Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov 2019 - Jan 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
June 25, 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

The study design was as requested in ECHA decision TPE-D-2114453298-41-01/F:

"Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method OECD TG 443) in rats, oral route with the registered substance, specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation; [...]
- Cohort 1A (Reproductive toxicity); and
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation."


Test material

Constituent 1
Reference substance name:
Octene, hydroformylation products, high-boiling
EC Number:
271-237-7
EC Name:
Octene, hydroformylation products, high-boiling
Cas Number:
68526-89-6
Molecular formula:
Unspecified
IUPAC Name:
Octene, hydroformylation products, high-boiling
Details on test material:
Identification: Octene, Hydroformylation Products, High Boiling;
CAS 68526-89-6
Alternate Identification: Oxo oil N or Oxo oil HS 9
CAS Number 68526-89-6
Batch (Lot) No.: Tank25128022019
Expiration Date: 27 Feb 2021
Physical Description: Liquid
Density: 0.8611 g/mL
Storage Conditions: Ambient / Nitrogen Headspace

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Han Wistar rat was chosen as the animal model for this study as it is a rodent species accepted by regulatory agencies for reproductive toxicity testing.
The total number of animals used in this study was considered to be the minimum required to properly characterise the effects of the test item. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.
Sex:
male/female
Details on test animals or test system and environmental conditions:
On 12 Nov 2019, 115 male (including 3 spares) and 115 female (including 3 spares) Han Wistar CRL:WI (Han) rats were received from Charles River UK Limited, Margate, Kent, UK. At the initiation of dosing the animals were 5 to 8 weeks old and weighed between 109 and 180 g (males, target approximately 200 to 350 g) and 135 and 189 g (females, target approximately 125 to 225 g). The excursion from the target weight was considered likely an error in the protocol; the actual weights were in-keeping with rats approximately 5-6 weeks old.

Animal Identification
At study assignment, each animal was identified using a subcutaneously implanted electronic cylindrical, ‘glass-sealed’ microchip. On Postnatal Day (PND) 1 the pups were identified by toe markings (tattooing). Selected F1 animals retained post weaning and allocated to Cohort 1A, 1B or Cohort 1 (Surplus) were subcutaneously implanted and identified by an electronic cylindrical, ‘glass-sealed’ microchip.

Environmental Acclimation
The F0 animals were allowed to acclimate to the Test Facility rodent toxicology accommodation for a period of 13 days before the commencement of dosing.

Selection, Assignment, Replacement and Disposition of Animals
Cages were racked by treatment group and vertically throughout the rack. Control animals were housed on a separate rack to test item groups which were housed on the same racks by group.
During the week before the commencement of dosing, individual body weights were checked to ensure all F0 animals were within ± 20% of the mean weight of each sex. The spare animals (3 males and 3 females), were not required and were returned to Test Facility stock.
In Cohort 1B, low dose group, only 22 pups per sex were selected rather than 25 as per the protocol.

Husbandry
Husbandry practices and environmental enrichment were carried out as per Test Facility SOPs and protocol. Each batch of diet, bedding, and all environmental enrichment items were supplied with a Certificate of Analysis. Water from the public supply is routinely analysed for quality (including microbiological burden). It was considered that there were no contaminants in any of these materials that influenced the outcome of this study.

Housing
Animals were initially socially housed 2 or 3 per cage by sex (F0 generation) or later between 2-4 per cage by sex (for the F1 generation) in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms.
Bedding material was sterilised white wood shavings.
A few days prior to mating, F0 males were transferred to individual cages with solid bottoms. F0 females were transferred to these cages for mating.
Mated females were transferred to individual solid bottomed cages. White paper tissue was supplied as nesting material from Gestation Day (GD) 20. They were retained in this type of cage until termination.
On a suitable day after completion of mating, the males were re-housed with their original cage mates.

Environmental Conditions
Temperatures of 18 to 23°C (target 19 to 23°C) with a relative humidity of 28 to 91% (target 40 to 70%) were maintained. A 12-hour light/12-hour dark cycle was maintained.
There were occasions where the target environmental conditions for temperature and humidity were not maintained. These occasions were transient and the health of the animals was unaffected on any occasion, therefore, these excursions were considered not to impact the outcome or integrity of the study.
At least ten air changes per hour were maintained in the animal rooms.

Food
SDS VRF-1 breeder diet was provided ad libitum throughout the study, except during designated procedures.

Water
The animals had access to water ad libitum from the public supply from water bottles which were changed as necessary throughout the course of the study.

Animal Enrichment
Animals were socially housed for psychological/environmental enrichment and were provided with items such as a device for hiding in and an object for chewing, except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments were documented in the study records and reviewed by the Study Director.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly, stored ambiently or delivered directly to the animal unit, and dispensed daily.
Details on mating procedure:
Mating Procedure – F0 Animals
Pairing was on a 1 male to 1 female basis. Females were housed with their llocated co-group male partner during the evening (after 5 pm) on Study Day 71, except Animal 2525F who was paired with a proven male on Study Day 72 due to the unscheduled death of this animal’s partner (Animal 2025M).
Vaginal lavages were taken early each morning from the day of pairing until mating had occurred and the stage of estrous observed in each vaginal lavage was recorded. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated GD 0.
The pairing period for each pair of animals was a maximum of 21 nights. If evidence of mating was not observed by the end of the pairing period, the female was separated from the male during the morning following the last night of pairing and treated as if mating had occurred during that night. Procedures for that female continued as if it had mated on the last night of pairing.
For each female the time taken to show a positive mating sign and the number of failed opportunities to mate (estrouses passed without a sign of mating) was evaluated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate top, middle, and bottom samples (middle only for control) were sent to the analytical laboratory for analysis. Triplicate top, middle, and bottom samples (middle only for control) were retained at the Test Facility as backup samples. Sample volumes were collected as follows; 2 mL into 4 mL volumetric flasks (control group), 2 mL into 10 mL volumetric flasks (low dose group), 1.66 mL into 25 mL volumetric flasks (mid dosed group) and 0.5 mL into 25 mL volumetric flasks (high dose group) and stored at ambient temperature.

All results were within acceptance criteria for concentration (mean sample concentrations within or equal to ± 15% of theoretical concentration) and homogeneity (relative standard deviation (RSD) of concentrations of ≤ 10%).
Duration of treatment / exposure:
The test or control item was administered once daily by oral gavage to appropriate animals.
The first day of dosing was designated as dosing Day 1 for each animal. The control and test item dosing formulations were stirred for at least 30 minutes before and continuously during dosing.

Treatment
F0 Males: 10 weeks prior to mating and throughout mating until the day before termination.
F0 Females: 10 weeks prior to mating, throughout mating and gestation until at least Lactation Day (LD) 21.
Cohort 1A: From PND 21 until the day before termination (at least PND91).
Cohort 1B Males: From PND 21 until the day before termination.
Cohort 1B Females: From PND 21 until the day before termination.
Cohort 1 (Surplus): Animals were not dosed.

Dosing Events
F0 Generation Animal 2025M was given a dosing holiday on Study Day 60 due to clinical signs and was sent for unscheduled euthanasia the following day.
Animal 2014M was given a dosing holiday on Study Day 106 due to convulsions observed at the pre-dose check. Dosing resumed the following day.
Animal 3525F was not dosed on LD 17 in error.
F1 Generation Animals 4724F and 4725F were not dosed until PND 24 in error.

The volume for each animal was determined on the most recent body weight measurement. The doses were given using a syringe with attached gavage cannula. F0 females that were found to be in the process of littering, or had recently littered, at the time of scheduled dose administration, were not dosed on that day and dosing re-commenced the following day for these animals.
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0: 28
F1A: 20
F1B: 25
Control animals:
yes, concurrent vehicle
Details on study design:
- Basis for dose level selection: A dose range finding study was conducted prior to the main study. Administration of the test substance was tolerated in Wistar (Han) rats at the limit dose level 1000 mg/kg/day without adversity over gestation, lactation and in the subsequent generation after direct dosing following weaning.

Examinations

Parental animals: Observations and examinations:
F0 Animals

Mortality/Moribundity Checks
Animals were observed twice daily, once at the start and once towards the end of the working day throughout the study for general health/mortality and moribundity.

Clinical Observations

Detailed Clinical Observations
Animals were subjected to detailed clinical observations at least once weekly, beginning Week -1. The animals were removed from the cage for examination. The examinations included, but were not limited to, changes in skin, fur, eyes, mucous membranes, palpebral closure, vocalisation, rearing, arousal, stains and autonomic activity (lacrimation, salivation, piloerection, unusual respiratory pattern). Changes in gait and posture, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour were assessed. These reflected normal procedures and no separate recordings were made for these specific signs.

Pre and Postdose Observations
Animals were observed once prior to dosing each day. During the first week of dosing, animals were observed immediately postdose, 0-1 hours postdose, 1-2 hours postdose and 2-4 hours postdose. After the first week of dosing, observations were reduced to immediately postdose and 0-1 hour postdose.
All the animals were examined for reaction to treatment. The onset, intensity and duration of these signs was recorded (if appropriate), with particular attention being paid to the animals during dosing and for the first hour after dosing.

Body Weights
Males were weighed weekly beginning Week -1. Females were weighed weekly beginning Week -1 until pairing for mating and then on GD 0, 7, 14 and 20 and on LD 1, 4, 7, 14 and 21. Females that completed parturition (and were considered fit for dosing) may have had a LD 0 body weight taken for the purposes of calculating an accurate dose volume. These weights are retained in the study records but have not been reported. Pups were weighed individually on PND 1, 4, 7, 14 and 21.
The final in-life body weight was recorded on the day of scheduled necropsy.

Food Consumption
Food consumption was quantitatively measured for both sexes weekly, beginning Week -1 until pairing for mating, and for the mated females on the GD periods 0 to 7, 7 to 14 and 14 to 20 and on LD periods 1 to 7, 7 to 14 and 14 to 21. Food consumption resumed being recorded weekly for the males from Study Day 79.

Water Consumption
Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles.


F1 Animals

Mortality/Moribundity Checks
Animals were observed twice daily, once at the start and once towards the end of the working day throughout the study for general health/mortality and moribundity.

Clinical Observations

Detailed Clinical Observations
Clinical Observations for Cohort 1A and 1B animals started nominally during the 4th week after the first litters being born (i.e. the animals were up to a maximum of 5 weeks old at the commencement of detailed observations). Once detailed observations started they were performed on the animals weekly.

Pre and Postdose Observations
Animals were observed once prior to dosing each day. During the first week of dosing, animals were observed immediately postdose, 0-1 hours postdose, 1-2 hours postdose and 2-4 hours postdose. After the first week of dosing, observations were reduced to immediately postdose and 0-1 hour postdose.
All the animals were examined for reaction to treatment. The onset, intensity and duration of these signs was recorded (if appropriate), with particular attention being paid to the animals during dosing and for the first hour after dosing.

Body Weights
Animals were individually weighed weekly, starting on a suitable day within one week of weaning of the majority of litters (Nominal Week 4).
The final in-life body weight was recorded on the day of scheduled necropsy.

Food Consumption
Food consumption was quantitatively measured weekly, starting on a suitable day within one week of weaning of the majority of litters (Nominal Week 4).

Water Consumption
Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles.

Clinical Pathology
Sample Collection
Blood was collected via the orbital sinus under non-recoverable isoflurane anaesthesia. Urine was collected in ascending animal order over 6 hours with absence of food and presence of water.
Animals were fasted prior to blood sampling. Samples were collected according to the following Table.


Animals Time Point Haematology Coagulation Clinical Urinalysis
Chemistry
F0 animals
(10 rats/sex/group) At Necropsy X X X -
F0 animals Last Week
(10 rats/sex/group) of Dosing - - - X
Cohort 1A animals At Necropsy
(up to 10 rats/sex/group) X X X -
Cohort 1A animals Last Week
(10 rats/sex/group) of Dosing - - - X
Unscheduled Before
euthanasia euthanasia X X X -
X = sample collected; - = not applicable

After collection, samples were transferred to the appropriate laboratory for processing.

Haematology
Blood samples (0.5 mL) were collected, transferred into tubes containing K2EDTA and analysed for the parameters specified below:
Red blood cell count
Haemoglobin concentration
Haematocrit
Mean corpuscular volume
Red blood cell distribution width
Mean corpuscular haemoglobin concentration
Mean corpuscular haemoglobin
Reticulocyte count (absolute)
Platelet count
White blood cell count
Neutrophil count (absolute)
Lymphocyte count (absolute)
Monocyte count (absolute)
Eosinophil count (absolute)
Basophil count (absolute)
Large unstained cells (absolute)

A blood smear was prepared from each haematology sample. Blood smears were labelled, stained, and stored. Blood smears were evaluated as required to confirm analyser results as per Test Facility SOPs.

Coagulation
Blood samples (0.5 mL) were collected, transferred into tubes containing 3.2% (w/v) trisodium citrate and processed for plasma, which was analysed for the parameters listed below:
Activated partial thromboplastin time
Fibrinogen
Prothrombin time
Sample quality

Clinical Chemistry
Blood samples (0.7 mL) were collected, transferred into tubes containing lithium heparin and processed for plasma, which was analysed for the parameters specified below:
Alanine aminotransferase
Aspartate aminotransferase
Alkaline phosphatase
Gamma-glutamyltransferase
Creatine kinase
Total bilirubin*
Urea
Creatinine
Calcium
Phosphate
Bile acids
Total protein
Albumin
Globulin
Albumin/globulin ratio
Glucose
Cholesterol
Triglycerides
Sodium
Potassium
Chloride
Sample quality

*Total bilirubin was <8.55μmol/L, therefore indirect and direct bilirubin were not measured.

Urinalysis
Urine samples were analysed for the parameters listed below:
Colour
Appearance/Clarity
Specific gravity
pH
Sediment
Volume
Protein
Glucose
Bilirubin
Ketones
Blood

Thyroid Stimulating Hormone (TSH) and Thyroxine (T4)
Blood Collection
Samples were collected according to the following table.
Animals Time Point TSH and T4
F0 animals
(10 rats/sex/group) Day of Necropsy X
F1 Culled offspring
(up to 4 rats/sex/litter,
where possible)* PND 4–At Necropsy X (analysed for T4 only)
Cohort 1A
(at least 10 rats/
sex/group) Day of Necropsy X
Cohort 1 (Surplus)
(10 rats/sex/group) Day of Necropsy X
X = sample collected
* Total of 25 pooled samples. Pooled samples were identified by their dam number.

Blood Collection – F0, Cohort 1A and 1 (Surplus) Animals
Blood (1 mL) was collected from the jugular vein from adult animals (F0 and Cohort 1A animals) or from the orbital sinus, under general anaesthetic without recovery (Cohort 1A surplus) within a short timeframe nominally between 8am and 10am on the morning of the day of necropsy. Animals were fasted prior to sampling.

Blood Collection – Culled PND 4 Pups
Blood samples were collected from available culled pups on PND 4 via cardiac puncture (under non-recoverable isoflurane anaesthesia) into uniquely labelled tubes, without anticoagulant. At least 0.5 mL of blood per litter (from up to
4 pups per sex, per litter, as necessary) was collected. Samples were collected from all litters at each dose level with sufficient numbers of culled pups. Samples were taken within a short timeframe on the morning of necropsy, nominally between 8am and 10am.

Sample Processing and Analysis
Blood samples were processed to serum. Samples were analysed for T4 and TSH using validated analytical procedures. T4 was measured on Advia Centaur CP Immunoassay System by using solid phase, competitive chemiluminescent enzyme immunoassays. TSH was measured by using a sold phase enzyme immunometric assay - ELISA kit manufactured by BioVendor, Cat. No. RTC007R.
Any residual/retained analytical samples were discarded.
Oestrous cyclicity (parental animals):
F0
Vaginal lavages were taken early each morning and the stages of estrous observed were recorded from 2 weeks prior to pairing (Study Day 57) until the day of detection of a copulatory plug in situ and/or of sperm in the lavage.
Vaginal smears were examined on the morning of necropsy to determine the stage of the estrous cycle to allow correlation with histopathology of the ovaries.

Cohort 1A
Vaginal lavages were taken early each morning and the stages of estrous
observed were recorded from the day after vaginal patency, continuing until 1 estrous cycle had been identified and then from PND 75 for at least 14 consecutive days. Vaginal smears were examined on the morning of necropsy to determine the stage of the estrous cycle on the day of necropsy.

Cohort 1B
Not monitored
Sperm parameters (parental animals):
F0 and Cohort 1A Males

Computer Aided Sperm Assessment (CASA)
From all F0 and Cohort 1A males only, the right cauda epididymis was placed in 0.3% BSA in Medium 199 as per Test Facility SOPs, and the sperm were allowed to “swim out” into the medium. An appropriate dilution of the sperm suspension was prepared and examined using a Hamilton Thorne sperm motility analyser.

Sperm Count and Morphological Analysis
The cauda epididymis was minced and suspended. Dilutions of this sperm suspension were counted using a haemocytometer to obtain a total sperm count which was expressed per cauda epididymis and per gram of cauda epididymis.
From all samples of the sperm suspension described above, a sperm smear was prepared and stained with eosin Y solution. For the control and the high dose group, at least two hundred sperm per animal were evaluated for morphological abnormalities using criteria described by Wyrobek and Bruce (1975).

Spermatid Count
The right testis was decapsulated and homogenised. The homogenate was sonicated to reduce tissue debris etc., if required. The number of homogenisation resistant spermatids in dilutions of this suspension was counted using a haemocytometer to obtain a total spermatid count which was expressed per testis and per gram of testis.
Litter observations:
Observations of F0 Females and Litters During Lactation
The females were allowed to litter normally. Any observed difficulty or prolongation of parturition was recorded. The day of birth of the litter (day on which first pups are born) was designated LD 0. The duration of gestation in days was calculated.
The numbers of live and dead pups born in each litter were recorded as soon as possible after completion of parturition on LD 0. The live pups were counted, sexed, weighed individually on PND 1 and examined for the presence of milk in the stomach and for any externally visible abnormalities daily up to and including PND 4. On PND 4, litter size was standardised to 8 pups, where possible 4 males and 4 females, by culling of extra pups via random selection. Extra pups were necropsied according to Section 4.15. Where 4 males or 4 females were not available, extra pups of the opposite sex were retained to ensure a total number of 8 pups. When the total number of pups in a litter on PND 4 was ≤ 8 pups, no litter size adjustment occurred. From PND 5, the total live pups were counted daily, and were sexed and examined for abnormality again on PND 7, 14 and 21. These pups were weighed individually, on PND 1, 4, 7, 14 and 21.
Where practicable, any pups that were found dead or were killed during lactation were sexed and appropriately examined as above. Any externally abnormal decedent pup was preserved in tissue fixative (e.g. 10% Neutral Buffered Formalin); externally normal pups were discarded.
Deficiencies in maternal care was recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding. White paper tissue was supplied to each mother for incorporation in the nest. This was replaced when it had become soiled.
The following females failed to produce a litter by their expected GD 24 and were sent for necropsy; Animals 1501F, 3528F, 4503F, 4512F and 4516F.
Animal 4509F had not shown any indication of mating and did not appear pregnant but had been acyclic and was displaying a high number of days in estrous. Therefore this animal was sent for scheduled euthanasia on Study Day 103 with the stage of estrous measured, samples for TSH and T4 collected and necropsied.

Pre-Weaning Physical Development of F1 Pups
Assessment of ano-genital distance and an individual body weight were measured for both sexes on PND 1. Ano-genital distance was measured using a calibrated calliper from the caudal margin of the genital tubercule. Measurements were recorded to the nearest 0.01 mm.
Nipple retention was assessed in males on PND 13.

Weaning and Selection of F1 Animal’s for Cohorts 1A and 1B and 1 (Surplus)
From each group, up to 85 males and 85 females were selected at random on PND 20 and identified on that day for post-weaning assessments, nominally by selecting up to 4 males and 4 females from each litter, where possible. Where fewer than 85 pups were weaned, the necessary additional animals were obtained by selecting an additional pup from appropriate litters; these appropriate litters would normally be selected arbitrarily but attention was paid to the retention of as wide a genetic pool as possible.
These pups were removed from their mother on PND 21 and housed in their new cages. Pups that were not selected for post-weaning assessments (Cohorts 1A and 1B or up to 20 surplus pups per sex per group) remained with their mother until termination.

Assessment of Sexual Maturation (Cohorts 1A and 1B)
Commencing at PND 28, females were examined daily for vaginal opening. The day on which the vagina became open was recorded, as was the body weight on that day. Commencing at PND 35, males were examined daily for balano-preputial separation. The day on which separation occurred was recorded, as was the body weight on that day.

Clinical Pathology
see examinations parental animals
Postmortem examinations (parental animals):
Terminal Procedures
Terminal procedures are summarised in the tables in section "Any other information on materials and methods incl. tables"

Unscheduled Deaths
When necessary for humane reasons, animals were euthanised as per Test Facility SOPs. The body weight was recorded and samples for evaluation of clinical pathology parameters and hormone analysis were obtained if possible. These animals underwent necropsy, and specified tissues were retained. When necessary, animals were euthanised without exsanguination and refrigerated before necropsy to minimise autolysis.

Scheduled Euthanasia
Animals 10 days of age or more, were euthanised by exposure to an increasing level of carbon dioxide, had a terminal body weight recorded, followed by exsanguination. Animals less than 10 days old were killed by an overdose of anaesthetic (for example sodium pentobarbitone) or by cervical dislocation followed by exsanguination. When possible, the animals were euthanised in a rotating order across dose groups such that similar numbers of animals from each group, including controls were necropsied throughout the day. Animals were fasted overnight before their scheduled necropsy.

Litter/Pup Examinations
Offspring Found Dead or Euthanised Before PND 14
Where practicable, these animals were sexed and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any externally abnormal pups were fixed in 10% formalin for optional further examination. Externally normal pups were discarded.

Offspring Found Dead or Euthanised After PND 14
These animals were subjected to a gross necropsy. An external examination was followed by an inspection of the cranial, thoracic and abdominal contents. Representative samples of any abnormal tissues were taken and fixed in neutral buffered 10% formalin. These carcasses were then discarded.

Scheduled Necropsy of Non-Selected Pups PND 4
Non-selected pups on PND 4 were necropsied. This consisted of external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved in 10% formalin or other appropriate fixative. The carcasses were discarded.

Necropsy
Adults and Surplus Pups
F0, Cohort 1A and 1B animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
The reproductive tracts of all F0 females were examined for signs of implantation with the number of any implantation sites being recorded. The total number of corpora lutea graviditatis was recorded for each female. The uteri of all non-pregnant females were fixed in buffered formalin and stained using 10% (aq) (v/v) ammonium sulfide solution and examined for implantation sites.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Offspring
Where practicable, offspring found dead or killed (unscheduled) were sexed internally, and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any abnormal pups were, where practicable, fixed in 10% formalin or methylated ethyl alcohol, as appropriate, for optional further examination. Externally normal pups were discarded.

Organ Weights

The organs identified in tables "Tissue Collection and Preservation" in section "Any other information on materials and methods incl. tables" were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organs were weighed before fixation unless otherwise noted. Organ to body weight percentages (using the terminal body weight) and organ to brain weight percentages were calculated.

Tissue Collection and Preservation
Summarised in the tables in section "Any other information on materials and methods incl. tables"

Immunophenotyping Sample Collection, Processing, and Analysis
Spleen samples (approximately half of the spleen) were taken from 10 rats per sex per group from the F1 Cohort 1A adults at necropsy and were collected into tubes containing RPMI (Roswell Park Memorial Institute) medium and stored immediately on wet ice. The remainder of the spleen was preserved in 10% neutral buffered formalin. The samples were transferred on the day of collection to the Test Facility analytical laboratory where upon receipt at the flow cytometry laboratory, the samples were stored on wet ice and were analysed using a validated analytical method (Campbell, 2019, 996094).

Histology
Tissues identified in tables "Tissue Collection and Preservation" in section "Any other information on materials and methods incl. tables" were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin.
In Cohort 1A, additional slides of the testes were prepared to examine staging of spermatogenesis for all Group 1 and Group 4 males, as well as any Group 2 or Group 3 males suspected of being infertile, or which died before mating. The testes were processed, sectioned at 5 micrometers, and stained with periodic acid–Schiff (PAS)/haematoxylin.

Histopathology
Histopathological evaluation was performed by a board-certified veterinary pathologist.
A detailed qualitative examination of the testes 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 were noted.

Bone Marrow Smear Evaluation
Two bone marrow smears were collected from the femur at scheduled and unscheduled necropsies (for possible examination). Bone marrow smears were allowed to air dry, fixed in methanol and as soon as practical after necropsy, were stained using May-Grunwald-Giemsa. Bone marrow smears were not evaluated.

Ovarian Follicle Counts (Cohort 1A)
The examination of the ovaries included quantification of the primordial and growing follicle and the confirmation of the presence or absence of the corpora lutea.
For Cohort 1A only, each ovary, 5 step serial sections at circa 20 µm (a small amount into the formalin-fixed ovary e.g. 25 µm before the next section was taken) were taken. One section was stained with haematoxylin and eosin for routine evaluation and 5 sections stained for immunohistochemistry (IHC) using proliferating cell nuclear antigen (PCNA) marker for enumeration of primordial and primary follicles.
Postmortem examinations (offspring):
See section "postmortem examinations (parental animals)"
Statistics:
Inferential Statistical Methods
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and have been reported at the 1% and 5% levels, unless otherwise noted.

The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Analyses were performed according to the matrix below when possible, but exclude any group with less than 3 observations.

Statistical Matrix

Variables for Inferential Analysis** Statistical Method
Parametric/ Non-Parametric
Body Weight* X
Body Weight Gains* X
Food Consumption* X
Haematology Variables X
Coagulation Variables X
Clinical Chemistry Variables X
Urinalysis Variables X
Thyroid Hormone Variable X
Organ Weights X
Organ Weight relative to Body Weight X
Organ Weight relative to Brain Weight X
Ovarian Scoring (total number of
oocytes per animal)*** X

X = performed
* Excludes animals not pregnant from the gestation phase summarisation and statistical analysis.
** Excludes animals euthanised preterminal from summarisation and statistical analysis.
*** Parameter was analysed using SAS v9.4.

Parametric/Non-parametric
Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Reproductive indices:
See "Reproductive and Survival Indices" in section "Any other informations on materials and methods incl. tables".
Offspring viability indices:
See "Reproductive and Survival Indices" in section "Any other informations on materials and methods incl. tables".

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Administration of the test item was associated with a dose-related increase in the incidence of salivation and ploughing behaviour post-dosing in both the F0 and F1 animals at all dose levels but particularly at 300 and 1000 mg/kg/day. These findings were considered to be most likely a result of palatability of the dosing formulations and were not evidence of adverse toxicity.
All other clinical observations occurred at low frequency, did not follow a dose-related trend or are commonly observed in this species, consequently they were considered unrelated to administration of the test item, including a convulsion observed on one occasion in one F0 female animal at 1000 mg/kg/day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were two unscheduled deaths during the course of this study, none considered test item-related. F0 animals 2014M and 2025M (both receiving 100 mg/kg/day) were euthanised prematurely on Days 129 and 61, respectively.
The cause of death of Animal 2014M was not established from gross or histological evaluation, although it had displayed convulsions on Day 4 and from Day 106 and respiratory abnormalities on Day 106. The animal had no noteworthy or test item-related histological findings.
Animal 2025M had displayed signs of respiratory abnormalities and swelling of the neck before euthanasia. The animal had no test item-related gross or histological findings. At necropsy, there was oesophageal dilatation. Histologically, noteworthy findings comprised mild degeneration of thyroid follicular cells, mild mononuclear inflammatory cell infiltration in the heart, minimal mixed inflammatory cell infiltration in the lung, including in the terminal bronchioles, moderate degeneration in the epithelium of the ileum (that may have occurred shortly before death) and evidence of stress (cortical hypertrophy in the adrenal and decreased cellularity in the thymus and mandibular lymph node). These findings did not enable a cause of death to be established.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In male animals body weight gains were slightly lower, leading to statistically significant differences in group mean body weights at 1000 mg/kg/day being observed. However, statistical significance was intermittent (on Day 64, 92, 99 and 127 only) with average differences being small in magnitude (never exceeding 10%).
Conversely, in female animals prior to mating, administration of the test item was associated with higher weight gains. Similarly, these gains led to statistically different group mean body weights at 1000 mg/kg/day that were intermittent (noted on Day 22-57 and 71) and were small (<10%). In lactation minor differences on Lactation Day (LD) 7 or 14 attained statistical significance at 1000 mg/kg/day, but were small (<10%) and were not toxicologically relevant.
Due to the effects on body weight and body weight gains being minor and not consistent between males and females, it was considered that there was no toxicologically relevant effect of the test item on animal body weight in the F0 generation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In general, a slightly higher food consumption was observed in both male (up to 15%) and female (prior to mating; up to 20%) animals administered 1000 mg/kg/day. In female animals a slightly higher food consumption was also evident compared to control animals over gestation (up to 15%), but a similar food consumption was observed in lactation.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No intergroup differences were noted.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects on haematology parameters following administration of the test item.

Coagulation
A trend in lower concentration of fibrinogen was observed in male animals in the F0 generation, however as the differences were not statistically significant at any dose level and a trend was not repeated in the F0 female or F1 generation males. In the F1 generation females there were statistically significant lower concentrations of similar magnitudes, however there was no dose-related trend. The fibrinogen findings were considered to be incidental and not test item related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In F0 male animals only at 1000 mg/kg/day, a statistically significant difference in sodium concentration was observed, however the magnitude of the difference to control (1.01X) was considered insufficient to be biologically relevant.
A statistical difference was noted in bile acids of F0 female animals at 300 mg/kg/day and F1 male animals at 1000 mg/kg/day and in F1 female animals there was an apparent dose-related trend, (although not statistically significant). It is noted that concentrations of bile acids in this study show very high levels of variability and do not show reproduceable differences and any change is unlikely to be attributed to the test item.

Differences in some other cases attained statistical significance, however they were either not part of a dose-related trend and/or were minor and of insufficient magnitude to be considered toxicologically relevant.
Endocrine findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related effects on urinalysis parameters. All values were considered to be within normal biological variation.
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):
Test Item-related Microscopic Findings
In the liver, there was minimal or mild centrilobular hepatocyte hypertrophy at all doses in males (with a dose-level related trend in incidence and severity) and at 1000 mg/kg/day in females.
In the thyroid, compared with controls, there was increased minimal to moderate follicular cell hypertrophy at 1000 mg/kg/day and in 1 animal at 100 mg/kg/day in males and at all doses in females.
In the kidney, compared with controls, there was increased minimal or mild hyaline droplet accumulation at all doses in males only (with a dose-level related trend in incidence and severity). This was accompanied by minimal or mild granular medullary casts in some affected males at 1000 mg/kg/day.

Additional Microscopic Findings
Other microscopic findings observed were of the nature commonly observed in this strain and age of rat, or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item-related.

The small number of protocol-required tissues that could not be examined were spread throughout the treatment groups and therefore considered not to have impacted the quality or integrity of the study. In F0 females, the olfactory bulbs of the brain were not available for histological evaluation in 23/28 control animals and 22/28 animals at 1000 mg/kg/day. This was considered not to have impacted the quality or integrity of the study because the olfactory bulbs were available, where the brain was required to be examined histologically, in the other F0 females and in all of the F0 males, and in all of the F1 Cohort 1A animals, and because there were no test item-related changes in any parts of the nervous system evaluated.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
There were no evident changes to the estrus cycle of female animals in the test item dosed groups.
Animal 4509 (which was not pregnant) was noted as being acyclic, with very prolonged periods of estrous. Given the absence of any indication of estrous cycle disruption in the other female rats at 1000 mg/kg/day it was considered likely that Animal 4509 was an anomalous finding not associated with administration of the test item.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no test item related effects on sperm motility parameters (% Motile, % Progressive motility and straight line velocity), morphological abnormalities, epididymal sperm or testicular spermatid reserves, in either the F0 or F1 males.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no test item related effects on mating, fertility or pregnancy rates. Gestation length was normal in all dose groups.
There were no effects on litter survival with birth, live birth, viability (Days 0-4) and lactation (Day 4-21) indices similar over all dose groups. Maternal and pup observations indicated there were no changes in maternal care.
There were no effects on pup body weight at any dose level.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Administration of the test item was associated with a dose-related increase in the incidence of salivation and ploughing behaviour post-dosing in both the F0 and F1 animals at all dose levels but particularly at 300 and 1000 mg/kg/day. These findings were considered to be most likely a result of palatability of the dosing formulations and were not evidence of adverse toxicity.
All other clinical observations occurred at low frequency, did not follow a dose-related trend or are commonly observed in this species, consequently they were considered unrelated to administration of the test item.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 Cohort 1B animal 2218M (receiving 100 mg/kg/day) was euthanised prematurely on PND 94. It had displayed respiratory abnormalities before euthanasia. No abnormalities were recorded at necropsy and there was no histological evaluation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In Cohort 1A, male animals administered 1000 mg/kg/day had a statistically significant lower body weight gain overall (-13% relative to control). A statistically significant lower body weight was observed compared with the control, from around Week 7. The differences were generally small and at the end of the dosing period animals at 1000 mg/kg/day were on average 11% lighter than control. In Cohort 1A, the statistically significant difference in body weight at 300 mg/kg/day in Week 4 was considered artefactual due to lack of dose response. In Cohort 1B, smaller effects on body weight were observed, and were significant only at Week 12-13.
In female animals (Cohort 1A and 1B) no effects on body weight were observed.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effect on food consumption was observed.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No intergroup differences were noted.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects on haematology parameters following administration of the test item.

Differences in some cases attained statistical significance (Cohort 1A only), however they were either not part of a dose-related trend and/or were minor and of insufficient magnitude to be considered toxicologically relevant.

Coagulation
A trend in lower concentration of fibrinogen was observed in male animals in the F0 generation, however as the differences were not statistically significant at any dose level and a trend was not repeated in the F0 female or F1 generation males. In the F1 generation females there were statistically significant lower concentrations of similar magnitudes, however there was no dose-related trend. The fibrinogen findings were considered to be incidental and not test item related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In F0 male animals only at 1000 mg/kg/day, a statistically significant difference in sodium concentration was observed, however the magnitude of the difference to control (1.01X) was considered insufficient to be biologically relevant.
A statistical difference was noted in bile acids of F0 female animals at 300 mg/kg/day and F1 male animals at 1000 mg/kg/day and in F1 female animals there was an apparent dose-related trend, (although not statistically significant). It is noted that concentrations of bile acids in this study show very high levels of variability and do not show reproduceable differences and any change is unlikely to be attributed to the test item.

Differences in some other cases attained statistical significance, however they were either not part of a dose-related trend and/or were minor and of insufficient magnitude to be considered toxicologically relevant.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on urinalysis parameters. All values were considered to be within normal biological variation.
Urine pH attained a statistically significant difference at 300 mg/kg/day (Females, Cohort 1A); this was considered spurious due to lack of dose response.
Sexual maturation:
no effects observed
Description (incidence and severity):
There was no effect on sexual maturation (vaginal opening or preputial separation), or on the time to commencement of the estrous cycles and first estrous cycle length.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
In male animals, anogenital distance was measured to be slightly longer at 1000 mg/kg/day (including when corrected for body weight), however this was considered not toxicologically relevant as it was longer and not shorter; furthermore the anogenital distance - body weight ratio was well within historical control data.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No males were found to have retained nipples on PND 13.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1 Cohort 1A Animals

Test Item-related Organ Weight Differences
Liver weight was higher than controls at 1000 mg/kg/day in both sexes by up to 21% for absolute weight. This correlated histologically with centrilobular hepatocyte hypertrophy. Thyroid weight was higher than controls at all dose levels in both sexes by up to 19% for absolute weight, showing some dose level-relationship. This correlated histologically with increased follicular cell hypertrophy. Kidney weight was higher than controls at 1000 mg/kg/day in males by 10% for weight relative to body weight. This correlated histologically with hyaline droplet accumulation.

Additional Organ Weight Differences
Kidney weight was higher than controls at 1000 mg/kg/day in females by 6% for weight relative to body weight. Because of the small difference from controls and lack of a histological correlate in this sex, this was considered not test item-related. Other organ weight differences, including those with statistical significance, were also considered not test item- related, as they had no histological correlate.

F1 Cohort 1B Animals

Test Item-related Organ Weight Differences
Liver weight was higher than controls at 1000 mg/kg/day in both sexes by up to 23% for absolute weight. Thyroid weight was higher than controls at 1000 mg/kg/day in males and at 100 and 1000 mg/kg/day in females, by up to 17% for absolute weight. Kidney weight was higher than controls at 1000 mg/kg/day in males by 15% for weight relative to body weight.

Additional Organ Weight Differences
Kidney weight was higher than controls at 1000 mg/kg/day in females by 9% for absolute weight and weight relative to body weight. This and other organ weight differences, including those with statistical significance, were considered not test item-related because the organs concerned (thymus and adrenals in both sexes and kidneys in females) did not have correlating histological findings in the sexes concerned in F1 animals in Cohort 1A.

F1 Cohort 1 Surplus Animals (Postnatal Days 21-24)
There were no test item-related organ weight differences. Kidneys were not required to be weighed in these animals.

Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 Cohort 1A Animals

There were no test item-related gross findings.

The only frequent gross findings were prominent lobular architecture in the liver (that did not occur in a dose level-related pattern and was indeed absent at 1000 mg/kg/day) and dark discoloration in the thymus and some lymph nodes and discoloration of the lung, that correlated histologically with agonal haemorrhage (or macrophage foci in the lung) or had no correlate and also did not occur in a dose level-related pattern.

Other gross findings observed were of the nature commonly observed in this strain and age of rat, or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item-related.

F1 Cohort 1B Animals

There were no test item-related gross findings.

The only frequent gross findings were prominent lobular architecture in the liver and dark discoloration in the thymus, that did not occur in a dose level-related pattern.
Other gross findings observed were of the nature commonly observed in this strain and age of rat, or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item-related.

F1 Cohort Surplus Animals

There were no test item-related gross findings.

One control female had a mass in the liver and one male pup at 300 mg/kg/day had dark discoloration of the thymus.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 Cohort 1A Animals

Test Item-related Microscopic Findings
In the liver, there was minimal or mild centrilobular hepatocyte hypertrophy at
1000 mg/kg/day in males and at 300 and 1000 mg/kg/day in females (minimal only, with a dose-level related trend in incidence).
In the thyroid, there was minimal follicular cell hypertrophy at all doses in males and females, with a dose-level related trend in incidence.
In the kidney, compared with controls, there was increased hyaline droplet accumulation at all doses in males only (minimal or mild, with a dose-level related trend in incidence and severity). This was accompanied by minimal or mild granular medullary casts in some affected males at 1000 mg/kg/day, although one male with mild casts did not have hyaline droplet accumulation.
Other microscopic findings observed were of the nature commonly observed in this strain and age of rat, or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item-related.

Other effects:
no effects observed
Description (incidence and severity):
Sperm Evaluation
There were no test item related effects on sperm motility parameters (% Motile, % Progressive motility and straight line velocity), morphological abnormalities, epididymal sperm or testicular spermatid reserves, in either the F0 or F1 males.

Ovarian Follicle Count
There was a no effect on the ovarian follicle count.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Immunophenotyping
The results demonstrated no dose related effects on any of the immune cell populations analysed. Although results showed a slightly lower percentage of T cells and B cells when compared to the concurrent control, these were not considered a direct test item or dose dependent effect.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
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

Thyroid Hormone Analysis


In general there were higher concentrations of Thyroxine (T4) observed in both male and female animals in the F0 generation at all dose levels and in the F1 animals at PND 21-24 at 1000 mg/kg/day compared with the concurrent control without attaining statistical significance in the F0 generation. Still, all F0 male animals were well within historical control data (HCD). The F0 female test group animals were slightly above HCD. However, the F0 female control group animals were already close to the upper HCD limit. HCD available consisted only of data from four studies and still it has (and hence T4 values in general) a high variability.


The change observed in the surplus F1 animals at PND 21-24 at 1000 mg/kg bw/day is furthermore not reflected in the F1 pups and PND 91-93 animals. There were no differences in the concentration of T4 in the unselected F1 pups culled on PND 4 and the PND 91-93 F1 generation (Cohort 1A), was, besides a weak negative trend in male animals, also well within historical control data.


Concentrations of TSH were generally low, with a proportion of samples below the reportable range.


Altogether, the changes reported are lacking a clear pattern and are considered to be of no toxicological relevance.


 


Thyroxine (T4) Concentrations (ng/mL)






























































Generation/Sex



Group 1


(0 mg/kg/day)



Group 2


(100 mg/kg/day)



Group 3


(300 mg/kg/day)



Group 4


(1000 mg/kg/day)



Historical control data


(n=4)



F0 Generation – Males



36.95



42.09


(+14%)



42.30


(+14%)



45.16


(+22%)



39.60 – 49.44



F0 Generation – Females



33.23



35.59


(+7%)



36.97


(+11%)



38.06


(+15%)



23.20 – 34.70



F1 Surplus – Males (PND 21-24)



38.04



37.98


(0%)



43.73


(+15%)



46.99** (+24%)



28.85 – 43.93



F1 Surplus – Females (PND 21-24)



39.24



38.65


(-2%)



39.66


(+1%)



46.32* (+18%)



27.51 – 34.43



F1 Generation – Males Cohort 1A



46.16



42.27


(-8%)



42.07


(-9%)



40.46


(-12%)



39.60 – 49.44



F1 Generation – Females Cohort 1A



28.03



30.41


(+8%)



31.74


(+13%)



30.46


(+9%)



23.20 – 34.70



 


*     p ≤ 0.05


** p ≤ 0.01


 


 

Applicant's summary and conclusion

Conclusions:
Administration of the test substance by once daily oral gavage was well tolerated in male and female rats at levels up to the limit dose of 1000 mg/kg/day, with no effects on development and reproductive function.
Executive summary:

The objective of this study was to determine the potential toxicity of Oxooel 9N, when given by oral administration to rats to assess the reproductive function in adult animals and their offspring. This study was designed to provide an evaluation of the pre- and postnatal effects of chemicals on development as well as a thorough evaluation of systemic toxicity in pregnant females, lactating females, young and adult offspring. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided information about the effects of the test substance on the integrity and performance of the adult male and female reproductive systems.


The study design was as follows (Control/vehicle = Corn Oil):


Experimental Design – F0 Animals

















































Group No.



Dose Level (mg/kg/day)



Dose Volume


(mL/kg)



Dose Concentration


(mg/mL)



Number of Animals



Males



Females



1



0 (Control)



4



0



28



28



2



100



4



25



28



28



3



300



4



75



28



28



4



1000



4



250



28



28



 


Experimental Design – F1 Animals Cohort 1A

















































Group No.



Dose Level (mg/kg/day)



Dose Volume (mL/kg)



Dose Concentration (mg/mL)



Number of Animals



Males



Females



1



0 (Control)



4



0



20



20



2



100



4



25



20



20



3



300



4



75



20



20



4



1000



4



250



20



20



 


Experimental Design – F1 Animals Cohort 1B

















































Group No.



Dose Level (mg/kg/day)



Dose Volume (mL/kg)



Dose Concentration (mg/mL)



Number of Animals



Males



Females



1



0 (Control)



4



0



25



25



2



100



4



25



22



22



3



300



4



75



25



25



4



1000



4



250



25



25



 


Experimental Design – F1 Animals Cohort 1 (Surplus)







































 


Group No.



Parental Dose Level (mg/kg/day)



Number of Animalsa



Males



Females



1



0 (Control)



20



20



2



100



20



20



3



300



20



20



4



1000



20



20



a Cohort 1 (Surplus) animals were not dosed.


The following parameters and end points were evaluated in this study: clinical observations, body weights, food consumption, estrous cycles, mating performance, fertility indices, duration of gestation and overall litter performance, litter survival indices, litter and pup weights, pre-weaning physical development of F1 pups, assessments of sexual maturation of F1 animals, clinical pathology parameters (haematology, coagulation, clinical chemistry, and urinalysis), thyroid stimulating hormone (TSH) and thyroxine (T4) analysis, gross necropsy findings, immunophenotyping analysis, organ weights, sperm evaluation, ovarian follicle counts and histopathological examinations.


Administration of the test item was associated with ploughing behaviour and salivation post dosing.


In F0 animals, histologically, in the liver there was minimal or mild centrilobular hepatocyte hypertrophy at all doses in males and at 1000 mg/kg/day in females, with a higher liver weight at 1000 mg/kg/day in both sexes. In the thyroid, there was increased minimal to moderate follicular cell hypertrophy at 1000 mg/kg/day and in 1 animal at 100 mg/kg/day in males and at all doses in females, with a higher thyroid weight in males at 300 and 1000 mg/kg/day and in females at 1000 mg/kg/day. In the kidney, there was increased minimal or mild hyaline droplet accumulation at all doses in males only, accompanied by minimal or mild granular medullary casts in some affected males at 1000 mg/kg/day. Kidney weight was higher at 1000 mg/kg/day in males.


In F1 Cohort 1A animals, histologically, in the liver, there was minimal or mild centrilobular hepatocyte hypertrophy at 1000 mg/kg/day in males and at 300 and 1000 mg/kg/day in females, with a higher liver weight at 1000 mg/kg/day in both sexes. In the thyroid, there was increased minimal follicular cell hypertrophy and a higher thyroid weight at all doses in males and females. In the kidney, there was increased minimal or mild hyaline droplet accumulation at all doses in males only, and minimal or mild granular medullary casts in males at 1000 mg/kg/day. Kidney weight was higher at 1000 mg/kg/day in males.


In F1 Cohort 1B animals, that were not examined histologically, liver weight was higher than controls at 1000 mg/kg/day in both sexes and thyroid weight was higher than controls at 1000 mg/kg/day in males and at 100 and 1000 mg/kg/day in females. Kidney weight was higher at 1000 mg/kg/day in males.


F1 Cohort 1 Surplus animals (euthanised on postnatal day {PND} 21-24 without being actively dosed) did not have test item-related organ weight differences in the liver or thyroid.


There were no other relevant effects observed on this study.


In conclusion, administration of Oxooel 9N by once daily oral gavage was well tolerated in male and female rats at levels up to the limit dose of 1000 mg/kg/day, with no effects on development and reproductive function.


Adaptive changes in pathology were observed in liver (males and females), thyroid (males and females). Differences in kidney pathology (males only) were also observed. Based on the minimal or mild severity of the findings the effects were considered not adverse.


Centrilobular hepatocyte hypertrophy in the liver often results from induction of microsomal metabolic enzymes, which is an adaptive response to xenobiotics (Maronpot et al 2010, Hall et al 2012). At the minimal or mild degree seen in this study, it was not injurious. In some affected females in this study, the enlarged hepatocellular cytoplasm was also slightly more eosinophilic than normal. Induction of hepatic microsomal metabolic enzymes in rats is liable to cause adaptive thyroid follicular cell hypertrophy as it increases thyroid hormone metabolic clearance, which results in a compensatory increase in thyroid hormone secretion. Rats are much more sensitive than humans to this effect on the thyroid gland (Zabka et al 2011).


In the kidney, hyaline droplet accumulation can be induced in male rats by xenobiotics (including some hydrocarbons) that disrupt the lysosomal catabolism of alpha-2u-globulin in the renal proximal tubules (Alden 1986, Frazier et al 2012). Because this globulin is synthesised in especially large amounts by the male rat liver and is not present in humans, the finding is not considered relevant to humans. In affected rats, debris from the droplets may be visible as granular casts at the junction of the outer and inner stripes of the outer medulla, where the S3 segment of the proximal tubule narrows into the descending limb of Henle, as seen in this study.


The presence of alpha-2u-globulin was demonstrated with Mallory-Heidenhain’s staining in a previous 90-Day study in Wistar rats performed (BASF SE/Evonik, 2014).


Based on the results of this extended one generation reproductive toxicity study (Cohort 1), the following no-observed-adverse-effect level (NOAEL) of Oxooel 9N were established:


Parental toxicity (F0 and F1): 1000 mg/kg/day


Reproductive NOAEL (F0): 1000 mg/kg/day


Post-Natal Developmental NOAEL (F0 and F1): 1000 mg/kg/day.