Vinyl neononanoate

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Justification for study design:

Required by ECHA under decision # TPE-D-2114449877-29-01/F.

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

Test item: Vinyl Neononanoate
Test item identity (including alternative names): VEOVA Vinyl Ester Monomer 9, Vinyl Neononanoate (Veova 9)
Intended use: Industrial chemical
Appearance: Clear, colorless liquid
Storage conditions: Ambient temperature (15 to 25 ºC), in the dark
Supplier: Sponsor
Batch number: RV8I0020
Expiry date: 09 September 2021
Purity: 100%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
Animals
Strain/Species: Crl:CD(SD) rat.
Supplier: Charles River (UK) Ltd.
Number of animals ordered: 106 males and 106 females; unrelated (males not related to females) Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization: Five days before commencement of treatment.
Age of the F0 animals at the start of treatment: Males 26 to 33 days old. Females 33 to 39 days old.
Weight range of the F0 animals at the start of treatment : Males 83 to 112 g. Females 103 to 143 g.
Allocation to Treatment Groups (F0 Generation)
Allocation: After a period of acclimatization. Animals showing signs of ill health were excluded. Animals at the extreme of the weight range were not
selected if alternatives were available. At commencement of the study the body weight of animals did not exceed ±20% of the mean for each sex.
Selection of Offspring to Form F1 Generation
Selection: On Day 18 to 21 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. Where possible, three males and three females were selected from each selected litter and were allocated to each of the four cohorts. If more were required, up to four males and four females were selected from each selected
litter. Selected animals were microchipped on Day 18-21 of age and separated from littermates on Day 21 of age. 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.
Identification
Identification of animals: Unique for each F0 animal and selected F1 offspring within study. All pre-weaning offspring were numbered individually within each litter on Day 1 of age.
Method: Microchip (F0 generation and selected F1 generation). Toe tattoo (pre-weaning offspring).
Identification of cages: Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).
Animal Replacement
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Animal Care and Husbandry
Environmental Control
Animal facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24°C and 40-70%. There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.
Animal Accommodation
Cages: 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.
Cage distribution: The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Study period Number of animals/cage Cage material Cage flooring
Male Female
Pre-pairing (acclimatization Up to 4 Up to 4 Polycarbonate Solid
and after selection) @ polycarbonate

Pairing 1 : 1 Polycarbonate/ Stainless steel grid
Stainless steel grid

Males to termination Up to 4 - Polycarbonate Solid
polycarbonate
Females after mating (from - 1 Polycarbonate Solid
Day 0 after mating) polycarbonate

Females during littering (from - 1 + litter Polycarbonate Solid
Day 20 after mating) polycarbonate

Females to termination (after - Up to 4 Polycarbonate Solid
weaning) polycarbonate

Offspring maturation (from Litter Polycarbonate Solid
weaning until selection) polycarbonate

@ Except when animals were separated into single housing overnight prior to urine collection (Section 3.6.12) or functional observational battery testing (Section 3.7.4). In both cases, following the completion of the investigations the animals were returned to group housing. See Section 4

Environmental Enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage
throughout the study (except for F0 females during lactation, when F1 Cohort 1A animals were separated into single housing overnight for urine collection and F1 Cohort 2A animals were separated into single housing overnight prior to functional observational battery testing) and replaced when necessary.
For F0 females chew blocks were returned on Day 21 of lactation after weaning of offspring.
Plastic shelter: Provided to each cage throughout the study (except during F0 pairing, for F0 females during lactation, when F1 Cohort 1A animals were separated into single housing overnight for urine collection and F1 Cohort 2A animals were separated into single housing overnight prior to functional observational battery testing) and replaced at the same time as the cages.
For F0 females shelters were returned on Day 21 of lactation after weaning of offspring.
Paper shavings: From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each F0 female cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

Diet Supply
Diet: SDS VRF1 Certified pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
A sample (100g) of each batch of diet used was retained within Pharmacy (frozen, -10 to -30°C) until finalization of the report. Samples were discarded after finalization of the
report; refer to Section 4
Availability Non-restricted (diet was removed overnight before blood sampling for hematology, blood chemistry or thyroid hormones and during the period of urine collection).
Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted (except during urine collection).
Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen chew blocks.
No specific contaminants were known that may have interfered with or prejudiced the
outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Dose Administration
Identity of Treatment Groups
The study consisted of one control and three treated groups identified as follows:

F0 generation
Group Treatment Dose # Number of animals Animal numbers
(mg/kg/day) Male Female Male Female
1 Control 0 25 25 1-25 201-225
2 Vinyl Neononanoate 66 25 25 26-50 226-250
3 Vinyl Neononanoate 200 25 25 51-75 251-275
4 Vinyl Neononanoate 600 25 25 76-100 276-300
# Expressed in terms of material as supplied

F1 generation
Cohort Group Treatment Dose # Number of animals Animal numbers
(mg/kg/day) Male Female Male Female
1A 1 Control 0 20 20 401-420 701-720
2 Vinyl Neononanoate 66 20 20 421-440 721-740
3 Vinyl Neononanoate 200 20 20 441-460 741-760
4 Vinyl Neononanoate 600 20 20 461-480 761-780

Cohort Group Treatment Dose # Number of animals Animal numbers
(mg/kg/day) Male Female Male Female
1B 1 Control 0 20 20 481-500 781-800
2 Vinyl Neononanoate 66 20 20 501-520 801-820
3 Vinyl Neononanoate 200 20 20 521-540 821-840
4 Vinyl Neononanoate 600 20 20 541-560 841-860

2A 1 Control 0 10 10 591-600 891-900
2 Vinyl Neononanoate 66 10 10 571-580 871-880
3 Vinyl Neononanoate 200 10 10 561-570 861-870
4 Vinyl Neononanoate 600 10 10 581-585, 881-890
587-590,
648

2B~ 1 Control 0 10 10 601-610 901-910
2 Vinyl Neononanoate 66 10 10 611-620 911-920
3 Vinyl Neononanoate 200 10 10 621-630 921-930
4 Vinyl Neononanoate 600 10 10 631-640 931-940
# Expressed in terms of material as supplied
~ No direct administration

Administration
Route: Oral gavage using a suitably graduated syringe and a flexible cannula inserted via the mouth.
Treated at: Constant doses in mg/kg/day.
Volume dose: 4 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1): Vehicle at the same volume dose as treated groups.
Frequency: Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
Formulation: A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Details on mating procedure:

F0 pairing commenced :After 10 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 and homogeneity: Homogeneity and stability of the test item in the vehicle was determined as part of this programme of work in Covance Study No. TP13QL. Stability was established for 24 hours when stored at ambient temperature (15 to 25°C) and 15 days when stored refrigerated (2 to 8°C).
Achieved concentration: Samples of each formulation prepared for administration in Week 1 (F0 generation) and Week 1 and the last week of treatment (F1 generation) were analyzed for achieved concentration of the test item.
Analysis: The method of analysis and results are presented in Attachment.

Duration of treatment / exposure:

F0 animals: For ten weeks before pairing until termination after litters are weaned.
F1 animals: From weaning until termination of respective cohort.
Oral gavage - Direct treatment of F1 offspring commenced at weaning (Day 21 of age); although direct treatment started at weaning, all offspring had potential for exposure in-utero and via the milk during lactation.
Unselected F1 offspring: No direct treatment - killed on Day 22 of age, organs retained.
Cohort 1A : General toxicity and pathology of the tissues of the male and female reproductive systems. Treated from weaning to approximately 13 weeks of age.
Cohort 1B : Spare Cohort Treated from weaning to approximately 14 weeks of age.
Cohort 2A : Developmental neurotoxicity (neurobehavioral testing followed by neurohistopathology assessment as adults) - treated from weaning up to approximately Day 75 of age.
Cohort 2B : Developmental neurotoxicity - no direct treatment, assigned to neurohistopathology assessment at weaning.

Frequency of treatment:

Daily

Details on study schedule:

Study initiation (Study Plan signed by Study Director) 17 September 2020
Experimental start date (animal arrival) 23 September 2020
Treatment of F0 animals commenced 28 September 2020
F0 necropsy
Males 03 to 05 February 2021
Females 26 January to 07 February 2021
F1 generation commenced 28 January 2021
F1 generation necropsy
Cohort 1A 06 to 09 April 2021
Cohort 1B 12 to 15 April 2021
Cohort 2A 15 to 20 March 2021
Cohort 2B 19 to 23 January 2021
Experimental completion date (Histopathology) 23 July 2021

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

F0: 25
F1A: 20
F1B: 20
F2A: 10
F2B: 10

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
Dose levels were selected in conjunction with the Sponsor based on the results of a
preliminary study conducted at this laboratory (Covance study no. XQ05LT).
In the preliminary study administration of Vinyl Neononanoate at dose levels of 150, 300 and 600 mg/kg/day was generally well tolerated. General condition (F0/F1), bodyweight (F0), food consumption (F0/F1), mating performance/fertility/reproductive performance (F0), litter size/survival (F1) sexual maturation (F1) and macropathology (F0/F1) were unaffected by treatment. Effects were limited to a dose related increase in water consumption for both F0 and F1 animals and low body weight gain for F1 offspring at 600 mg/kg/day from Day 7 to Day 18 of age resulting in low mean body weights for these animals at weaning and at the start of the F1 generation.
Based on the results of the preliminary study there was nothing to preclude the use of
600 mg/kg/day as the high dose on this extended one generation study (OECD443); low and intermediate dose levels of 66 and 200 mg/kg/day were selected to provide a 3-fold dose interval.

Examinations

Parental animals: Observations and examinations:

Serial Observations
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 timeof 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 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.
Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior.

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.
A complete necropsy was performed in all cases as described.

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 Days 21 23, 25, 27* and 29* of age
From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.
Before necropsy.
* Only applicable for selected F1 offspring before formal commencement of the F1 generation at nominal 4 Weeks of age (Day 28 of age ± 2 days).

Food Consumption - F0 and F1 Generation
The weight of food supplied to each cage, that remaining 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, 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.

Mating Procedure - F0 Generation
F0 pairing commenced: After 10 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.

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.

Oestrous cyclicity (parental animals):

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 (using pipette lavage): For at least three days prior to the start of the necropsy phase and on the day of termination.

Sensory Function - Quantitative - Cohort 2A
At Days 24 (±1) of age, the animals were tested in an automated system for auditory startle habituation:
Startle amplitudes were measured over five consecutive blocks of ten trials (total 50 trials).

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 (with the exception of F0 Group 1 Male 8, where the right vas deferens was sampled) into pre-warmed (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 analyzed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyser (CASA).
F0 Group 1 Male 24 vas deferen was not sampled in error.
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 (insufficient sperm on slide to assess 200 for F1 Group 1 animal 414).
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 followed by removal of the tunica and homogenization 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 homogenization-resistant spermatid count using CASA.
Groups 2 and 3 Samples frozen for possible future assessment.

Litter observations:

Records Made During Littering Phase - F0 Generation
The records maintained were as follows:
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 ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five 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 and 21 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, 1B and 2A
Males: Sexual maturation was assessed by daily examination from Day 38 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 28 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.

Neurobehavioral Screening - Cohort 2A
A functional observational battery (FOB) was performed on all Cohort 2A animals on
Day 70±1 of age. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Animals were caged individually the day before observations took place and the labels on these cages showed only the study, animal and cage numbers. Labels on cages of other animals of the same sex as those being tested were also changed such that they showed no group information. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Specific details on the assessments identified below are presented in Attachment 14.5.
In cage observations"
Abnormal motor movements (e.g. fasciculation, convulsions, stereotypy, excessive
sniffing, licking, grooming and twitches)
Palpebral closure
Posture
Tremor

In hand observations
Ease of removal from cage
Exophthalmos
Fur condition
Piloerection
Reactivity to handling
Salivation/lacrimation
Vocalization

Observations in the arena - two minute recording period.
Abnormal motor movements (e.g. fasciculation, convulsions, stereotypy, excessive
sniffing, licking, grooming and twitches)
Activity count
Arousal
Fecal count
Gait
Grooming
Palpebral closure
Posture
Rearing count
Tremor
Urination

Reactivity investigation
Approach response
Body temperature
Body weight
Grip strength: fore and hindlimb
Landing foot splay
Pupil closure reflex
Righting reflex
Auditory startle reflex
Tail pinch response
Touch response (pinna reflex)
Proprioception

Motor Activity - Cohort 2A
Before treatment, on Day 65±1 of age, the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Covance.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing

Cohort 2B
No specific investigations were performed in life; animals were dispatched to necropsy at weaning for histopathological examinations.

Postmortem examinations (parental animals):

Hematology, Peripheral Blood - F0 and F1 Cohort 1A Generation
Blood samples were collected after overnight withdrawal of food. Sampling for Cohort 1A was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum eriod of one hour prior to the commencement of blood sampling procedures. Samples were ollected at the following occasions:

Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Cohort 1A Ten male and ten female animals per group

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 (Hct)*
• Hemoglobin concentration (Hb)
• Erythrocyte count (RBC)
• Mean cell hemoglobin (MCH)*
• Mean cell hemoglobin concentration (MCHC)*
• Mean cell volume (MCV)
• Red cell distribution width (RDW)
• Total leucocyte count (WBC)
• Differential leucocyte count:
• Neutrophils (N)
• Lymphocytes (L)
• Eosinophils (E)
• Basophils (B)
• Monocytes (M)
• Large unstained cells (LUC)
• Platelet count (Plt)
* Derived values calculated in ClinAxys

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.
• 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. Sampling for Cohort 1A was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasions:

Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Cohort 1A Ten male and ten female animals per group

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 (ALP)
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Gamma-glutamyl transpeptidase (gGT)
• Total bilirubin (Bili)
• Bile acids (Bi Ac)
• Urea
• Creatinine (Creat)
• Glucose (Gluc)
• Total cholesterol (Chol)
• Sodium (Na)
• Potassium (K)
• Chloride (Cl)
• Calcium (Ca)
• Inorganic phosphorus (Phos)
• Total protein (Total Prot)
• Albumin (Alb)

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 at the following occasion:
Occasion Generation Animals
Termination F1 Cohort 1A Ten male and ten female animals per group

The individual samples were examined for the following characteristics:
• Using manual methods:
• Clarity and Color (App) - by visual assessment
• Volume (Vol) - using a measuring cylinder
• pH - using a pH meter
• Specific gravity (SG) - by direct refractometry using a SG meter

• Using Multistix reagent strips interpreted using the Clinitek®500 instrument:
• Glucose (Gluc)
• Ketones (Keto)
• Bile pigments (Bili)
• Blood pigments (UBld)

• Using a Cobas 6000 Analyzer:
• Protein - total (T-Prot) and concentration (Prot)
• Sodium - total (T-Na) and concentration (U-Na)
• Potassium - total (T-K) and concentration (U-K)
• Chloride - total (T-Cl) and concentration (U-Cl)

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 (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.
• Epithelial cells (Epi)
• Leucocytes (WBC)
• Erythrocytes (RBC)
• Casts
• Other abnormal components (A)

The slide was also examined for abnormalities in spermatozoa and crystals.

Thyroid Hormone Analysis - TSH and T4
Blood samples were collected at the following occasions:

Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Offspring Ten litters per group - pooled litter sample on Day 4
of age#
Ten male and ten female animals per group on Day
22 of age from as many litters as possible
F1 Adults - Cohort 1A Ten male and ten female animals per group (approx.
13 weeks of age)
# - Restricted to T4 only

Animal numbers are documented in Attachment 14.3 and Attachment 14.4.
Conditions: Adults: Following overnight deprivation of food. Blood sampling of F1 Cohort 1A animals coincided with urine collection, animals were therefore deprived of water overnight but had access to water for a minimum period of one hour prior to blood sampling. Offspring: No overnight deprivation of food.
Blood sample site: Adults: Sublingual vein. Offspring Day 4 of age: Decapitation Offspring Day 22 of age: Orbital sinus
Anaesthetic Adults and offspring on Day 22 of age: Isoflurane. Offspring Day 4 of age: not required
Anticoagulant: None.
Tubes Greiner Minicollect - with clot activator.
Blood volume Adults and offspring Day 22 of age: 1.0 mL. See Section 4. Offspring Day 4 of age: maximum possible.
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 2000g for ten minutes at 4°C.
Number of aliquots: Adults and offspring Day 22 of age: Two per animal.
• Aliquot 1: 0.2 mL serum for T4
• Aliquot 2: residual serum for TSH
Offspring Day 4 of age: single aliquot.
• Maximum possible for T4
Final storage conditions Deep frozen (approximately -60°C to -90°C).
Fate of samples: Aliquot 1 (T4): dispatched to the Department of Bioanalysis, Covance.
Aliquot 2 (TSH): dispatched to the Department of Immunology and Immunotoxicology, Covance.
T4 Performed by the Department of Bioanalysis, Covance.
TSH Performed by the Department of Immunology and Immunotoxicology, Covance.

Terminal Investigations
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 and those with total litter loss: Terminated with first cohort of females with live litters.
Unselected offspring: Culled on Day 4 and Day 22 of age.
F1 Cohort 1A animals: At approximately 13 weeks of age.
F1 Cohort 1B animals: At approximately 14 weeks of age.
F1 Cohort 2A animals: At approximately Day 75 of age.
F1 Cohort 2B animals: At Day 21of age.

Method of Kill
F0 generation, F1 Cohorts 1A and 1B : Animals 14 days and older: Carbon dioxide asphyxiation with subsequent exsanguination.
Animals less than 14 days of age: Subcutaneous injection of sodium pentobarbitone.
F1 Cohort 2A and 2B: Intraperitoneal injection of a lethal dose of barbiturate. Heart exposed to permit perfusion of fixative via the aorta.
Sequence: To allow satisfactory inter-group comparison.

Macroscopic examination
All animals, including surplus offspring culled on Day 4 of age 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. Decedent offspring ≤21 days of age, (found dead or welfare kill), where possible, were examined and carcass retained.
For F0 females the number of implantations were counted.
For females of Cohort 1A, counts were performed for the number of ovarian follicles and corpora lutea.
The organs weighed, tissue samples fixed and sections examined microscopically
(if applicable) are detailed as follows:

Pathology procedures - F0 (Parental) Animals
Tissue and regions examined Necropsy Histology Pathology
Weigh Fix Light microscopy
Abnormalities * * *
Adrenals * * * *
Brain (cerebellum, cerebrum, midbrain) * * * *
Cecum * * *
Colon * * *
Duodenum * * *
Epididymides L+R R R R
Esophagus * * *
Eyes * * *
Femurs - (longitudinal section through joint) a) * *
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 * *b) * *
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 L+R R R R
Thymus * * * *
Thyroid with parathyroids c) * * *
Trachea * * *
Urinary bladder * * *
Uterus with cervix * * * *
Vagina * *d) *
Vas deferens † † †
a) Both hindlimbs retained, one sectioned where appropriate
b) Weighed together
c) Weighed after partial fixation
d) Section approximately 5mm from vulva
* Organs weighed, samples fixed or sections examined microscopically
† Only one examined
L Left
R Right.
Animal ID retained

Pathology procedures - F1 Cohort 1A Adult Animals
Tissue and regions examined Necropsy Histology Pathology
Weigh Fix Light microscopy
Abnormalities * * *
Adrenals * * * *
Brain (cerebellum, cerebrum, midbrain) * * * *
Cecum * * *
Colon * * *
Duodenum * * *
Epididymides L+R R R R
Esophagus * * *
Eyes * * *
Femurs - (longitudinal section through joint) a) * *
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 * * * *
- left axillary * * * *
Optic nerves * * *
Ovaries with oviduct L+R L+R L+R b) L+R
Pancreas * * *
Pituitary * * * *
Prostate - dorsolateral and ventral combined * * *c) *
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 L+R R R R
Thymus * * * *
Thyroid with parathyroids d) * * *
Trachea * * *
Urinary bladder * * *
Uterus with cervix * * * *
Vagina * *e) *
Vas deferens † † †
a) Both hindlimbs retained, one sectioned where appropriate
b) Fixed identified as L+R. Five sections cut at approximately 100 micron intervals from the inner third of each ovary
c) Weighed together
d) Weighed after partial fixation.
e) Section approximately 5mm from vulva
* Organs weighed, samples fixed or sections examined microscopically.
† Only one examined
# 3-5mm section of spleen preserved for histopathological examination; remaining used for splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes and natural killer cells)
L Left
R Right
Animal ID retained

Pathology procedures - F1 Cohort 1B Adult Animals
Tissue and regions examined Necropsy Histology
Weigh Fix
Abnormalities * *
Adrenals *
Brain (cerebellum, cerebrum, midbrain) *
Cecum *
Colon *
Duodenum *
Epididymides * * *
Esophagus *
Eyes *
Femurs - (longitudinal section through joint) a)
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 *
- left axillary *
Optic nerves *
Ovaries with oviduct L+R L+R L+R
Pancreas *
Pituitary * * *
Prostate – dorsolateral and ventral combined * *b) *
Rectum *
Sciatic nerves *
Seminal vesicles (with coagulation 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 * c) *
Vas deferens *
a) Both hindlimbs retained, one sectioned where appropriate.
b) Weighed together
c) Section approximately 5mm from vulva
* Organs weighed, samples fixed or sections examined microscopically.
L Left
R Right
Animal ID retained

Pathology procedures - F1 Cohort 2A Animals
Tissue and regions examined Necropsy Histology Pathology
Weigh Fix Paraffin Resin Light Microscopy
Abnormalities * * *
Brain - Five coronal sections: Olfactory lobes, Forebrain,
Cerebrum (hippocampus, thalamus, hypothalamus),
Cerebrum (tectum, tegmentum), Medulla oblongata,
Mid-sagittal: Cerebellum, Pons * * * *
Dorsal and ventral root fibers with Dorsal root ganglia -
cervical (C3-C6) and lumbar (L1–L4) * * *
Eyes * * *
Optic nerves * * *
Sciatic nerves - Longitudinal and transverse sections,
sciatic notch * † †
Sciatic nerves - Longitudinal and transverse sections,
mid-thigh. * † †
Skeletal muscle - gastrocnemius * † †
Spinal cord - Longitudinal and transverse sections cervical
(C3-C6) and lumbar (L1-L4) * * *
Tibial nerves - Longitudinal and transverse sections, knee * † †
Tibial nerves - Longitudinal and transverse sections, calf
muscle branch(es) * † †
Carcass was retained in fixative
* Organs weighed, samples fixed or sections examined microscopically
† Only one examined
Animal ID retained

Pathology procedures - F1 Cohort 2B Animals
Tissue and regions examined Necropsy Histology Pathology
Weigh Fix Paraffin Resin Light Microscopy
Abnormalities * * *
Brain - Five coronal sections: Olfactory lobes, Forebrain,
Cerebrum (hippocampus, thalamus, hypothalamus),
Cerebrum (tectum, tegmentum), Medulla oblongata,
Mid-sagittal: Cerebellum, Pons * * * *



Pathology procedures - Unselected F1 offspring on Day 22 of age
Ten male and ten females per group; one male or one female from each litter to ensure that all litters are represented.
Tissue and regions examined Necropsy
Weigh Fix
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 *
Animal ID retained
The retained tissues were checked, where possible, carcass retained for decedent offspring ≤21 days of age.

Postmortem examinations (offspring):

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.

Immunophenotyping of Spleen Leucocytes - Cohort 1A
Ten males and ten females per group from Cohort 1A, from as many litters as possible, 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 portions of the spleen was then weighed, placed into 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 Immunology and immunotoxicology,
Covance. A copy of the whole spleen and partial spleen weights were provided to the
department.

Fixation
F0 animals, unselected F1 animals, F1 Cohorts 1A and 1B
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes - F0 and F1 adults only: Initially in modified Davidson’s fluid.
Eyes: In Davidson’s fluid.
F1 Cohort 2A and 2B
Standard: Glutaraldehyde:paraformaldehyde fixation by in situ perfusion followed by immersion.
Peripheral nerves Left nerve specimens were retained in situ in the carcass.

Histology
F0 animals and F1 Cohort 1A and 1B
Processing to slide: 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.
Full list: All adult animals killed or dying prematurely.
All terminal F0 and F1 Cohort 1A adult animals of
Groups 1 and 4 killed at a scheduled interval.
Reproductive organs: All suspect fertility F0 animals of Groups 2 and 3.
Abnormalities: All terminal F0 and F1 Cohort 1A adult animals of Groups 2 and 3 killed at a scheduled interval.
All F1 Cohort 1B animals.
Cecum, kidneys, liver, thyroid and urinary bladder: All terminal F0 and F1 Cohort 1A adult animals of Groups 2 and 3 killed at a scheduled interval.
Routine staining Sections were stained with hematoxylin and eosin.
Processing to block - Reproductive organs: All F1 Cohort 1B animals.
Tissue samples were dehydrated and embedded in paraffin wax.
F1 Cohorts 2A and 2B (Cohort 2B Brain only)
Routine staining: Paraffin wax: 4-5 µm sections stained with haematoxylin and eosin.
Resin: 2 µm sections stained with toluidine blue.
Full list: All terminal adult animals of Groups 1 and 4 killed at a scheduled interval.
Abnormalities only: All terminal adult animals of Groups 2 and 3 killed at a scheduled interval.
Brain (to block only): All terminal adult animals of Groups 2 and 3 killed at a scheduled interval.

Light Microscopy - F0 Animals and F1 Cohort 1A and 1B
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Premature deaths F0, F1 Cohort 1A All animals from all groups. All specified above.
and 1B
Scheduled kill F0 animals All animals of Groups 1 and 4 All specified above
All animals of Groups 2 and 3 Cecum, kidneys, liver, thyroid
urinary bladder and abnormalities
All animals of Groups 2 and 3 Reproductive organs
with suspect fertility (including pituitary)

F1 Cohort 1A All animals of Groups 1 and 4 All specified above
All animals of Groups 2 and 3 Cecum, kidneys, liver, thyroid
urinary bladder and abnormalities
F1 Cohort 1B All animals from all groups. Abnormalities only

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/Cohort 1A - Qualitative evaluation of one section from each ovary
F1 Cohort 1A - Qualitative evaluation of one section from each ovary.
Quantitative evaluation of five sections from the middle third from each ovary for the assessment of primordial follicle and small growing follicle population as well as evaluation of corpora lutea numbers in one section from each ovary.
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.

Light Microscopy - F1 Cohort 2A and 2B
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Premature deaths All animals of all groups All specified above
Scheduled kill All animals of Groups 1 and 4. All specified above
All animals of Groups 2 and 3. Abnormalities only

Brain morphometry: Estimation of thickness of the neocortex, corpus callosum,
hippocampus and folia of cerebellum (pyramis) in standardized sections.

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Description (incidence and severity):

Signs associated with administration were limited to a low incidence of increased salivation in one male and for females receiving 600 mg/kg/day.
There were no signs at routine physical examination that could be attributed to administration of the test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test article-related mortality occurred in the F0 generation. There were 4 unscheduled deaths among the F0 animals that were all considered unrelated to treatment.
Animal 1M 6 was euthanized for welfare reasons on Day 66. Macroscopic examination revealed a perforated esophagus, adhesions and abnormal contents in the thoracic cavity and dark lumbar lymph nodes. Microscopic evaluation revealed moderate necrosis in the liver, moderate adhesions and inflammatory cell infiltration in the pleura of the lungs, moderate epicardial fibrosis and inflammatory cell infiltration of the myocardium of the heart, moderate inflammatory cell infiltration of the peri-thymic tissue and involution/atrophy of the thymus, slight granuloma and moderate adhesions of the thoracic cavity and moderate intrasinusoidal erythrocytes in the lumber lymph node. The major factor contributing to death was gavage injury, recorded as “accidental death”.
Animal 2M 27 was euthanized for welfare reasons on Day 113. Macroscopic examination revealed dark areas and perforated esophagus, and adhesions with abnormal contents in the thoracic cavity. Microscopic evaluation revealed moderate inflammatory cell infiltration and perforation of the esophagus, moderate inflammatory cell infiltration in the pleura of the lungs, slight epicardial inflammatory cell infiltration in the heart, moderate inflammatory cell infiltration of the peri-thymic tissue and slight increased tingible body macrophages in the thymus, and moderate inflammatory cell infiltration and adhesions of the thoracic cavity. The major factor contributing to death was gavage injury, recorded as “accidental death”.
Animal 2M 30 was euthanized for welfare reasons on Day 93. Macroscopic examination revealed dark areas and perforated esophagus, dark areas in the lungs, pale areas in the heart, dark areas and distension in the stomach and abnormal contents in the thoracic cavity. Microscopic evaluation revealed slight inflammatory cell infiltration in the serosa of the esophagus, moderate necrosis in the liver, moderate inflammatory cell infiltration in the pleura of the lungs, moderate hemorrhage and myocardial degeneration of the myocardium and epicardial inflammatory cell infiltration in the heart, moderate erosion of the glandular region of the stomach and moderate inflammatory cell infiltration of the peri-thymic tissue and involution/atrophy in the thymus. The major factor contributing to death was gavage injury, recorded as “accidental death”.

Animal 2M 37 was found dead on Day 91. Macroscopic examination was unremarkable.
Microscopic examination revealed moderate granular casts and tubular basophilia in the
kidneys, considered as test item related, and moderate inflammatory cell infiltration in the
serosa of the esophagus. The major factor contributing to death was undetermined.

Description (incidence and severity):

Body weight and bodyweight change for males at dose levels up to and including 600 mg/kg/day was unaffected by treatment.
Before pairing the overall body weight gain for females receiving Vinyl Neononanoate at all dose levels was high at approximately 109/110% of Controls (p<0.05); the resultant mean body weight on Day 71 of treatment was slightly high with the difference attaining statistical significance at 600 mg/kg/day (p<0.05).
During gestation the mean bodyweight for females at 600 mg/kg/day was statistically significantly high when compared with Controls on GD0-8, 12 and 14 (p<0.05). Overall, the bodyweight gain for females receiving the test item was similar to Controls and showed no effects of treatment.
The overall body weight gain for females during lactation and the anticipated body weight loss after weaning was unaffected by treatment.

Description (incidence and severity):

During the ten week treatment period before pairing males and females receiving 600 mg/kg/day showed occasions of statistically significantly high food consumption when compared with Controls; males during Weeks 5, 6 and 10, and females during Weeks 2-6, 8 and 10 (p<0.05/0.01). Females at 66 or 200 mg/kg/day also showed slightly high consumption during Week 4 of treatment (p<0.05).
During gestation there was no adverse effect on food consumption when compared with Controls, with females receiving 600 mg/kg/day showing slightly high mean values on GD8-10 and GD12-14 (p<0.01).
During lactation food consumption for females receiving 600 mg/kg/day during LD7-14 was sightly low when compared with Controls (p<0.01); consumption at 66 or 200 mg/kg/day was similar to Control values.

Description (incidence and severity):

Neutrophil counts were high for both males and females at 600 mg/kg/day when compared with Controls (p<0.05).
Females that received 600 mg/kg/kg day also showed slightly low hemoglobin concentration (p<0.05) and low mean cell hemoglobin concentration (p<0.01) when compared with Controls. Females that received 200 or 600 mg/kg/day also showed high mean reticulocyte counts (p<0.01).

Description (incidence and severity):

Males and females that received Vinyl Neononanoate showed high cholesterol levels when compared with Controls; these differences attained statistical significance for males at all dose levels (p<0.05) and for females at 600 mg/kg/day (p<0.01).
Males that received 200 or 600 mg/kg/day showed low alanine amino-transferase and aspartate amino-transferase activity when compared with Controls (p<0.01). Males at 600 mg/kg/day showed high urea (p<0.01), high creatinine (p<0.05) and low bilirubin levels (p<0.05). Calcium levels, phosphorous levels and total protein for males at all dose levels were high when compared with Controls (p<0.05/0.01), with males at 600 mg/kg/day also showing low potassium levels (p<0.01) and a low mean albumin/globulin ratio (p<0.05).
Females that received 200 or 600 mg/kg/day had slightly but statistically significantly high gamma glutamyl transpeptidase activity (p<0.05). Females at all dose levels had high mean potassium levels (p<0.05) and females that received 600 mg/kg/day had high calcium levels.

Description (incidence and severity):

In the male kidneys, increased incidence of tubular basophilia (minimal to marked severity) hyaline droplets accumulation (minimal to moderate severity) and granular casts (minimal to moderate severity) were seen in animals at all dose levels; increased incidences of tubular dilatation (slight to marked severity), inflammatory cell infiltration (slight to moderate severity) and cortical scars (slight to moderate severity) were seen in animals administered 200 and 600 mg/kg/day plus cysts (slight severity) in rats administered 600 mg/kg/day. In addition, an increased incidence of pelvic dilatation (slight to moderate severity), the presence of intratubular crystal (slight to moderate severity) and urothelial hyperplasia (slight to moderate severity) in the kidneys and an increased incidence of urothelial hyperplasia (minimal to moderate severity) in the urinary bladder, considered to be related to deposition of the test article, were seen in animals administered 600 mg/kg/day.
In the female kidneys, an increased incidences of tubular basophilia (minimal to moderate severity) and inflammatory cell infiltration (minimal to slight) were seen at all dose levels.
Centrilobular hepatocyte hypertrophy was seen at minimal to moderate severity in both sexes at all dose levels. The change identified in this study is of a minor severity and not accompanied by other test article changes in the liver (degeneration or necrosis for example).
Increased incidence of follicular cell hypertrophy (minimal to slight severity) was seen in the thyroid of both sexes administered 600 mg/kg/day.
Increased incidence of mucosal hyperplasia (slight to moderate severity) was seen in the cecum of both sexes administered 200 and 600 mg/kg/day and in a single female administered 66 mg/kg/day.

Reproductive function / performance (P0)

Description (incidence and severity):

Estrous cycles before mating and prior to scheduled termination were unaffected by treatment with Vinyl Neononanoate at dose levels up to and including 600 mg/kg/day.

Description (incidence and severity):

Sperm count, motility and morphology were unaffected by administration of Vinyl Neononanoate at dose levels up to and including 600 mg/kg/day.

Description (incidence and severity):

Pre-Coital Interval
Pre-coital interval was unaffected by treatment with the majority of animals mating within four days of pairing.

Mating Performance and Fertility
Mating performance and fertility was unaffected by treatment.

Gestation Length and Gestation Index
The gestation length for the majority of females was within the expected range of 22 to 23.5 days, with the exception of one female (no. 287) at 600 mg/kg/day that showed a gestation length of 24 days.
Female no 287 was observed with one live offspring on Day 1 post-partum; this offspring was subsequently found dead on Day 2 of age. In the absence of similar findings at the high dose this female/litter were considered atypical and findings considered to be unrelated to treatment.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Results: F1 generation

General toxicity (F1)

Description (incidence and severity):

The general condition of offspring was unaffected by administration of Vinyl Neononanoate
to parental animals.
There were no signs at routine physical examination or in association with dose
administration that could be attributed to treatment with Vinyl Neononanoate.

Description (incidence and severity):

No test article-related mortality occurred in the F1 generation. There were 4 unscheduled
deaths among the F1 animals that were all considered unrelated to treatment.
Animal 1M 495, assigned to Cohort 1B, was euthanized for welfare reasons on Day 26.
Macroscopic examination revealed dark areas on the adrenals, bilateral pale areas and
granular appearance, and unilateral pelvic dilatation in the kidneys and edematous
prostate, enlarged mesenteric lymph node and abnormal contents in the thoracic cavity.
Microscopic evaluation revealed slight cystic/hemorrhagic degeneration in the adrenal,
moderate myocardial inflammatory cell infiltration in the heart, marked
glomerulosclerosis and moderate pelvic dilatation in the kidneys, slight intrasinusoidal
erythrocytes and moderate dilated/cystic sinuses in the mesenteric lymph node, moderate
colloid reduction and slight edema in the prostate, marked colloid reduction in the seminal
vesicles and slight edema in the adipose tissue. The major factor contributing to death was
kidney lesion.
Animal 4M 586, removed from the study, was euthanized for welfare reasons on Day 23.
Macroscopic examination revealed perforated esophagus, bilateral pelvic dilatation in the
kidneys and abnormal contents and adhesions in the thoracic cavity. The animal was not
subjected to histopathological examination.
Animal 2F 733, assigned to Cohort 1A, was euthanized for welfare reasons on Day 58.
Macroscopic examination revealed pale color in the duodenum and the jejunum. There
was no significant microscopic findings. The major factor contributing to death was not
determined.
Animal 2F 806, assigned to Cohort 1B, was euthanized for welfare reasons on Day 50.
Macroscopic examination revealed a perforated esophagus, edematous thymus and
adhesions and abnormal contents in the thoracic cavity. Microscopic evaluation revealed
slight mucosal hyperplasia in the cecum, slight pleural inflammatory cell infiltration in the
lungs, slight edema of the peri-thymic area, and slight abscess and moderate adhesions of
the thoracic cavity. The major factor contributing to death was gavage injury, recorded as
“accidental death”.

Description (incidence and severity):

Offspring bodyweight on Day 1 of age and subsequent body weight gain up to weaning on
Day 21 of age showed no adverse effects of treatment.
Absolute mean body weights for selected F1 males receiving 600 mg/kg/day were
statistically significantly low when compared with Controls from Day 21 of age up to
nominal Day 43 of the F1 generation. From Day 21 to 25 of age body weight gain at
600 mg/kg/day was slightly low (p<0.01), however following the start of the F1 generation
the mean body weight gains (including overall body weight gain) were generally similar to
Controls at all dose levels.
Body weights for selected F1 females at all dose levels from Day 21 and Day 25 of age
showed no adverse effects of treatment; however, body weight gain at 600 mg/kg/day was
marginally low over this period (p<0.05).
On Day 1 of the F1 generation at nominal 28 ±2 days of age the mean bodyweight for
females at 600 mg/kg/day was low when compared with Controls (p<0.01). From Day 8 at
66 mg/kg/day and from Day 15 of the F1 generation at 200 or 600 mg/kgday absolute mean
bodyweights for selected females were high when compared with Controls; there was no
evidence of a dose response. From Day 1 of the F1 generation the body weight gain of
selected F1 females were generally high when compared with Controls and the overall body
weight gain was statistically significantly high for females receiving Vinyl Neononanoate
(p<0.05/0.01).

Description (incidence and severity):

Food consumption for males was generally similar to Controls. During Days 29-57 of the
F1 generation, food consumption for males receiving 600 mg/kg/day was marginally high
when compared with Controls (p<0.05/0.01)
Females at 200 or 600 mg/kg/day showed slightly but statistically significant high food
consumption from Day 4 of the F1 generation this was also apparent at 66 mg/kg/day from
Days 4 to 36 of the F1 generation.

Description (incidence and severity):

Males that received 600 mg/kg/day showed slightly low hemoglobin concentration (p<0.05)
and a high neutrophil count (p< 0.05); these differences were not apparent in females at this
dose level.
At 600 mg/kg/day females had a slightly low erythrocyte count (p<0.05) and females at all
dose levels of Vinyl Neononanoate had slightly low platelet counts with statistical
significance attained at 200 and 600 mg/kg/day (p<0.05); there was no evidence of a dose
response.

Description (incidence and severity):

Males at 200 or 600 mg/kg/day had slightly low alanine aminotransferase activity (p<0.01)
and males at 600 mg/kg/day had slightly low alanine phosphatase activity (p<0.05); these
differences were not apparent in female animals.
Urea plasma levels for males at 200 or 600 mg/kg/day were high when compared with
Controls (p<0.05 and p<0.01, respectively). Conversely, in females receiving Vinyl
Neononanoate, urea concentration was low when compared with Controls at 200 and
600 mg/kg/day (p<0.05); there was no evidence of a dose response.
Females that received Vinyl Neononanoate had high mean bile acid levels when compared
with Controls, with the difference at 600 mg/kg/day attaining statistical significance (p<0.01)
and creatinine levels were low (p<0.05 at 66 mg/g/day and p<0.01 at 200 and 600
mg/kg/day); no dose response was apparent.
The mean plasma cholesterol level was statistically significantly high for females that
received 600 mg/kg/day (p<0.01) and the albumin to globulin ratio was slightly low for males
at 600 mg/kg/day(p<0.05).

Description (incidence and severity):

Urinary output was high for males that received 600 mg/kg/day (p<0.01).
The urinary pH of all animals was low when compared with controls, with a dose relationship
apparent in both males and females.
Total protein and protein concentrations were statistically significantly high in males at all
dose levels (p<0.01). For females total protein was high at 600 mg/kg/day (p<0.01) and
protein concentration was high at all dose levels (p<0.05/0.01)
Urinary sodium, potassium and chloride concentrations in males were low at all dose levels
with statistically significance attained for each electrolyte at 200 and 600 mg/kg/day and for
sodium at 66 mg/kg/day. However total sodium was similar across the groups whilst the total
potassium and chloride levels at 600 mg/kg/day were high when compared with Controls.
Specific gravity of urine from treated females was slightly high when compared with the
Controls with the difference attaining statistical significance at 200 and 600 mg/kg/day.

Description (incidence and severity):

The age and body weight at attainment of vaginal opening and balano preputial separation
was unaffected by treatment with Vinyl Neononanoate.
For Cohort 1A females the interval between completion of vaginal opening and the first
estrus smear was similar across the groups showing no adverse effect of treatment

Description (incidence and severity):

Ano-genital distance for both male and female offspring on Day 1 of age was unaffected by
parental treatment.

Description (incidence and severity):

Examination of male offspring on Day 13 of age did not reveal any nipples.

Description (incidence and severity):

Assessment of brain, spleen and thymus organ weights for unselected offspring on PND22
showed no differences that could be attributed to parental treatment.

The absolute mean liver weights were statistically significantly higher than control in females
at 200 or 600 mg/kg/day (16% and 36% increase respectively) and the body weight relative
mean liver weights were statistically significantly higher than control in both sexes that
received 600 mg/kg/day (18% increase in males and 30% in females).
The absolute and body weight relative mean kidney weights were statistically significantly
higher than control in both sexes that received 600 mg/kg/day (29% and 35% increase
respectively in males and 18% and 12% increase respectively in females) and in males at
66 or 200 mg/kg/day (10% and 7% increase respectively at 66 mg/kg/day and 16% and 14%
increase respectively at 200 mg/kg/day).
The absolute and body weight relative mean spleen weights were statistically significantly
higher than control in both sexes at 600 mg/kg/day (14% and 19% increase respectively in
males and 18% and 12% increase respectively in females).
All other differences in organ weight parameters, statistically significant or not, were
consistent with normal variation and considered incidental. These differences were
characterized by one or more of the following: inconsistency between sexes; presence only in
absolute weight or in relative to body weight ratios but not both; lack of a dose relationship or
correlative findings; and/or the magnitude was considered small.

Description (incidence and severity):

Macroscopic examination of offspring that were either culled on Day 4 of age, died prior to
scheduled termination or on PND22 did not reveal any findings that could be attributed to
parental treatment.

Abnormal color of the kidneys and a higher incidence of pale areas were observed for males
that received 600 mg/kg/day. A higher incidence of dilated pelvis was also observed for
males and females that received 600 mg/kg/day.

Neuropathology - Cohort 2A and 2B
Cohort 2B (PND22)
There were statistically significant increases (both p≤0.05) in the greatest dorsal-ventral
thickness of the hippocampus (Group 1 = 1.483 mm vs Group 4 = 1.631 mm) and the width
of the pyramis folia of the cerebellum (Group 1 = 0.678 mm vs Group 4 = 0.770 mm) in male
offspring born to the parents receiving 600 mg/kg/day. Both of these measurement fell within
the historical control range 1.5 mm to 1.9 mm n = 13 and 0.64 mm to 0.86 mm n = 12
respectively. In addition, slight decreases in both of these measurement were recorded in the
females. Therefore, these changes were considered to be within background variation.
Cohort 2A (PND75)
There were no changes in any of the brain morphometry measurements in the selected
F1 animals that received 600 mg/kg/day, from weaning to Day 75 of age, when compared
with the control animals.
There was no test article related neuropathological alterations, following the
histopathological examination of the Cohort 2A and Cohort 2B animals.

Description (incidence and severity):

In the male kidneys, an increased incidence of tubular basophilia (minimal to marked
severity), hyaline droplets accumulation (slight to moderate severity) and granular casts
(minimal to moderate severity) were seen in animals at all dose levels. Increased incidences
of tubular dilatation (slight to moderate severity), inflammatory cell infiltration (slight to
moderate severity) and cortical scars (slight to moderate severity) were seen in the animals
administered 600 mg/kg/day. These changes were similar in nature to those recorded in the
F0 animals. In addition, presence of intratubular crystals (minimal to slight severity) in
animals administered 600 mg/kg/day and urothelial hyperplasia (slight to moderate severity)
and an increased incidence of pelvic dilatation (slight to marked severity), and an increased
incidence of urothelial hyperplasia (minimal to slight severity) in the urinary bladder in
animals administered 200 or 600 mg/kg/day.
In the female kidneys, increased incidences of tubular basophilia (minimal to marked
severity), inflammatory cell infiltration (minimal to slight severity) and cortical scars (slight
to moderate severity), plus tubular dilatation (minimal to moderate severity), were seen in the
animals administered 600 mg/kg/day. In addition, there was an increased incidence of pelvic
dilatation (slight to marked severity), urothelial hyperplasia (minimal to slight severity) and a
single case of minimal presence of intratubular crystal in the kidneys.
Centrilobular hepatocyte hypertrophy was seen at minimal to slight severity in both sexes
administered 200 or 600 mg/kg/day and in males administered 66 mg/kg/day.
An increased incidence of follicular cell hypertrophy (minimal to slight severity) was seen in
the thyroid of both sexes administered 600 mg/kg/day and in males administered
200 mg/kg/day.
Minimal mucosal hyperplasia was seen in the cecum of both sexes administered 200 or
600 mg/kg/day.
There was no test article related finding seen in the spleen to account for the statistically
significantly higher spleen weight recorded in both the F0 and F1 animals.
All other microscopic findings were considered spontaneous and/or incidental because they
occurred at a low incidence, were randomly distributed across groups (including concurrent
controls), and/or their severity was as expected for this age of Sprague-Dawley rats.
Consequently, they were considered not test article related.
The testes revealed normal progression of the spermatogenic cycle, and the expected cell
associations and proportions in the various stages of spermatogenesis were present. The test
article has no effect on the ovaries and the stages of vaginal estrus.

Description (incidence and severity):

Litter size, offspring survival and sex ratio showed no adverse effects of treatment.

Ovarian follicle and corpora lutea counts for females that received 600 mg/kg/day were similar to Controls and unaffected by treatment.

Sperm count, motility and morphology were unaffected by treatment.

Developmental neurotoxicity (F1)

Description (incidence and severity):

Auditory Startle Response Habituation - Cohort 2A
Auditory startle response habituation for F1 Cohort 2A animals at Day 24±1 of age, both in
terms of latency to peak amplitude values and peak amplitude values, was unaffected by
treatment with Vinyl Neononanoate at all dose levels investigated.

Motor Activity - Cohort 2A
Both ambulatory (low beam) and rearing (high beam) activity was unaffected by treatment.

Neurobehavioural Screening - Cohort 2A
A detailed functional observational battery was performed for all Cohort 2A animals on
nominal Day 70±1 of age.

In Cage Observations
There were no test item-related changes evident in the behaviour of the Cohort 2A animals
during the in-cage observations.

In Hand Observations
During the in-hand observations there were no findings observed which represented
neurological changes related to administration of Vinyl Neononanoate.

Arena Observations
The 2-minute arena observation of the Cohort 2A animals did not reveal any findings that
could be attributed to treatment

Reactivity Investigations
Reactivity investigations did not reveal any neurological changes in the Cohort 2A animals at
any dose level investigated

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental neurotoxicity

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

Oral administration of Vinyl Neononanoate to Sprague Dawley rats for at 66, 200 or
600 mg/kg/day was generally well-tolerated.
Treatment with Vinyl Neononanoate was associated with Alpha 2u-globulin nephropathy
which is an adverse male rat specific toxicological syndrome. In addition, there were
histopathological findings in the liver (hepatocellular hypertrophy) that were attributed to
adaptive induction of metabolising enzymes in the liver that resulted in secondary rodent-
specific hypertrophic changes in the follicular cells of the thyroid glands. Therefore, the
kidney and thyroid pathology are rodent specific and not considered to be relevant in man.
The liver pathology is considered to be an adaptive response to administration of a xenobiotic
and is considered non-adverse.
Reproductive performance (encompassing mating performance, fertility and offspring
development), neurobehavior and neuropathology were unaffected by treatment.
It is therefore concluded that 600 mg/kg/day was the no observed effect level (NOEL) for
reproductive performance/offspring development and the no observe adverse effect level
(NOAEL) for general systemic toxicity relevant to human health. The NOAEL for
developmental neurotoxicity is also concluded to be 600 mg/kg/day.

Executive summary:

Introduction

The purpose of this study was to assess the influence of Vinyl Neononanoate on reproductive performance when administered continuously by oral gavage to Sprague Dawley rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity and potential effects on sexual maturation and estrous cycles and developmental neurotoxicity. 

In the F0 generation, three groups of 25 male and 25 female rats received Vinyl 

Neononanoate at dose levels of 66, 200 or 600 mg/kg/day at a volume dose of 4 mL/kg/day. 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. In the F1 generation, 50 males and 50 females were treated from weaning to their scheduled termination at the same dose levels and volume-dose as the F0 generation. Similarly constituted Control groups received the vehicle, corn oil, at the same volume dose and throughout the same relevant period. 

For the F0 generation data were recorded on clinical observations, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance. Clinical pathology (hematology and 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 of age. Serum samples that were collected from selected offspring on Day 22 of age were analyzed for thyroid-related hormones. 

At weaning the F1 generation was split into four cohorts: 

For Cohort 1A, data were recorded on clinical condition, detailed physical examination and arena observations, body weight, food consumption, sexual maturation and estrous cycles. 

Clinical pathology (hematology, blood chemistry and urinalysis) and thyroid-related hormones, sperm assessment, ovarian follicle and corpora lutea counts, organ weight, macroscopic pathology, full microscopic pathology and immunophenotyping investigations were performed. 

For Cohort 1B, data was recorded on clinical condition, body weight, food consumption, sexual maturation and estrous cycles. Organ weight and macroscopic investigations were performed. 

For Cohort 2A, data was recorded on clinical condition, body weight and food consumption. Neurobehavioural screening, brain weight, macroscopic pathology and microscopic investigations were performed. 

For Cohort 2B, animals received no direct treatment and no specific in-life investigations were performed. Animals were dispatched to necropsy at weaning for microscopic pathology investigations of the brain. 

 

Results 

F0 Responses 

There was no test article-related mortality and the general condition of animals was unaffected by treatment. 

F0 females receiving Vinyl Neononanoate had slightly high bodyweight and bodyweight gain during the ten week treatment period before pairing; this was not evident in F0 males.  Body weight gain during gestation and lactation was unaffected by treatment. 

During the ten week treatment period before pairing males and females receiving 600 mg/kg/day showed occasions of high food consumption: the incidence being greater in females. At 600 mg/kg/day food consumption during gestation was slightly high from GD8-10 and 12-14 and during lactation consumption was slightly low from LD7-14. Food consumption at 66 or 200 mg/kg/day was essentially similar to Controls. 

Estrous cycles, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by treatment.  

Circulating levels of thyroid-related hormones were unaffected by treatment.   

Hematological assessment at termination revealed high neutrophil counts for both males and females that received 600 mg/kg/day. Females at 600 mg/kg/day also showed low hemoglobin concentrations, low mean cell hemoglobin concentration and high mean reticulocyte counts when compared with Controls; females at 200 mg/kg/day also had a high mean reticulocyte count. 

Plasma cholesterol levels for both treated males and females were high when compared with Controls. Other differences included: 

• Low alanine and aspartate amino-transferase activity for males at 200 and 

600 mg/kg/day, 

• High urea, high creatinine, low bilirubin for males at 600 mg/kg/day, 

• High calcium for males at all dose levels and females at 600 mg/kg/day, 

• High phosphorous and total protein for males at all dose levels, 

• Low potassium and albumin/globulin ratio for males at 600 mg/kg/day, 

• High gamma glutamyl transpeptidase activity for females at 200 or 600 mg/kg/day, 

• High potassium levels for females at all dose levels. 

Sperm motility, counts and morphology were unaffected by treatment. 

Body weight relative liver weight was high for both males and females at 200 or 

600 mg/kg/day.   

Body weight relative kidney and spleen weight was high for both males and females at 

600 mg/kg/day and for males at 200 mg/kg/day.   

Macroscopic examination revealed an increase in the incidence of kidneys of abnormal colour/with pale areas in males that received 600 mg/kg/day.  

Vinyl Neononanoate related microscopic findings were noted in the kidneys, urinary bladder, liver, thyroid, and cecum. 

In kidneys, changes characteristic of alpha 2u-globulin nephropathy syndrome (tubular basophilia, tubular dilatation, inflammatory cell infiltration, cortical scars, cysts, hyaline droplets accumulation and granular casts) were seen in the males administered 66, 200 and 600 mg/kg/day. In addition, changes related to the accumulation of test article were seen in the kidneys (pelvic dilatation, presence of intratubular crystals and urothelial hyperplasia) and the urinary bladder (urothelial hyperplasia). These changes are considered as adverse. 

Slight increase in the incidences of tubular basophilia and inflammatory cell infiltration were seen in the kidneys of females administered 66, 200 and 600 mg/kg/day. These changes are considered as non-adverse. 

The hypertrophic changes noted in the liver and the thyroids of both sexes administered 600 mg/kg/day and in the liver of animals administered 66 and 200 mg/kg/day, are suggestive of adaptive responses, and are considered to be non-adverse. 

The mucosal hyperplasia noted in the cecum of both sexes administered 200 and 

600 mg/kg/day, and a single female at 66 mg/kg/day, are suggestive of adaptive responses, and are considered to be non-adverse. 

F1 Litter responses 

General condition of offspring was unaffected by parental treatment. 

Litter size, offspring survival, sex ratio, ano-genital distance, offspring bodyweight, offspring organ weights and macropathology were unaffected by parental treatment. 

No nipples were apparent for F1 male offspring on Day 13 of age. 

F1 Responses 

There was no test article-related mortality and the general condition of animals was unaffected by treatment. 

Overall, the selected F1 females receiving Vinyl Neononanoate showed high bodyweight gain and food consumption when compared with Controls; this was not evident for selected F1 males. 

The age and body weight at attainment of vaginal opening and balano preputial separation was unaffected by treatment with Vinyl Neononanoate. 

For Cohort 1A females the interval between completion of vaginal opening and the first estrus smear was similar across the groups showing no adverse effect of treatment 

Estrous cycles were unaffected by treatment at dose levels up to and including 

600 mg/kg/day. 

Assessment of sensory function and motor activity, with neurobehavioral screening did not reveal any adverse effects of treatment. 

Circulating levels of thyroid-related hormones were unaffected by treatment.   

At 600 mg/kg/day males showed low hemoglobin and high neutrophil counts, whilst females showed low erythrocyte counts. Females at all dose levels of Vinyl Neononanoate had slightly low platelet counts. 

Males at 200 or 600 mg/kg/day had slightly low alanine aminotransferase activity (p<0.01) and males at 600 mg/kg/day had slightly low alanine phosphatase activity (p<0.05); these differences were not apparent in female animals. 

At 200 and 600 mg/kg/day urea plasma levels for males at 200 or 600 mg/kg/day were high whilst females receiving Vinyl Neononanoate had low urea concentrations.   

Females that received Vinyl Neononanoate had high mean bile acid and low creatinine levels.   

At 600 mg/kg/day the mean plasma cholesterol level was high for females and the albumin to globulin ratio was slightly low for males. 

Urinary output was high for males that received 600 mg/kg/day. 

The urinary pH of all groups of treated animals was low when compared with Controls. 

Specific gravity of urine from treated females was slightly high. 

Urinary total protein was high for males at all dose levels and protein concentration was high for both males and females at all dose levels. 

Urinary sodium, potassium and chloride concentrations in males were low at all dose levels; total sodium for males was similar across the groups whilst the total potassium and chloride 

levels at 600 mg/kg/day were high.   

Fluctuations were observed in the immunophenotyping parameters across the different treatment groups for both males and females. These differences were not related to administration of Vinyl Neononanoate, as the values of the immunophenotyping parameters obtained in spleen leukocytes (percentages and cells/spleen) were within the ranges observed in the Control animals and not statistically significant. 

Body weight relative liver weight was high for both males and females at 600 mg/kg/day.   

Body weight relative kidney and spleen weight was high for both males and females at 600 mg/kg/day and relative kidney weights were high for males at 66 and 200 mg/kg/day.   

Macroscopic examination revealed an increase in the incidence of kidneys of abnormal colour/with pale areas in males that received 600 mg/kg/day. A higher incidence of dilated pelvis was also observed for males and females that received 600 mg/kg/day. 

Vinyl Neononanoate related microscopic findings were noted in the kidneys, urinary bladder, liver, thyroid, and cecum. 

In kidneys, changes characteristic of alpha 2u-globulin nephropathy syndrome (tubular basophilia, tubular dilatation, inflammatory cell infiltration, cortical scars, hyaline droplets accumulation and granular casts) were seen in the males administered 66, 200 and 600 mg/kg/day. In addition, changes related to the accumulation of test article were seen in the kidneys (pelvic dilatation, presence of intratubular crystals and urothelial 

hyperplasia) and the urinary bladder (urothelial hyperplasia). These changes are 

considered as adverse. 

Slight increase in the incidences of tubular basophilia, inflammatory cell infiltration and cortical scar, and tubular dilatation were seen in the kidneys of females administered 600 mg/kg/day. In addition, changes related to the accumulation of test article were seen in the kidneys (pelvic dilatation, presence of intratubular crystals and urothelial hyperplasia) and the urinary bladder (urothelial hyperplasia). These changes are 

considered as non-adverse. 

The hypertrophic changes noted in the liver and the thyroids of both sexes administered 600 mg/kg/day are suggestive of adaptive responses and are considered to be non-adverse. 

The mucosal hyperplasia noted in the cecum of both sexes administered 600 mg/kg/day are suggestive of adaptive responses and are considered to be non-adverse. 

There was no test article related neuropathological alterations or changes in brain morphometry measurements, following the histopathological examination of the Cohort 2A and Cohort 2B animals. 

 

Conclusion  

Oral administration of Vinyl Neononanoate to Sprague Dawley rats for at 66, 200 or 600 mg/kg/day was generally well-tolerated.   

Treatment with Vinyl Neononanoate was associated with Alpha 2u-globulin nephropathy   which is an adverse male rat specific toxicological syndrome. In addition, there were histopathological findings in the liver (hepatocellular hypertrophy) that were attributed to adaptive induction of metabolising enzymes in the liver that resulted in secondary rodent-specific hypertrophic changes in the follicular cells of the thyroid glands. Therefore, the kidney and thyroid pathology are rodent specific and not considered to be relevant in man. The liver pathology is considered to be an adaptive response to administration of a xenobiotic and is considered non-adverse. 

Reproductive performance (encompassing mating performance, fertility and offspring development), neurobehavior and neuropathology were unaffected by treatment. 

It is therefore concluded that 600 mg/kg/day was the no observed effect level (NOEL) for reproductive performance/offspring development and the no observe adverse effect level (NOAEL) for general systemic toxicity relevant to human health. The NOAEL for developmental neurotoxicity is also concluded to be 600 mg/kg/day.