECHA Guidance on Information Requirements and Chemical Safety Assessment R.7a, chapter R.7.6 (version 4.1, October 2015). have been identified for the inclusion of Cohort 3 (developmental immunotoxicity).
- Route of administration: Oral, as this is a relevant and possible route of human exposure and a suitable route of administration as specified in OECD TG 443.
- Other considerations: The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan™:WIST strain was used because of the historical control data available at this laboratory.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited.
- Age at study initiation: (P) 28 to 34 days
- Weight at study initiation: (P) Males: 61 to 115 g; Females: 69 to 95 g
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used throughout the study except during pairing. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily. The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
- Environmental Enrichment: Aspen chew products: A soft white untreated wood product; provided to each cage throughout the study and replaced when necessary; Plastic shelter: Provided to each cage throughout the study [except during pairing or lactation (from Day 20 after mating)] and replaced at the same time as the cages; Paper shavings: From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting
material was changed at the same frequency as the cage bedding.
- Number of animals per cage: Acclimatization, treatment and selected F1 maturation: up to four animals of one sex; Pairing: one male and one female; Males to termination: up to four animals; Females after mating (from Day 0 after mating): one animal; Females during littering (from Day 20 after mating): one animal + litter; Females to termination (after weaning): up to four animals; Offspring maturation (from weaning until selection): litter
- Diet: SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals, ad libitum
- Acclimation period: Six days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. A series of formulations at the required concentrations were prepared in ascending order by dilution of individual weighings of the test item.
- Frequency of preparation: Weekly.
- Storage of formulation: Refrigerated (2 to 8°C).

VEHICLE
- Concentration in vehicle: 0, 7.5, 25, and 75 mg/mL at 0, 30, 100, and 300 mg/kg bw, respectively.
- Amount of vehicle: 4 mL/kg
Details on mating procedure:
- F0 pairing commenced: After ten weeks of treatment.
- Male/female ratio: 1:1 from within the same treatment groups (sibling pairing was not permitted).
- Duration of pairing: Up to two weeks.
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
- Day 0 of gestation: When positive evidence of mating was detected.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability: The stability of the test item in the liquid matrix was investigated in Envigo Study Number MN84RG. Specimen formulations at 1 and 200 mg/mL were analyzed. Formulations were confirmed to be stable for one day at ambient temperature (15 to 25°C) and for 15 days after refrigerated storage (2 to 8°C).
- Achieved concentration: The formulations for Week 1 (F0 Generation), Week 1 (F1 generation) and Last week (F1 generation) for all groups were sampled, 4 × 1 mL (accurately weighed), from the middle of the formulation by Pharmacy personnel. Two samples from each group were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.
Frequency of treatment:
Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
Details on study schedule:
Selection of Offspring to Form F1 Generation
- Selection: On Day 18 to 20 of age.
- Allocation - formal start of F1 generation: Nominally Day 28 of age (direct dose administration commenced on Day 21 of age).
- Method: The offspring with the lowest within-litter identification per sex from each selected litter were selected to form the F1 generation, after exclusion of grossly atypical animals. or F1 Cohort 1A and 1B where possible, two males or two females were allocated to each of the two cohorts. Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age. Formal commencement of the F1 generation was on a nominal Day 28 of age (where possible 28±2 days of age for selected F1 animals). Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
P: 24; F1A: 20; F1B: 20
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Clinical Observations - F0 and F1 Generation:
- Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
- Signs Associated with Dosing: Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
-- F0 generation: Week 1 - Daily; Weeks 2 to 4 - twice weekly (middle and end of the week); Week 5 onward - once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 21 of lactation for F0 females).
-- F1 generation Week 1 - Daily; Weeks 2 to 4 - twice weekly (middle and end of the week); Week 5 onward - once each week.
-- Detailed observations were recorded at the following times in relation to dose administration: Prior to dosing; One to two hours after completion of dosing; As late as possible in the working day.
- Clinical Signs: A detailed physical examination was performed on each animal to monitor general health according to the following schedule: Physical examination: Once each week. After mating of F0 females: Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation. A detailed physical examination was performed at nominally the same time of day on each occasion by an observer. After removal from the home cage, animals were assessed for physical condition and behavior during handling. Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior. Any deviations from normal was recorded with respect to nature, and, where appropriate, degree of severity.

Mortality - F0 and F1 Generation
- A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

Body Weight - F0 and F1 Generation:
- The weight of animals was recorded as follows:
-- F0 males: Day that treatment commenced. Each week. Before necropsy.
-- F0 females: Day that treatment commenced. Each week until mating detected. Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating. Days 1, 4, 7, 14, 21 and 28 of lactation. Before necropsy.
-- F1 selected animals: From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter. Before necropsy.

Food Consumption - F0 and F1 Generation:
- The weight of food supplied to each cage, that remained and an estimate of any spilled was recorded as follows:
-- F0 males and females: Weekly from the day that treatment commenced until paired for mating. For females after mating food consumption was performed to match the body weight recording: Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17 and 18-19 after mating Days 1-3, 4-6, 7-13, and 14-20 of lactation.
-- F1 selected animals: From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.
- From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

Parturition Observations and Gestation Length - F0 Generation:
- Duration of gestation: Time that elapsed between mating and commencement of parturition.
- Parturition observations: From Day 20 after mating animals were checked three times daily for evidence of parturition. The progress and completion of parturition was monitored; numbers of live and dead offspring were recorded and any difficulties observed were noted.

Hematology, Peripheral Blood - F0 and F1 Cohort 1A Generation
- Blood samples were collected after overnight withdrawal of food at termination of 10 animals per sex per dose.
- Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer: Hematocrit, Hemoglobin concentration, Erythrocyte count, Reticulocyte count, Mean cell hemoglobin, Mean cell hemoglobin concentration, Mean cell volume, Total leucocyte count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelet count
- Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.
- Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of: Prothrombin time (PT) - using IL PT Fibrinogen reagent and
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry - F0 and F1 Cohort 1A Generation
- Blood samples were collected after overnight withdrawal of food at termination of 10 animals per sex per dose.
- Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche Cobas 6000 Analyzer in respect of: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyl transpeptidase, Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Sodium,
Potassium, Chloride, Calcium, Inorganic phosphorus, Total protein, Albumin
- Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

Urinalysis - F1 Cohort 1A Generation
- Urine samples were collected after overnight withdrawal of food and water of 10 animals per sex per dose.
- The individual samples were examined for the following characteristics: Clarity and Color, Volume, pH, Specific gravity, Glucose, Ketones, Bile pigments, Blood pigments, Protein (total and concentration), Sodium (total and concentration), Potassium (total and concentration), Chloride (total and concentration), Potassium (total and concentration).
- A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields was recorded in the raw data and entered onto the database and the number seen were derived from Epithelial cells, Leucocytes, Erythrocytes, Casts, Other abnormal components. The slide was also examined for abnormalities in spermatozoa and crystals.

Thyroid Hormone Analysis - TSH and T4
- Blood samples were collected at termination in Ten male and ten female animals per group of the F0 adults; Ten male and ten female animals per group on Day 22 of age from as many litters as possible of the F1 Offspring; Ten male and ten female animals per group (approximately 13 weeks of age) of the F1 Adults.
- Conditions: Adults: Following overnight deprivation of food; Offspring: No overnight deprivation of food.
- Blood sample site: Adults: Sublingual vein; Offspring: Orbital sinus
- Anaesthetic: Isoflurane.
- Anticoagulant: None.
- Tubes: Greiner Minicollect - with clot activator.
- Blood volume: 1 mL
- Treatment of samples: Samples were kept at ambient temperature (15 to 25˚C) for a minimum of 30 minutes prior to centrifugation
- Centrifugation conditions: At 2000 g for ten minutes at 4°C.
- Number of aliquots: Two per animal. Aliquot 1: 0.2 mL serum for T4; Aliquot 2: residual serum for TSH
- Final storage conditions: Deep frozen (approximately -60°C to -90ºC).
Oestrous cyclicity (parental animals):
Estrous Cycle Monitoring - F0 Generation
- Dry and wet smears were taken as follows:
-- Dry smears: For 15 days before pairing, using cotton swabs.
-- Wet smears: After pairing until evidence of mating confirmed.
- For four days before scheduled termination (nominally Days 25 to 28 post partum). Females that failed to litter were retained and smeared for four days starting on the day on which the first batch of ‘true’ Day 25 post partum females started smearing, and were then killed with that first batch of females.

Estrous Cycle Monitoring - Cohort 1A
- Dry and wet smears were taken as follows:
-- Wet smears (using pipette lavage) Following onset of vaginal patency until first cornified (estrus) smear was recorded. For at least three days prior to the start of the necropsy phase and on the day of termination.
-- Dry smears (using cotton swabs): For two weeks from approximately Day 75 of age.

Estrous Cycle Monitoring - Cohort 1B
- Wet smears were taken as follow:
-- Wet smears (using pipette lavage): For at least three days prior to the start of the necropsy phase and on the day of termination.
Sperm parameters (parental animals):
Immediately after scheduled sacrifice of each F0 and F1 Cohort 1A male and collection of blood, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed. The following tests were performed:

Sperm motility:
- All groups: A sample of sperm was expressed from the left vas deferens into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 μm depth cannula by capillary action and, at least 200 sperm per animal analysed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyser (CASA). For F0 - Group 1 Male 4 and Group 3 Male 52 unable to assess 200 sperm.
Sperm morphology:
- Groups 1 and 4: A 200 µL aliquot of the sperm/medium mixture (described above) was diluted with 800 µL of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male where possible. For F0 - Group 1: Males 4 and 19 and Group 4 Male 76 unable to assess 200 sperm. For F1A - Group 4: Male 480 unable to assess 200 sperm.
- Groups 2 and 3: Fixed samples retained for possible future assessment.
Sperm count:
- Groups 1 and 4: The left cauda epididymis of each male was weighed and then the tunica was removed, then homogenized for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to
a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.
- Groups 2 and 3: Samples frozen for possible future assessment.
Homogenization-resistant spermatid counts
- Groups 1 and 4: After removal of the tunica, the left testis of each male then homogenized for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenizationresistant
spermatid count using CASA.
- Groups 2 and 3: Samples frozen for possible future assessment.
Litter observations:
Records Made During Littering Phase - F1 Generation
- Clinical observations: Observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment. On Day 1 of age, all offspring received a qualitative assessment of body temperature, state of activity and reaction to handling.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age. On Day 4 of age, litters containing more than eight offspring were reduced to eight by random culling, leaving, whenever possible, four male and four female offspring in each litter.
- Sex ratio of each litter: Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.
- Individual offspring body weights: Recorded on Days 1, 4 (before culling), 7, 14, 21, 23 and 25 of age. Selected F1 generation: Days 27 and 29 of age. Unselected F1 offspring: Day 22 of age.
- Weaning of offspring: The dam was removed from the litter cage and offspring were weaned on Day 21 of age.
- Ano-genital distance: Day 1 of age - all offspring.
- Nipple/areolae count: Day 13 of age - male offspring.

Sexual Maturation - F1 Generation - Cohorts 1A and 1B
- Males: Sexual maturation was assessed by daily examination from Day 35 of age until balano-preputial separation occurred. Body weight was recorded on the day of completion of separation.
- Females: Sexual maturation was assessed by daily examination from Day 25 of age until vaginal opening occurred. Body weight was recorded on the day of vaginal opening. For Cohort 1A: a wet smear was taken daily from the day of vaginal opening until first estrus was detected.
Postmortem examinations (parental animals):
Time of necropsy
- F0 males: After weaning of the F1 animals, after confirmation that no further mating required
- F0 females: Day 28 post partum
- F0 females failing to produce a viable litter: Terminated with first cohort of females with live litters
- Unselected offspring: F1 litters: Culled on Day 4 and Day 22 of age
- Cohort 1A animals: At approximately 13 weeks of age
- Cohort 1B animals: At approximately 14 weeks of age

Method of kill:
- Animals 14 days and older: Carbon dioxide asphyxiation with subsequent exsanguination
- Animals less than 14 days of age: Intraperitoneal injection of sodium pentobarbitone.
- All animals, including surplus offspring culled on Day 4 of age and Day 21/22 of age unselected offspring were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. Decedents offspring ≤21 days of age, (found dead or welfare kill), where possible, were examined and carcass retained. For females of Cohort 1A, counts were performed for the number of ovarian follicles and corpora lutea.

Organ weights
- For bilateral organs, left and right organs were weighed together, unless specified in the relevant pathology procedures table. Requisite organs were weighed for animals killed at scheduled intervals.
- For unselected F1 offspring on Day 22 of age, organs were weighed from ten males and ten females per sex per group from as many litters as possible.
- The following organs were weighted:
-- F0 (Parental) Animals: Adrenals, Brain (cerebellum, cerebrum, midbrain), Epididymides, Heart (including auricular and ventricular regions), Kidneys, Liver (section from two lobes), Ovaries with oviduct, Pituitary, Prostate - dorsolateral and ventral combined, Seminal vesicles (with coagulating gland), Spleen, Testes, Thymus, Thyroid with parathyroids, Uterus with cervix
-- Cohort 1A: Adrenals, Brain (cerebellum, cerebrum, midbrain), Epididymides, Heart (including auricular and ventricular regions), Kidneys, Liver (section from two lobes), Lymph nodes (mesenteric and left axillary), Ovaries with oviduct, Pituitary, Prostate - dorsolateral and ventral combined, Seminal vesicles (with coagulating gland), Spleen, Testes, Thymus, Thyroid with parathyroids, Uterus with cervix
-- Cohort 1B: Epididymides, Ovaries with oviduct, Pituitary, Prostate - dorsolateral and ventral combined, Seminal vesicles (with coagulating gland), Testes, Thyroid with parathyroids, Uterus with cervix

Fixation: F0 animals, Cohorts 1A and 1B
- Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of Testes - F0 and F1 adults only In modified Davidson’s fluid and Eyes In Davidson’s fluid.
- The following organs were fixated:
-- F0 (Parental) Animals: Abnormalities, Adrenals, Brain (cerebellum, cerebrum, midbrain), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Femurs - (longitudinal section through joint), Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Liver (section from two lobes), Lungs (section from two major lobes including bronchi), Optic nerves, Ovaries with oviduct, Pancreas, Pituitary, Prostate - dorsolateral and ventral combined, Rectum, Sciatic nerves, Seminal vesicles, (with coagulating gland), Skeletal muscle, Skin with mammary glands (inguinal area), Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), Spleen, Sternum - bone marrow, Stomach, Testes, Thymus, Thyroid with parathyroids, Trachea, Urinary bladder, Uterus with cervix, Vagina, Vas Deferens
-- Cohort 1A and 1B: Abnormalities, Adrenals, Brain (cerebellum, cerebrum, midbrain), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Femurs - (longitudinal section through joint), Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Liver (section from two lobes), Lungs (section from two major lobes including bronchi), Lymph nodes (mesenteric and left axillary), Optic nerves, Ovaries with oviduct, Pancreas, Pituitary, Prostate - dorsolateral and ventral combined, Rectum, Sciatic nerves, Seminal vesicles (with coagulating gland), Skeletal muscle, Skin with mammary glands (inguinal area), Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), Spleen, Sternum - bone marrow, Stomach, Testes, Thymus, Thyroid with parathyroids, Trachea, Urinary bladder, Uterus with cervix, Vagina, Vas Deferens

Histology - Adults
- Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining
tissues required. Sections were stained with hematoxylin and eosin.
- The following organs were processed:
-- All organs stated at fixation: All adult animals killed prematurely. All F0/F1A terminal adult animals of Groups 1 and 4 killed at a scheduled interval.
-- Abnormalities only: All F0/F1A terminal adult animals of Groups 2 and 3 killed at a scheduled interval. All F1B terminal adult animals.
-- Reproductive organs only: The reproductive organs were examined from F0 animals in Groups 2 and 3 that showed reduced fertility. This included males that failed to sire a pregnancy and females that were not pregnant and failed to litter.
Histology - Cohort 1B
- Tissue samples were dehydrated and embedded in paraffin wax.
- The following organs were processed: Abnormalities, Epididymides, Ovaries with oviduct, Pituitary, Prostate - dorsolateral and ventral combined, Seminal vesicles (with coagulation gland), Testes, Thyroid with parathyroids, Uterus with cervix, Vagina

Immunophenotyping of Spleen Leucocytes - Cohort 1A
- Ten males and ten females per group from Cohort 1A were selected for immunophenotyping. Where possible, one male or one female was assigned from each selected litter. The whole spleen was weighed. After weighing, a 3-5 mm mid transverse section was removed and retained for histopathological evaluation. The remaining portion of the spleen was then weighed, placed in to a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held in wet ice until processing for analysis. Samples were sent via courier to the Department of Bioanalysis, Biomarkers and Clinical Sciences (BBC), Covance. A copy of the whole spleen and partial spleen weights were provided to BBC.

Light Microscopy - Adult Animals
- Tissues preserved for examination were examined as follows:
-- Premature deaths: All adult animals and all animals from all groups. All specified organs under fixation
-- Scheduled kill: F0 animals and Cohort 1A: All animals and all specified organs under fixation of Groups 1 and 4. Abnormalities, kidneys and liver of all animals of Groups 2 and 3. Reproductive organs of all F0 animals of Groups 2 and 3 with suspect fertility. Abnormalities of all animals of Cohort 1B.
- Right testis: A detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.
- Ovaries F0 - Qualitative evaluation of one section from each ovary Cohort 1A - Qualitative evaluation of five sections from each ovary with quantitative assessment of primordial follicle and small growing follicle populations as well as corpora lutea.
- Vagina: The stage of vaginal estrus was evaluated based on vaginal epithelial morphology (and appearance of the uterus and endometrial glands).
- All other findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
Postmortem examinations (offspring):
Organ weights:
- For unselected F1 offspring on Day 22 of age, organs were weighed from ten males and ten females per sex per group from as many litters as possible.
- The following organs were weighted: Brain (cerebellum, cerebrum, midbrain), Spleen, Thymus

Fixation:
- Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of Testes - F0 and F1 adults only In modified Davidson’s fluid and Eyes In Davidson’s fluid.
- The following organs were fixated: Abnormalities, Brain (cerebellum, cerebrum, midbrain), Epididymides, Ovaries, Pituitary, Prostate, Seminal vesicles, Skin with mammary glands (inguinal area), Spleen, Testes, Thymus, Uterus with cervix and oviducts, Vagina
Statistics:
See any other information on materials and methods
Reproductive indices:
Percentage mating, Conception rate, Fertility index, Gestation index
Offspring viability indices:
Post implantation survival index, Live birth index, Viability index, Lactation index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
- Throughout the 10-week pre-pairing treatment period there were no clinical signs observed at routine examination considered to be related to treatment with OTNE.
- Throughout Days 0 to 20 of gestation, and Days 1 to 28 of lactation, there were no treatment-related clinical signs or signs associated with the administration of OTNE.
- An overview of the clinical signs can be found in tabular form in the attached study report in Table 1, 2, and 3, and in Appendix 1 and 2.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- During Week 9 of the 10-week pre-pairing treatment period, Control Male No. 12 was euthanized for reasons of animal welfare. Control Male No. 12 was observed to have swollen areas of the ventral surface, encrustation of the ventral surface and whole body pallor, the severity of which resulted in premature euthanasia. Macroscopic investigation revealed a ventrocranial palpable mass which correlated microscopically with a malignant basal cell tumor. Moderate increase of extramedullary hematopoiesis was present in the spleen and was consistent with a secondary adaptive response due to the presence of the neoplasia. The major factor contributing to death was the presence of the malignant basal cell tumor.
- On Day 1 of lactation, the female No.267 treated with OTNE at 100 mg/kg/day presented decreased activity, dyspnea and abnormally cold body temperature. The severity of the clinical signs resulted in premature euthanasia. The macroscopic examination revealed abnormal (dark) contents of the cecum, abnormal (clear gas and soft pale) contents of the esophagus, abnormal color (pale) of the jejenum which were not correlated with microscopic lesions. Additionally, the macroscopic thickening of the uterine horns correlated with microscopic placental retention which was considered to be the major factor contributing to death (urogenital lesions).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- For males and females receiving 30, 100 or 300 mg/kg/day, body weight gain was similar to that of the Controls throughout the 10 Week pre-pairing treatment period. Body weight for males receiving 30, 100 and 300 mg/kg/day was similar to that of controls from week 11 to week 18 of treatment.
- For females receiving 30, 100 or 300 mg/kg/day, body weight gain was similar to that of the Controls throughout Days 0 to 20 of gestation and from Day 1 to 28 of lactation.
- An overview of the body weight can be found in tabular form in the attached study report in Table 4, 5, and 6, and in Appendix 3, 4, and 5.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- For males and females receiving OTNE at 30, 100 or 300 mg/kg/day, group mean food intake was generally similar to that of the Controls throughout the 10 Week pre-pairing treatment period.
- For females receiving 30, 100 or 300 mg/kg/day, group mean food intake was similar to that of the Controls throughout Days 0 to 20 of gestation.
- For females receiving OTNE at 30, 100 or 300 mg/kg/day, group mean food intake was generally similar to that of the Controls throughout Days 1 to 14 of lactation. When receiving 100 or 300 mg/kg/day, group mean food intake for females from Days 14 to 21 of lactation was marginally high when compared to the Control.
- An overview of the food consumption can be found in tabular form in the attached study report in Table 7, 8, and 9, and in Appendix 6, 7, and 8.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- At scheduled termination hematocrit levels were slightly but statistically significantly low for males receiving 300 mg/kg/day (95% of control). Hemoglobin levels for males receiving 300 mg/kg/day were also slightly but statistically significantly low (96% of control).
- Hemoglobin levels for F0 females receiving 30, 100 and 300 mg/kg/day were slightly but statistically significantly high compared to control group (104.5%, 104% and 103%). Mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and mean corpuscular volume were all slightly but statistically significantly high for females receiving 300 mg/kg/day when compared to respective controls (105.7%, 102.4% and 103.1%). Neutrophil levels were slightly low for females receiving 100 mg/kg/day when compared to control (67.8%). As the figure did not attain statistical significance, and does not appear to be related to dose level, it is unlikely to be related to treatment.
- An overview of the haematological findings can be found in tabular form in the attached study report in Table 15, and in Appendix 10.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- At scheduled termination alkaline phosphatase levels were marginally low (85.7% and 84.3%) but did not attain statistical significance for males receiving 100 and 300 mg/kg/day. Aspartate aminotransferase was statistically significantly low for males receiving 300 mg/kg/day compared to control (82.6%). Gamma-Glutamyl Transpeptidase was statistically significantly high for males receiving 300 mg/kg/day compared to controls. Phosphate was statistically significantly low for males receiving 300 mg/kg/day compared to controls (85.9%). Total protein was statistically significantly high for males receiving 300 mg/kg/day compared to control (105.9%). Albumin was statistically significantly high for males receiving 300 mg/kg/day compared to control (105.1%).
- Alkaline phosphatase levels were slightly low for females receiving 30 and 100 mg/kg/day compared to control (86.3% and 90.2%) and statistically significantly low for females receiving 300 mg/kg/day compared to control (60.8%). Bilirubin was statistically significantly low for females receiving 30, 100 and 300 mg/kg/day compared to control (50% compared to control for all treated groups). Bile Acids were low for females receiving 30 and 100 mg/kg/day compared to control (54% and 52.3%) but did not attain statistical significance. Bile acids for females receiving 300 mg/kg/day were statistically significantly low compared to control (27.2%). Cholesterol was slightly high for females receiving 30 and 100 mg/kg/day compared to control (109.9% and 112.9%). Cholesterol for females receiving 300 mg/kg/day was statistically significantly high compared to control (116.3%). Chloride was slightly but statistically significantly low for females receiving 300 mg/kg/day compared to control (98.1%). Total protein was statistically significantly high for females receiving 300 mg/kg/day compared to control (105.7%).
- An overview of the clinical biochemistry findings can be found in tabular form in the attached study report in Table 16, and in Appendix 11.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Kidneys: In the majority of males treated with OTNE at 300 mg/kg/day and in more than half of males treated with OTNE at 100 mg/kg/day, the proximal convoluted tubules exhibited minimal to moderate increase of intracytoplasmic hyaline droplets. Additionally, almost half of the males treated with OTNE at 300 mg/kg/day exhibited minimal tubular basophilia.
- Liver: The majority of the females and some of the males treated with OTNE at 300 mg/kg/day exhibited minimal centrilobular hypertrophy. A few males treated with OTNE at 300 mg/kg/day exhibited minimal general hypertrophy.
- Incidental Findings: The incidence and distribution of all other microscopic findings were consistent with the common background pathology observed in Han Wistar rats at these laboratories.
- An overview of the histopathological findings can be found in tabular form in the attached study report in Table 33 and 34, in Appendix 24, and in Annex 6.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis - TSH and T4
- Mean serum TSH concentrations were seen to increase in treated F0 adult male rats when compared to the Control group with no dose response. Mean serum TSH concentrations in female rats were not seen to increase substantially in F0 adult rats. There was no effect of treatment on serum T4 concentrations in F0 animals.
- An overview of the Thyroid Hormone Analysis can be found in tabular in the attached study report in Annex 3 and 4.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
- Estrous cycles were considered to be unaffected by treatment. Irregular, extended and acyclic estrous cycles were seen in both control and treated groups. Nine control animals (38%), eight animals receiving 30mg/kg/day (33%), seven animals receiving 100 mg/kg/day (29%) and nine animals receiving 300 mg/kg/day (38%) had irregular cycles.
- Four control animals (17%), two animals receiving 30 mg/kg/day (8%), five animals receiving 100 mg/kg/day (21%) and one animal receiving 300 mg/kg/day (4%) had extended estrus cycles.
- Four control animals (17%), one animal receiving 30 mg/kg/day (4%) and two animals receiving 300 mg/kg/day (8%) were acyclic. Abnormal cycles were consistent across all dose groups when compared to controls – the cause is thought to be due to a light cycle error therefore the abnormal estrous cycles are not considered to be treatment related. This did not have an effect on mating performance or pre coital interval.
- An overview of the oestrus cycle can be found in tabular form in the attached study report in Table 10 and in Appendix 9.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- No adverse effects were observed following treatment with OTNE with doses up to 300 mg/kg/day.
- An overview of the sperm assessment can be found in tabular form in the attached study report in Table 22, 23, and 24 and in Appendix 18, 19, and 20.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance (pre coital interval) and fertility were unaffected by treatment at all dose levels. Gestation length and gestation index were unaffected by treatment at all dose levels.
- An overview of the pre coital interval, mating performance and fertility, and gestation length and gestation index can be found in tabular form in the attached study report in Table 11, 12, and 13 and in Appendix 9.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Adults
- From Day 21 to 28 of age and after the formal commencement of the F1 generation (from nominal Week 4 of age) through to termination, there were no test-item related changes in general clinical condition observed which were related to treatment with OTNE.
- An overview of the clinical signs can be found in tabular form in the attached study report in Table 35 and in Appendix 27.

Offspring
- There were no clinical signs observed among the F1 offspring that were considered to be related to parental treatment with OTNE.
- An overview of the clinical signs can be found in tabular form in the attached study report in Appendix 12.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Offspring
- Post implantation survival, the mean number of offspring born and the number of live offspring on Day 1 was essentially similar across all groups – offspring survival after birth was unaffected by treatment.
- An overview of the Litter Size and Survival Indices can be found in tabular form in the attached study report in Table 17 and 18, and in Appendix 13 and 14.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Adults
- From Day 21 up to nominal week 4 of age, the group mean body weight gain for F1 males and females was similar to that of the Controls.
- On Days 4-8 after the formal commencement of the F1 generation group mean body weight gain was slightly but statistically significantly high for males receiving 100 mg/kg/day. On Days 8 to 15, 22-29, 36-43 and 43-50 after the formal commencement of the F1 generation, group mean body weight gain was statistically significantly low for males receiving 300 mg/kg/day and as a result, the overall body weight gain from Days 1 to 57 was marginally but statistically significantly low when compared to the controls. For males receiving 30 or 100 mg/kg/day, group mean body weight gain for was essentially similar to the Controls throughout the duration of study with the exception of low body weight gain from days 43-50 when compared to controls. The toxicological relevance was considered minimal.
- For females receiving 30, 100 or 300 mg/kg/day, group mean body weight gain was marginally low from Days 4 to 8 after the formal commencement of the F1 generation when compared to the Controls. Overall body weight gain from Day 1 to 57 was statistically significantly low for females receiving 30 mg/kg/day only, which is due to chance and unrelated to treatment.
- An overview of the Body Weight can be found in tabular form in the attached study report in Table 36 and in Appendix 28.

Offspring
- The group mean body weight of male and female offspring on Day 1 of age, and subsequent body weight gain of the offspring to weaning on Day 21 of age, was unaffected by parental treatment with OTNE at all dose levels investigated.
- An overview of the Offspring Body Weight can be found in tabular form in the attached study report in Table 21 and in Appendix 17.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Adults
- Group mean food intake for males and females that received 30, 100 and 300 mg/kg/day was similar to that of controls throughout the duration of treatment after the formal commencement of the F1 generation.
- An overview of the Food consumption can be found in tabular form in the attached study report in Table 37 and in Appendix 29.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Adults: Cohort 1A
- At scheduled termination the hematological examination in males receiving 30, 100 and 300 mg/kg/day revealed no toxicologically significant differences from control.
- Red blood cell count was slightly but statistically significantly low in females receiving 300 mg/kg/day compared to control (95.3%). Blood platelets were statistically significantly high in females receiving 300 mg/kg/day (117%) compared to control. These findings were not clearly test-item related.
- An overview of the hematology findings can be found in tabular form in the attached study report in Table 43 and in Appendix 33.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Adults: Cohort 1A
- Alanine Amino Transferase was statistically significantly low in males receiving 300 mg/kg/day compared to controls (81.8%). Bile acids were low in males receiving 30 and 100 mg/kg/day (71.3% and 62.3%) compared to controls. Bile acids were statistically significantly low in males receiving 300 mg/kg/day (45.7%). Glucose was statistically significantly low in males receiving 300 mg/kg/day (90.6%) compared to controls.
- Alkaline phosphatase was low in females receiving 300 mg/kg/day (77.6%) compared to controls. Bilirubin was statistically significantly low in females receiving 300 mg/kg/day (0%) compared to controls. Bile acids were low in females receiving 30, 100 and 300 mg/kg/day (62%, 44.7% and 38.4%) compared to controls. Cholesterol was statistically significantly high in females receiving 100 and 300 mg/kg/day (116.8% and 130.4%) compared to controls. Calcium levels were slightly but statistically significantly high in females receiving 300 mg/kg/day (102.7%) compared to controls.
- An overview of the blood chemistry findings can be found in tabular form in the attached study report in Table 44 and in Appendix 34.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Adults: Cohort 1A
- pH was statistically significantly low in males receiving 300 mg/kg/day (87%) compared to controls. Specific gravity was slightly but statistically significantly high in males receiving 300 mg/kg/day (101.2%) compared to controls. Absolute and total protein was statistically significantly high for males receiving 100 and 300 mg/kg/day (154.4% to 275%) compared to controls. Total sodium levels were statistically significantly low in males receiving 300 mg/kg/day (62.8%) compared to controls.
- Urine volume was low in females receiving 300 mg/kg/day (64.3%) compared with controls. pH was low in females receiving 300 mg/kg/day (84.7%) compared with controls. Specific gravity was slightly but statistically significantly high in females receiving 300 mg/kg/day (100.9%) compared with controls. Total protein and total sodium was low in females receiving 300 mg/kg/day (77.9% and 66.6%) compared with controls. Protein was statistically significantly high in females receiving 300 mg/kg/day (126.6%) compared to controls. Urine potassium was statistically significantly high in females receiving 300 mg/kg/day (136.3%) compared to controls. Urine chloride was statistically significantly high in females receiving 300 mg/kg/day (156.5%) compared to controls.
- An overview of the urinalysis findings can be found in tabular form in the attached study report in Table 45 and in Appendix 35.
Sexual maturation:
no effects observed
Description (incidence and severity):
Adults
- Sexual maturation was considered to be unaffected by treatment at 30, 100 or 300 mg/kg/day.
- An overview of the sexual maturation can be found in tabular form in the attached study report in Table 38 and Appendix 30.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
- Ano-genital distance in male and female offspring was unaffected by maternal treatment at 30, 100 or 300 mg/kg/day.
- An overview of the Anogenital Distance can be found in tabular form in the attached study report in Table 20 and Appendix 16.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There was no effect of treatment on offspring nipple counts in males.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Adults
- Relative kidney weights were high for cohort A males receiving 30 mg/kg/day (103.5%) and statistically significantly high for males receiving 100 and 300 mg/kg/day (106.7% and 115.9%) compared to controls.
- Relative liver weights were high for cohort A males receiving 30 mg/kg/day (104.6%) and statistically significantly high for males receiving 100 and 300 mg/kg/day (111.6% and 121.1%) compared to controls.
- Relative thyroids and parathyroid weights were statistically significantly high for cohort A males receiving 300 mg/kg/day (124.0%) compared to controls.
- Relative prostate weight was statistically significantly high in cohort B males that received 300 mg/kg/day (119.4%) compared to controls. Thyroids and parathyroid weights were statistically significantly high for cohort B males receiving 100 and 300 mg/kg/day (120.9 and 137.2%) compared to controls.
- Relative kidney weights were marginally high for cohort A females receiving 30 and 100 mg/kg/day (102.4% and 103.8%) compared to controls, but did not attain statistical significance.
- Relative liver weights were high for cohort F1A females receiving 30, 100 and 300 mg/kg/day (110.3%, 109.8% and 128.4%) compared to controls. Thyroids and parathyroid weights were marginally high for cohort A females receiving 30, 100 and 300 mg/kg/day (105.4%, 105.4% and 110.8%) compared to controls, but did not attain statistical significance.
- Absolute ovaries and oviduct weights were marginally high for cohort F1A females receiving 30 mg/kg/day (108.1%) compared to controls but did not attain statistical significance. Ovaries and oviduct weights were statistically significantly high for cohort A females receiving 100 and 300 mg/kg/day (109.8% and 110.8%) compared to controls.
- Thyroid and parathyroid weights were statistically significantly high for cohort B females receiving 30, 100 and 300 mg/kg/day (115.9%, 115.9% and 117.5% compared to controls.
- Relative ovaries and oviduct weights were high for cohort B females receiving 30, 100 and 300 mg/kg/day (110.8%, 111.2% and 111.7%) compared to controls.
- An overview of the Organ Weights can be found in tabular form in the attached study report in Table 50, 51, 52, and 53 and Appendix 40 and 41.

Offspring
- There was no effect of treatment on the absolute or body weight relative brain, spleen or thymus weights.
- An overview of the Offspring Organ Weights can be found in tabular form in the attached study report in Table 29 and 30 and in Appendix 23.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Adults
- There were no macroscopic differences in males or females from Control that were attributable to treatment at 30, 100 or 300 mg/kg/day.
- An overview of the Macropathology findings can be found in tabular form in the attached study report in Table 54 and 55, in Appendix 42 and 43, and in Annex 6.

Offspring
- There were no findings considered to be related to treatment.
- An overview of the Offspring Macropathology can be found in tabular form in the attached study report in Appendix 25 and 26.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Adults
- Kidneys: In all males treated with OTNE at 300 mg/kg/day, the proximal convoluted tubules exhibited minimal to moderate increase of intracytoplasmic hyaline droplets. In more than half of the males treated with OTNE at 100 mg/kg/day, the proximal convoluted tubules exhibited minimal increase of intracytoplasmic hyaline droplets. Almost half of the males treated with OTNE at 300 mg/kg/day and a few males treated with 100 mg/kg/day exhibited minimal to slight, generally multifocal tubular basophilia. Males treated with 30 mg/kg/day also exhibited tubular basophilia but the lesion was minimal and focal in all cases, and was considered likely to be background.
- Liver: More than half of the females and some of the males treated with OTNE at 300 mg/kg/day exhibited minimal centrilobular hypertrophy. One male treated with OTNE at 300 mg/kg/day exhibited minimal general hypertrophy.
- The incidence and distribution of all other microscopic findings were consistent with the common background pathology observed in Han Wistar rats at these laboratories.
- An overview of the Histopathology findings can be found in tabular form in the attached study report in Table 56 and 57, in Appendix 42 and 43, and in Annex 6.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Adults: Cohort 1A: Thyroid Hormone Analysis - TSH and T4
- Mean serum TSH concentrations were seen to increase in F1 adult male rats when compared to the Control group. Mean serum TSH concentrations in female rats were not seen to increase substantially. There was no effect of treatment on serum T4 concentrations in F1A animals and selected F1A adult males.
- An overview of the Thyroid Hormone Analysis results can be found in tabular form in the attached study report in Annex 3 and 4.

Adults: Cohort 1A: Spleen Cell Immunophenotyping
- The oral gavage administration of OTNE had no treatment related effects on the immunophenotyping parameters measured in spleen leukocytes. See Annex 5 of the attached study report.
- An overview of the Thyroid Hormone Analysis results can be found in tabular form in the attached study report in Annex 5.

Adults: Vaginal Opening to First Estrus and Estrous Cycle
- The period between vaginal opening and first estrous was unaffected by treatment at 30, 100 and 300 mg/kg/day. Estrous cycles from day 75 of age and the stage of estrous cycle at termination were unaffected by treatment at 30, 100 and 300 mg/kg/day.
- An overview of the time of Vaginal Opening to First Estrus and Estrous Cycle can be found in tabular form in the attached study report in Table 39, 40, and 42 and in Appendix 31 and 32.

Adults: Cohort 1A: Ovarian Follicle Counts and Corpora Lutea
- Ovarian follicle counts for females that received 300 mg/kg/day were slightly low when compared to the control group. There was no effects of treatment on corpora lutea counts.
- An overview of the Ovarian Follicle Counts and Corpora Lutea can be found in tabular form in the attached study report in Table 46 and in Appendix 36.

Adults: Cohort 1A: Sperm Assessment
- At 300 mg/kg/day there was a slight increase in cauda epididymal weight and sperm concentration compared to concurrent control. This could indicate a potential blockage in the reproductive tract but as no affect was observed on motility or morphology, is not considered adverse.
- No adverse effects were observed following treatment with OTNE with doses up to 300 mg/kg/day.
- An overview of the Ovarian Follicle Counts and Corpora Lutea can be found in tabular form in the attached study report in Table 47, 48, and 49 and in Appendix 37, 38, and 39.

Offspring: Thyroid Hormone Analysis - TSH and T4
- F1 male offspring mean serum concentrations in treatment groups remained similar to that of the Control group. Mean serum TSH concentrations in female rats were not seen to increase substantially. There was no effect of treatment on serum T4 concentrations in F1 offspring at Day 22.
- An overview of the Thyroid Hormone Analysis results can be found in tabular form in the attached study report in Annex 3 and 4.

Offspring: Sex Ratio
- There was no clear effect on offspring sex ratio when treated with OTNE at all dose levels investigated.
- An overview of the sex ratio can be found in tabular form in the attached study report in Table 19 and in Appendix 15.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

F1 Litter Responses
- One female in the 100 mg/kg/day group (No. 267) was prematurely killed due to animal welfare concerns. In addition, two Control females (F0 female No. 204 and 211), one female receiving 30 mg/kg/day (F0 female No. 229), one females receiving 100 mg/kg/day (F0 female No. 252) and three females receiving 300 mg/kg/day (F0 female No. 285, 287 and 276) were not pregnant. Therefore, the following assessment is based on 22, 23, 22 and 21 litters at 0, 30, 100 and 300 mg/kg/day respectively.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Formulation Analysis: The mean concentrations of OTNE in test formulations were within 8% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 4%, confirming precise analysis. See Annex 2 of the study report.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test (OECD 443, GLP), the systemic NOAEL was determined to be >=300 mg/kg bw. The fertility and developmental toxicity NOAELs are >= 300 mg/kg bw.
Executive summary:

Introduction:

The test substance was investigated, in a study performed according to OECD TG 443, on systemic and reproductive toxicity when administered continuously by oral gavage to Han Wistar rats. In the F0 generation, 24 Han Wistar rats per sex received OTNE at dose levels of 30, 100 or 300 mg/kg/day. The dose was based on all repeated dose toxicity studies showing increase relative liver weights with hypertrophy at 500 mg/kg bw of ca. 50% indicative for overloading the metabolic pathway. The NOAEL for this effect was set at 120 mg/kg bw and therefore 300 mg/kg bw was anticipated to show some liver toxicity, which could be well tolerated. Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation (resulting in exposure up ca 120 days. In the F1 generation, 40 rats per sex were treated from weaning to their scheduled termination (relevant to each cohort) at the same dose levels and as the F0 generation resulting in approximately 70 exposure days for the F1A cohort and 77 exposure days for the F1B cohort. A similarly constituted Control group received the vehicle, corn oil.

Measured parameters:

For the F0 generation data were recorded on clinical condition, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance. Clinical pathology (haematology, blood chemistry and thyroid-related hormones), sperm assessment, organ weight, macroscopic pathology and microscopic pathology investigations were performed.

For F1 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, organ weights and macropathology were assessed. Nipple counts were performed on male offspring on Day 13 and 20 of age. Blood samples were collected from selected offspring on Day 4 and 22 of age for biomarker investigations.

For F1 generation - Cohort A, data were recorded on clinical condition, body weight, food consumption, sexual maturation, vaginal opening and estrous cycles. Clinical pathology (haematology, blood chemistry, thyroid hormones and urinalysis), sperm assessment, ovarian follicle counts, organ weight, macroscopic pathology, full microscopic pathology and spleen cell immunophenotyping investigations were performed.

For F1 generation - Cohort B, data was recorded on clinical condition, body weight, food consumption, estrous cycles, sexual maturation, organ weight, and a targeted set of macroscopic pathology investigations were performed.

Results – systemic toxicity:

F0 adults (and F1 offspring up to weaning)

Mortality: There were two deaths throughout the duration of study; Control male No. 12 and Female No. 267 that received 100 mg/kg/day were euthanized prematurely on animal welfare grounds during the 10-week pre-pairing treatment period and on Day 1 of lactation respectively. Female No. 267 had shown signs comprising decreased activity, irregular breathing and was abnormally cold to touch, macropathological examination revealed abnormal contents of the gastrointestinal tract and thickened uterus horns. When compared to the animals killed at scheduled termination, these findings were atypical and therefore the death of female No. 267 was not considered related to treatment.

Clinical signs: There were no clinical signs considered to be related to treatment in F0 males or in the F0 females which reared their litters to weaning.

Body weight gain of males and females before pairing and of females during gestation and lactation was unaffected by treatment.

Food consumptionof both sexes before pairing, males after pairing and females during gestation and lactation was generally similar to that of the Controls. From Days 14 to 21 of lactation, food intake was marginally high for females that received 100 or 300 mg/kg/day.

Haematologyinvestigations revealed slightly low haematocrit and haemoglobin concentrations for males that received 300 mg/kg/day and an increase in mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and mean corpuscular volume for females that received 300 mg/kg/day. For females that received 30, 100 or 300 mg/kg/day, a slight increase in haemoglobin levels was evident. All these effects were <6% and considered incidental and not adverse.

Blood chemistryinvestigations revealed marginally low alkaline phosphatase levels for males that received 100 or 300 mg/kg/day and females that received 30, 100 or 300 mg/kg/day (< 20%). Low aspartate aminotransferase and phosphate levels were evident for males that received 300 mg/kg/day. Gamma-glutamyl transpeptidase increased from zero to 1. Total protein and albumin levels increased minimally for males that received 300 mg/kg/day and protein levels at this dose for females but <=6%. Bilirubin and bile acid levels were low and for females that received 30, 100 or 300 mg/kg/day. For females, cholesterol was slightly increased at the 300 mg/kg/day (+16%). The toxicological relevance decreases of alkaline phosphate levels, low aspartate transferase and bile acid levels is unclear, because increase is considered to be related to liver effects. Others liver related parameters such as, increase in gamma-glutamyl transferase and some increase in cholesterol are considered to be due to increased liver function.

Macropathology: There were no test item related macropathology findings in any of the organs.

Organ weights and Histopathology:Liver: Relative Liver weights were dose dependently statistically significantly high among males and females that received 100 or 300 mg/kg/day (9.5 and 29.5%, respectively). For females only, relative liver weights were dose dependently increased up to 26.5%. Treatment-related changes in the liver were present in both males and females of F0 generation. In some males and approximately half of the females treated with OTNE at 300 mg/kg/day, the hepatic changes were consistent with minimal centrilobular or general hepatocellular hypertrophy (males only). Some changes in blood chemistry values such as the increase in gamma glutamyl peptide transferase, increase in total protein, increase in cholesterol, increase in liver weight and slight liver hypertrophy is anticipated to be related to an adaptive increase of liver function and not considered adverse.Thyroid: Relative thyroid weights were also increased in males but less so in females. No histopathological findings were seen and therefore their increases were not considered adverse.Spleen: The higher mean weights of the spleen and in F0 males that received 300 mg/kg/day were not supported by any microscopic pathology changes and thus biological significance and any relationship to treatment is not evident.Kidneys: Treatment-related changes in the kidneys were present in males only. Increased amounts of hyaline droplets within the renal proximal tubular epithelium were considered to be treatment related in kidneys of males treated with OTNE at 300 mg/kg/day and at 100 mg/kg/day examined: 15/25 and 23/24 animals, respectively. Additionally, higher incidence of minimal focal to multifocal, tubular basophilia was observed in the kidneys of males treated with OTNE at 300 mg/kg/day: 11/24 animals. Hyaline droplets and the increase in tubular basophilia are considered to be related to alpha-2u-globulin hydrocarbon nephropathy, an adaptive response, male sex specific and not considered adverse for humans; but no immune staining was done.

Results Reproductive toxicity (F0)

Fertility P0: Irregular, extended, and acyclic estrous cycles were observed in both control and treatment groups. The cause was thought to be related due to a light cycle error in the animal facility room and therefore these abnormalities were not considered related to treatment. There was no effect of treatment on pre-coital interval, fertility, gestation length and gestation index. Sperm motility, counts and morphology were unaffected by treatment.

Developmental toxicity: At 30, 100 or 300 mg/kg/day, there was no effect of treatment on post implantation survival, the mean number of offspring born and the number of live offspring on Day 1. Offspring survival after birth was unaffected by treatment and there was no clear effect on offspring sex ratio. There was no effect of treatment on offspring ano-genital distance, nipple counts in males, organ weights or macropathological findings. Male and female reproductive organs were not affected. There was no effect of treatment on serum T4 levels in adults or offspring, and no conclusive effect on TSH levels.

Results: Selected F1 offspring - Cohorts 1A and 1B

Clinical signs: Treatment of the F1A or F1B males and females was generally well tolerated and there were no test-item related changes in clinical condition observed from weaning on Day 21 of age up until scheduled termination at approximately Week 13 or 14 of age.

Body weight: At 30, 100 or 300 mg/kg/day, body weight gain of both sexes between Days 21 to 25 of age was generally similar to that of the Controls. For males that received 300 mg/kg/day, group mean body weight gain from Day 1 to 57 after the formal commencement of the F1 generation was marginally low when compared to the controls (<=-5%). Food consumption was not affected.

Haematology investigationof the F1A cohort did not reveal any treatment related changes. Two significant finding in females were not considered toxicologically relevant: Hemoglobulin concentrations decrease of 5% and platelet count increase of 17% in absence of other related findings.

Blood chemistry investigationof the F1A cohort revealed low alanine amino transferase and glucose levels for males that received 300 mg/kg/day. For males and females that received 30, 100 or 300 mg/kg/day, bile acids were low when compared to the controls. Low alkaline phosphatase values were evident in females that received 300 mg/kg/day. Calcium was minimally but significantly increased (+3%). For females that received 100 or 300 mg/kg/day, cholesterol was high (+17 and +30%, respectively) when compared to the controls. Except for calcium most parameters are liver related.

Urinalysis investigationof the F1A cohort revealed low pH in males and females that received 300 mg/kg/day: from ca 7 to 6.1. For males that received 100 or 300 mg/kg/day, total protein and protein was high when compared to the controls 92 and 275%, respectively. Additionally, total sodium levels for males that received 300 mg/kg/day was low and for males that received 30 or 100 mg/kg/day, Females that received 300 mg/kg/day, urine volume, total protein and total sodium was low whereas, protein, urine potassium and urine chloride was high when compared to the controls. Urine sodium levels were high. These effects are considered minor and not considered adverse in absence of related finding in kidney.

Macropathology: There were no test item related macropathology findings on all organs.

Organ weight: Relativeliverweights were high for cohort 1A males and females that received 30, 100 or 300 mg/kg/day (<=28% for the high dose). For cohort 1A and 1B males that received 300 mg/kg/day and cohort 1A and 1B females that received 300 mg/kg/day.Thyroid and parathyroidweights were high in F1A and 1B males when compared to the controls (<=37%); in females it was also increased but not significantly (<11%), which are anticipated to be related to increase in liver function. RelativeKidneyweights were statistically significantly high among cohort 1A males and females that received 300 mg/kg/day (<=16%) and slightly high for males that received 100 mg/kg/day (+7%).

Histopathology, Liver: Treatment-related changes in the liver were present in both males and females of the F1 generation. In some males and approximately half of the females treated with OTNE at 300 mg/kg/day, minimal centrilobular hypertrophy was apparent with one male showing minimal general hypertrophy. These are similar to what is seen in the F0 generation.

Microscopic pathology changes related to treatment with OTNE were seen in the kidneys and the liver but not in any other organs. Increases in the weight of some organs in some cohorts e.g. thyroid, prostate and oviduct were therefore not considered adverse.Kidney: Treatment-related changes in the kidneys were present in males only. In males of the F1 generation, increased amounts of hyaline droplets within the renal proximal tubular epithelium was considered to be treatment related in kidneys of males treated with OTNE at 300 and 100 mg/kg/day. Additionally, higher incidence of minimal to slight, focal to multifocal, tubular basophilia was observed in the kidneys of males treated with OTNE at 100 or 300 mg/kg/day (number of animals). These effects are considered to be related to alpha-2u-globulin hydrocarbon nephropathy.

Reproductive toxicity (F1A and F1B): Reproductive parameters for the F1 group, there was no adverse effect of treatment on sperm motility, counts or morphology. At 300 mg/kg/day there was a slight increase in cauda epididymal weight and sperm concentration, which is not considered toxicological relevant. Age at sexual maturation, time between vaginal opening and first estrous and oestrus cycles and ovarian follicle counts were unaffected by treatment. For cohort 1A females that received 100 or 300 mg/kg/day and cohort 1B females that received 300 mg/kg/day, ovaries and oviduct weights were high when compared to the controls just <=11%. No histopathological findings were seen and therefore biological significance or any relationship to treatment is uncertain. Prostate weights of cohort 1B males that received 300 mg/kg/day was high when compared to the controls (+19%). No histopathological findings were seen.

Conclusion: Considering systemic toxicity for F0 and F1 animals, minor effects were seen on haematology and blood chemistry of which the latter can be related to increased liver function. In the liver the increased relative liver weights and increased minimal hypertrophy is considered adaptive. Relative kidney weights and histopathological changes in the kidneys of F0 and F1 Cohort 1A males, and changes in the liver of F0 and F1 Cohort 1A males and females were not considered adverse within the context of this study and for males these were related to alpha 2u-globulin hydrocarbon nephropathy. The NOAEL for systemic toxicity in the F0 and F1 A adult animals was concluded to be the high dose of 300 mg/kg/day. Based on the results and absence of effects obtained in this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance of the F0 and F1 animals was the high dose of 300 mg/kg/day for males and females.