Registration Dossier

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-16 to 2018-12-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Emerald Kalama Chemical Limited (Dans Road, WA8 0RF Widnes, Cheshire, United Kingdom); Batch/lot number: A170421E
- Expiration date of the lot/batch: 2020-06-06
- Purity test date: 2018-02-07

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤70 RH%), under inert gas, protected from humidity (stored in a tightly closed container)
- Stability under storage conditions: stable for at least 15 day when stored at room temperature
- Stability under test conditions: stable for at least 15 day when stored at room temperature
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: formulation samples in the 15-220 mg/mL concentration range (using PEG 400 as vehicle) were proven as being stable for at least 15 day when stored at room temperature.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear, pale yellow liquid

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Wistar)

Details on species / strain selection:

The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for Dose Range Finding study (study code: 17/181-220PE)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) x approximately 14 weeks old at start of treatment and approximately 16 weeks old at mating
- Weight at study initiation: (P) Males: 458 – 525 g; Females: 254 – 296 g
- Fasting period before study: Not specified
- Housing: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage (Type II and III polycarbonate) with the exception of the mating and gestation/delivery period when they were paired or individually housed, respectively
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH (D-59494 Soest Germany) (Batch number: 840 33675 / 639 38520, Expiry date: 31 January 2019 / 30 April 2019), ad libitum
- Water (e.g. ad libitum): tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum
- Acclimation period: 34 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 24.9 °C (target range 22 ± 3 °C)
- Humidity (%): 25 – 60 % (target range 30-70 %)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2018-09-13 To: 2018-12-23

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

polyethylene glycol 400 (PEG 400)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was formulated at appropriate concentrations in the vehicle (as a visibly stable homogenous formulation) in the Pharmacy of the Test Facility. Formulations were prepared at appropriate intervals to be used within three days after preparation.

Stability of the test material in the vehicle was assessed in the conditions employed on the study during the analytical method validation. In that study, the formulation samples in the 15-220 mg/mL concentration range (using PEG 400 as vehicle) were proven as being stable for at least 15 day when stored at room temperature.

Dosing solutions were administered to the test item or vehicle-treated (control) animals daily on a 7 days/week basis, by oral gavage using a bulb-tipped gavage needle attached to a syringe. A constant volume of 5 mL/Kg bw were administered to all animals. The actual volume to be administered were calculated and adjusted based on each animal’s most recent body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on results of a short solubility test performed at the Test Facility, polyethylene glycol 400 (PEG 400) was selected as vehicle for this study in agreement with the Sponsor based on the formulation and analytical trials. The same vehicle was used in the Dose Range Finding (DRF) study.
- Concentration in vehicle: 0, 15, 50, and 200 mg/mL for the control, low-, mid-, high-dose groups, respectively.
- Amount of vehicle (if gavage): 5 mL/Kg bw
- Lot/batch no. (if required): Supplier: ACROS; Batch number: A0392628
- Purity: Not specified

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Females remained with the same male until copulation occurred (the mating period lasted for 5 days)
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): Sperm positive females were housed individually

Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male from the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred (the mating period lasted for 5 days in this study). A vaginal smear was prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope. The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were housed individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test material formulations for concentration and homogeneity was performed using a validated HPLC-UV method. Duplicate samples were taken from the top, middle and bottom of the test item formulations three times during the study (during the first and last weeks and approximately midway during the treatment), one set to analyse and one set as a backup, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Formulation samples (both sets) were kept on room temperature until shipment. Samples were shipped on ambient temperature within three days after sample collection for concentration and homogeneity measurement to the Principal Investigator (PI).

Formulation analysis was conducted under the control of the Principal Investigator in compliance with the Study Plan and the Test Site relevant SOPs.

Acceptance criteria of the concentration analysis were set according to the analytical method validation; it was 100 ± 10 % of the mean nominal concentration.

Acceptance criteria of the homogeneity was that the coefficient of variation (CV) of replicates (top, middle and bottom of test item formulations) should be less than 10%.

Duration of treatment / exposure:

Males: dosed for 28 days (14 days pre-mating and 14 days during the mating / postmating period)

Females: dosed for 14 days pre-mating, during the mating period, through gestation and parturition, until the day before the necropsy (13-days of post-partum dosing)

Frequency of treatment:

once daily 7 days/week

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected by the Sponsor in consultation with the Study Director based on available acute oral toxicity data (LD50 over 2000 mg/Kg bw in rats) and results of the Dose Range Finding (DRF) study performed at the Test Facility (Test Facility Study code: 17/181-220PE), where no clearly adverse effects were seen up to 1000 mg/Kg bw/day.

The aim was to use a maximum of 1000 mg/Kg bw/day or to induce toxic effects, but ideally without death or suffering and a NOAEL at the lowest dose. Based on the results from the preliminary DRF study, the lower dose levels were set using a ratio of 4 (1000, 250, 75 mg/Kg/day). The oral route was selected as it is one of the possible routes of human exposure.

- Rationale for animal assignment (if not random):
All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges on the day of start of treatment. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.

- Fasting period before blood sampling for clinical biochemistry: Yes (overnight period of food deprivation, in case of dams it was the night after the litter had been culled).

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: rats were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed once a day, during the pre-treatment and treatment period (in the afternoon (pm) or after treatment at approximately the same time with minor variations as practical during the working day), as no peak period of effects was noted after dosing during the first few days of treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed at the start of the pre-exposure period and once before the first exposure (to allow for within-subject comparisons), then weekly (in the morning (am) or before treatment) and before necropsy.

Animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

On Gestation Day 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).

All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with an accuracy of 1 g at least weekly during the pre-exposure period, then on Day 0 for randomisation purposes, afterwards at least weekly and at termination.

Parental females were weighed on Gestation Days (GD) 0, 3, 7, 10, 14, 17 and 20 and on Post-Partum Days (PPD) 0 (within 24 hours after parturition), 4, 7, 10, 13 and 14 (before termination). The body weight of the female animals measured on GD3, 10 and 17 as well as PPD7 and PPD10 were only additional measurements as aid for the calculation of accurate treatment volumes, thus these data were not evaluated statistically.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g weekly (on the days of body weight measurements). The daily intake per animal was calculated (During pairing, while one male and one female were in a single cage, the food intake was measured, but the data was not statistically analysed since it is not appropriate to use the mean value, because males eat more than females. The data presented in the summary tables represents the average daily food intake from the animals available for data analysis) for the statistical data analysis and reporting.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OTHER:

Functional observation battery (FOB) and locomotor activity measurement
Five males and five females of the parental generation were randomly selected in each group for these measurements.

Assessment of potential test material-related neurotoxicity was performed during the last exposure week (males on Day 27; females on PPD9-12). Selected animals were subjected to the functional observation battery including quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength.

To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots for the hind limbs was measured. This step was performed 3 times for each animal.

Fore/hind limb grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of the test material. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal. The procedure was repeated with the hind limbs (3 times for each animal) and the appropriate grip support.

Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted, and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.

Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmia, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated using a scoring system, where score “0” was given when the behaviour or reaction of the animal was considered normal, and -1 or -2, or +1 and +2 was given if the response was less than or more than expected in an untreated animal.

Locomotor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field of 50 x 50 cm for a 1-hour observation time, when DVD recording of activity/movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made, to produce the appropriate parameters. The data from all groups was evaluated for total travelled distance in 5-minute segments. The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls and comparing the treatment groups.

Clinical Pathology
All adult animals, including the selected animals for blood sampling (5 animals per sex per group – the same animals were used), were fasted (overnight period of food deprivation, in case of dams it meant the night after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.

For terminal blood sampling in all selected animals (5 males and 5 females/group), 3 samples were taken from each animal: one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

Haematology and blood clotting times:
The parameters listed in Table 2 were evaluated in selected animals.

Clinical Chemistry:
The parameters listed in Table 3 were evaluated in selected animals.

Urinalysis
Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours. The evaluation of the urine samples was performed as indicated in the Table 4.

Thyroid Hormone Analysis
For thyroid hormone analysis, blood samples were taken by cardiac puncture (or decapitation in case of pups) into tubes containing K3-EDTA as anticoagulant as follows:
- from up to two pups per litter on PND4,
- from all dams and up to two pups per litter on PND14 (females) / PND13 (pups),
- from all adult males at termination.

Pup blood was pooled by litter.

Blood samples were kept on ice from sampling until centrifugation (within 30 minutes of collection), then centrifuged rapidly (1600 g / approx. 3000 rpm, 10 minutes, 4ºC). The resulting plasma was divided into at least two aliquots (volume target of at least 125 μL for the first aliquot and at least 75 μL for the second aliquot, if possible; any leftover material was also retained for safety reason as a third aliquot) and stored in an ultrafreezer (-80 ± 10 °C) until shipping for analysis. Samples (Set #1 and Set #2) were shipped for hormone analysis on dry ice to the Principal Investigator.

Samples (Set #1) of adult males were assessed for T4 levels. The measurement of the T4 hormone levels in adult females, or measurement of other thyroid hormone (TSH) were not performed as was not deemed necessary by the Study Director and Sponsor.

The thyroid hormone analysis was conducted using a validated method under the control of the Principal Investigator in compliance with Study Plan, its amendment, and the relevant SOPs of the Test Site.

Analytical procedure
The method for T4 measurement was based on a solid phase extraction from plasma followed by an analysis of the extract by reverse phase liquid chromatography (HPLC) with MS/MS detection after ionization using an Electro Spray Interface (ESI, positive mode).

The analytical procedure for the determination of T4 in rat plasma by LC-MS/MS was developed at the Test Site (Charles River Laboratories, Evreux). Complete validation was carried out prior to the analysis of the study samples. The validated concentration range for T4 was 4.00 to 500 ng/mL.

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored for each female (all females including spare animals) by vaginal smears daily for 15 days during the pre-exposure period. Females that failed to show a 4 or 5-day cycle was not included in the study, twelve females showing regular oestrus cycles were treated and used for mating in each group (the rest of the females were moved back to the spare colony after the mating process had been finished). Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

Parameters examined in all P male parental generations:
Special attention will be paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, all pups/litter as nearly as possible

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect the presence of gross abnormalities. Additionally, any abnormal behaviour of the offspring was recorded.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND0) and on PND4 and PND13, with accuracy of 0.01 g. All litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized or autolyzed) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded. The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND0). Presence of nipples/areolae in male pups were recorded on PND13. All pups were examined externally at weighing on PND4. One male and one female pup (where possible) was allocated randomly for culling for blood sampling on PND4. All pups were culled on PND13.

Samples of PND13 pups were assessed for T4 levels. The measurement of the T4 hormone levels in PND4 pups, or measurement of other thyroid hormone (TSH) were not performed as was not deemed necessary by the Study Director and Sponsor.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not specified

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not specified

Postmortem examinations (parental animals):

SACRIFICE
At termination, surviving adult rats (male and female) were euthanized under pentobarbital anaesthesia, followed by exsanguination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

Gross necropsy was performed on all animals, irrespective of the date of death. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened, and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.

The pre-terminally euthanized and the found dead animals were examined similarly to the terminal animals.

Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and to allow correlation with histopathology of the reproductive organs. The number of implantation sites and of corpora lutea were recorded in female animals as applicable.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 5 and Table 6 were weighed and prepared for microscopic examination, respectively.

Organ weight measurements: Individual and/or paired absolute organ weight was recorded for each animal (note: paired organ weight was only recorded for testes and epididymides). Paired organ weights were summarised as applicable. Relative organ weight (to body and brain weight) were calculated and reported.

The organ weights of the pre-terminally euthanized animal were measured similarly to the terminal animals. However, no organ weight measurement was performed in the found dead animal.

Histopathology: The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerves and the testes with epididymides were retained in modified Davidson’s fixative, and all other organs in 10% buffered formalin solution.

For microscopic examination, the retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

For the adult animals, a detailed histological examination was performed as follows:
• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group), plus the liver of all remaining males and females, and kidneys and thyroids of all remaining males,
• in found dead animals (#1012, #2512, #4003, #4005) and in one Control female (#1507) that was pre-terminally euthanized,
• all organs where macroscopic findings (abnormalities) were seen at necropsy,
• retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups and of all males / females in the Mid and Low dose groups that failed to sire / deliver healthy pups (#2003/#2503, #2012/#2512, #3005/#3505).

Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes and bone marrow). Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.

Postmortem examinations (offspring):

SACRIFICE
- The anaesthetic product (pentobarbital) was diluted for pups’ euthanasia as required. All pups were culled on PND13.

GROSS NECROPSY
- Dead pups and pups euthanized on PND4 and/or PND13 were carefully examined externally for gross abnormalities, where possible. Particular attention was paid to the external reproductive organs which were examined for signs of altered development. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs. Presence of nipples/areolae in PND13 pups (if any) was also recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid glands from one male and one female PND 13 pup selected by the Study Director from each litter were preserved in 10% buffered formalin solution. In
this case, the thyroid weight (pooled) was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage. For microscopic examination, the retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

Statistics:

For information on statistics, please see the section 'Any other information on materials and methods incl. tables'.

Reproductive indices:

1) Male Mating Index: (Number of males with confirmed mating x Total Number of males cohabited) x 100

2) Female Mating Index: (Number of sperm − positive females / Total Number of females cohabited) x 100

3) Male Fertility Index: (Number of males impregnating a female / Total Number of males cohabited) x 100

4) Female Fertility Index: (Number of pregnant females / Number of sperm - positive females) x 100

5) Gestation Index: (Number of females with live born pups / Number of pregnant females) x 100

Offspring viability indices:

1) Survival Index %: (Number of live pups (at designated time) / Number of pups born) x 100
Note: Survival index on PND13 was calculated from number of pups after culling on PND4 instead of number of pups born.

2) Pre-implantation mortality %: (Number of corpora lutea-Number of implantations / Number of corpora lutea) x 100

3) Intrauterine mortality %: (Number of implantations-Number of liveborns / Number of implantations) x 100

4) Total mortality %: (Number of implantations-Number of viable pups (PND0/4/13) / Number of implantations) x 100

5) Sex ratio % (females): (Number of female pups (PND0/4/13) / Number of viable pups (PND0/4/13)) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Adverse treatment-related effects were observed in high dose male rats during the study. Hunched back, piloerection, and slightly decreased activity were observed in one high dose male (#4003) in the morning of Day 6. This animal was found dead that same afternoon. Similar signs were observed for another male rat (#4005) in the high dose group on Days 10 and 11, that was found dead on Day 12. Additionally, one surviving high dose male (#4001) also displayed hunched back, red discharge from the nose, and slightly decreased activity on Days 12-14. Piloerection was also recorded for this animal on Days 12-20 and soft/liquid faeces was observed from Day 12-Day 28.

The following clinical observations were made but not considered to be treatment-related:
Hyperactivity, piloerection and extreme vaginal prolapse were recorded in one control female rat (#1507) on Day 39. This female was pre-terminally euthanized on the same day.

Red liquid from the vulva was noted for one female rat in the mid dose group (#3505) on Day 33 and 35. Liquid faeces was seen on Day 33, piloerection was also recorded in the period of Days 33-35, while hunched back was observed on Day 35. The animal was symptom-free from Days 36 to Day 40 and euthanized on Day 41 (GD 26).

Hunched back and piloerection were recorded in one low dose female rat (#2503) on Day 6. Noisy respiration (slight/moderate) was observed in the period of Days 6-9. Nodule (1-2 cm) was also seen in this female at the left axillary site from Day 17 until the end of the observation period.

Slight noisy respiration was detected in one mid dose male rat (#3006) on Days 2-5 and on Day 16, and in two high dose female rats (#4505 and #4507) on Day 3. Soft faeces were recorded for all male rats including controls in the period of Days 12-28.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Two male rats in the high dose group died during the study. One male rat (#4003) was found dead on Day 6, while the other male rat (#4005) was found dead on Day 12. The cause of death could not be determined. No clear treatment-related sign was observed at necropsy or histopathology examination. However, based on the observed clinical signs of these animals plus another male rat in the high dose group, these deaths were considered to be most probably test material related.

Other, non-treatment-related mortalities were also observed. One control male rat (#1012) was found dead on Day 15, although no clinical signs were recorded previously for this animal. Based on the necropsy findings, this was confirmed to be a gavage accident. One female rat (#1507) in the control group was pre-terminally euthanized on Day 39 (GD22, day of delivery) for animal welfare reasons (due to vaginal prolapse). One female in the low dose group (#2512) was found dead on Day 5, although no clinical signs were recorded previously for this animal. Based on the necropsy findings, this was confirmed to be a gavage accident. One female in the mid-dose group (#3505) was terminated on GD 25. Previous signs of premature delivery were observed on GD19 (vaginal bleeding, hunched back and piloerection). The animal still had a hunched back, piloerection, and increased respiratory rate on GD21, but no pups were delivered within the expected timeframe.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A slight treatment-related effect on body weight or body weight gain was detected during the study in the high dose group male rats.

A slightly (not statistically significant) lower weight gain was observed in high dose male rats from Day 0 to Day 7 / Day 14. However, this was largely due to 2 high dose males (#4003 and #4005) which died (a third male showing severe clinical signs (#4001) might have also been involved in the effect). Overall there was no significant effect on the body weight or weight gain of surviving high dose male rats. High dose female rats clearly did not demonstrate any adverse effect of the treatment on body weight or body weight gain (they were statistically above the control mean values). There were no treatment-related effects on body weight or body weight gain observed in the mid- or low-dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A slight adverse, but transient treatment-related effect was observed on food consumption in the first week of the treatment in the high dose group male rats, with no significant difference in the overall food consumption for the 28-day treatment period. No effect was observed in other males or any female rats.

Slight but statistically significant (p<0.01) decreased food consumption was observed in the first week of the treatment in the high dose group male rats (by 16%) when compared to control animals. However, there was no statistical significance in the daily food consumption value of the high dose males when calculated for the entire treatment period. No similar trend was observed in female rats. The effect did correspond with the body weights of the high dose males in the first week, but the transient difference at the start of the treatment was considered to be a minor effect with no overall significance over the 28 days. There was no decrease in food intake observed in the mid- or low-dose group males, or in female rats in any dose group.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed in the haematology parameters. Statistically significant differences were considered to be incidental. There was no relationship with dose and/or no similar trend in the other sex. None of these differences were considered to reflect any effect of treatment with the test material.

Statistically significant increase in mean cell volume (by 8%, p<0.01) and statistically significant decrease in red blood cell count (by 7%, p<0.05) and in mean cell haemoglobin concentration (by 4%, p<0.05) were observed in male rats in the high dose group when compared to the control group. However, there was no clear dose response, and no similar trend seen in female rats. All the mean group values were within the historical control range and therefore, these findings were not attributed to treatment with the test material.

The relative amount of eosinophil leukocytes showed relatively large percentage differences in the treated groups when compared to control due to the low absolute values which is normal for this parameter. Statistical significance was also gained in high dose female rats. However, opposite trends were seen in the treated males and females and the observed values were within the historical control range. Thus, these differences were considered as animal variability, and not a treatment-related effect.

No biologically relevant, statistically significant changes were recorded for any other haematology parameters in the treated male and female rats.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant changes or biologically relevant effects on the serum chemistry parameters evaluated that could be ascribed to exposure to the test material.

Increased (statistically significant; p<0.01) phosphorus and chloride levels and decreased (statistically significant; p<0.05) glucose concentration were seen in high dose males when compared to control animals. A statistically significant decrease (p<0.01) in urea concentration was also observed in the high- and mid-dose group females when compared to control animals. Decreased urea concentration (p<0.05) was also recorded for the low-dose group female rats. However, mean values were within the historical control range or there was no dose response or no similar trend seen in the other sex in those cases. Therefore, the observed differences were considered as being incidental and not treatment-related.

Statistically increased albumin concentration (by 10%, p<0.05) and therefore increased albumin/globulin ratio was detected in high dose males when compared to the control group. Similar but smaller differences were observed in the high dose females (by 6%), but no statistical significance was achieved. However, since there was no dose response, and the values were well within the historical control range in both cases, these findings were considered to be animal variability, and not a treatment-related effect.

No other statistically significant changes were recorded for any other parameters in the treated male and female rats.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed in the urinalysis parameters. Significantly (at p<0.05) lower pH values of the collected urine samples were recorded in the high dose group males when compared to the control. However, the observed mean values were comparable with the pH of control
females, and all the individual values were within the historical control range. No similar trend was observed in female animals (although a minor decrease in the high dose group females was noted). Therefore, the observed differences were not considered to be treatment-related.

A slight increase (significant at p<0.05) in the semi-quantitative urinary leukocyte assessment was also observed in the high dose males when compared to the control animals. However, no similar trend was observed in female animals. Thus, the observed value was not considered to be treatment-related.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups. There was no effect of treatment observed during the assessment of grip strength, foot splay or motor activity.

All dose groups of male and female rats had a normal locomotor activity; in all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 15-30 minutes. There was no statistically significant difference between the treated and control animals when evaluating the overall distance travelled (0-60 minutes). The test material did not affect normal locomotor activity.

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):

Parental Generation (Found Dead)
For control male (#1012) found dead on Day 15 of the treatment, the histopathological examination confirmed correlating changes in the oesophagus which included marked inflammation and moderate haemorrhage in the adventitial part of the oesophagus. Chronic inflammation was also noted around the thyroids. In addition, focal mild ulceration, degeneration and necrosis of the muscle and mixed inflammation was noted at the tip of the tongue. Spleen had moderate extra medullary haematopoiesis. Gavage accident was considered as the factor contributing to the death of this animal.

For low-dose group female (#2512) found dead on Day 5 of the treatment, histopathological examination revealed correlating changes in the oesophagus consisting of multifocal degeneration/necrosis of the muscular layer, haemorrhage and inflammation. Gavage accident was considered as the factor contributing to the death of this animal.

For high dose male (#4003) found dead on Day 6 of the treatment, the macroscopic changes in the thymus correlated with reduced cellularity of the thymus, microscopically. Other microscopic changes included multifocal minimal erosion/ulcers in the glandular stomach, moderate bilateral increase in the porphyrins in the harderian gland and focal minimal infiltration of inflammatory cells in the pericardium. The factor contributing the death of this animal was not determined.

For the second high-dose group male (#4005) found dead on Day 12 of treatment, the microscopic
changes included multifocal minimal erosion/ulcers in the glandular stomach, decreased cellularity of the thymus, mild extramedullary haematopoiesis in the spleen and multifocal minimal infiltration of inflammatory cells in the heart. Necropsy or microscopic examination did not reveal any significant changes to identify the factor contributing to the death of this animal.

Parental Generation (Pre-terminal Euthanasia)
For control group female (#1507) pre-terminally euthanized on Day 39 post treatment, necropsy revealed protruding vagina, which correlated with moderate increase in vaginal mucification at microscopic examination. Other histopathological changes included mild bilateral retinal atrophy in the eye, moderate extramedullary haematopoiesis in the spleen, minimal focal erosion in the glandular stomach, minimal focal unilateral tubular basophilia in the kidney and marked decreased secretion in the mammary gland.

For mid-dose group female (#3505) terminated on GD 26, organs from the reproductive system (ovary, oviduct, uterus and cervix) when examined microscopically did not reveal any treatment-related changes. Organs from the reproductive system (testes, epididymis, prostate, seminal vesicle and coagulating gland) were microscopically examined from the mating partner of this animal (#3005) and did not reveal any treatment-related changes.

Parental Generation (Terminal - Day 28 or PPD14)
Treatment-related findings were observed in the liver of the mid-dose group males (250 mg/Kg bw/day) and in both sexes of the high-dose group (1000 mg/Kg bw/day) and in the kidney of the high-dose males.

Minimal centrilobular hepatocellular hypertrophy was observed in the liver of 3/12 mid-dose group males (#3001, #3002 and #3012), 5/10 high-dose group males (#4002, #4004, #4006, #4009 and #4010), and 5/9 high-dose group female rats (#4503, #4504, #4506, #4507 and #4509). The centrilobular hepatocellular hypertrophy observed in those animals were considered as a non-adverse adaptive response. This microscopic finding correlated with increased organ weight changes.

Minimal to mild focal/multifocal bilateral or multifocal unilateral tubular basophilia was observed in the kidney of 9/10 high-dose group male rats (#4001, #4002, #4004, #4006, #4008, #4009, #4010, #4011 and #4012). One control male (#1010), three low dose males (#2004, #2007 and #2009), and three mid-dose group male rats (#3001, #3004 and #3012) also had the same findings.

The increased incidence and severity of tubular basophilia observed in the kidney of the high-dose group male rats when compared to the control group was considered as an indication of tubular degeneration/regeneration. The changes observed in the kidney correlated with the slightly increased kidney weights in high dose males. No similar changes were observed in the high dose female rats. The incidence of basophilic tubules in the low- and mid-dose group males was of minimal severity and at comparable incidence to control males without a dose dependent trend. Therefore, this was not considered to be treatment-related.

Moderate focal erosion/ulcer of the glandular stomach was observed in one (1/5) high dose male rats (#4006). This finding correlated with focal red discolouration in the glandular stomach observed during necropsy. It should be noted that the high dose males that were found dead and the pre-terminally euthanized control female also had minimal multifocal erosion/ulcer in the glandular stomach. Thus, erosion/ulcer observed in the glandular stomach of one terminal high dose male rat was considered to have an uncertain relationship with test material treatment.

Two (2/5) high dose male rats had axonal degeneration in the sciatic nerve (minimal focal in male #4004 and mild multifocal in male #4006). In the absence of any correlating clinical symptoms and being confined only to males at minimal focal severity in one animal, this finding was considered incidental. The incidence of axonal degeneration in the sciatic nerve in this study is above the historical control data for this finding in the Test Facility (26 studies, 0.4% in control females (range 0 – 10%) and none in control males). However, it has been reported that incidence of nerve fibre degeneration as a background change ranges from 1% to 20% and even young adult rats may exhibit high spontaneous incidence (Pardo et al 2020).

Minimal unilateral/bilateral follicular cell hypertrophy observed in the thyroid in occasional treated males (1/12 low dose (#2001) and 2/12 mid dose (#3009 and #3011) males) were also considered within the common background. All other changes observed in the control and/or treated animals were without meaningful differences in severity and incidence, and therefore regarded as incidental or common background.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis
No endocrine disruptor effect of the test material was observed in the study based on the results of thyroid hormone analysis, thyroid gland weights and thyroid histopathology. Significantly reduced T4 (thyroxin) hormone concentration was detected for parental males of the mid- and high-dose groups, although the group mean values were within the historical control range. Furthermore, no similar difference was recorded in PND13 pups. The Phase Report of the thyroid hormone
analysis.

The measurement of the thyroid hormone levels in the parental females was not performed as it was not deemed necessary by the Study Director / Sponsor; based on the observed results of the thyroid hormone analysis or the additional histopathology results.

Additionally, no statistically significant increase compared to control was detected in the absolute or relative thyroid weights in parental animals. Some observed minor differences (without statistical significance and dose response) were considered as animal variability, not being a treatment-related effect.

The thyroid glands of all males were examined microscopically. The histopathology revealed no alteration in any of the high dose group males. Minor background findings were recorded in the mid- and low-dose groups. No alterations were recorded in female rats (5 control and 5 high dose) and all examined samples were normal. Thus, the histopathology evaluation confirmed the lack of effect on the T4 hormone post exposure to the test material.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No effect of test material was observed on oestrus cycles of the female rats.

Each female selected for the treatment showed acceptable cycles (mean cycle length was 4.00 days for each group) before starting the treatment period. There was no effect of treatment on the oestrus cycle of females (mean cycle length was in the 4.65-4.94 days for each treated group after the treatment, while a mean cycle length of 4.28 days was recorded for control females). No prolonged dioestrus and no pseudopregnancy was noted for any females after the treatment was started. Prolonged oestrus was recorded in some control and treated females, but the frequency was similar between the groups, and almost all of those females successfully mated later.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with treatment. The male fertility index was 91% in the control group and 91%, 100% and 90% in the low-, mid- and high-dose groups, respectively. The female fertility index was 83% in the control group and 91%, 100%, and 75% in the low-, mid-, and high-dose groups, respectively. The number of non-pregnant females in the treated groups was lower or comparable to the control group. The gestation index was in the normal range in all groups (92-100%). Exposure to the test material was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 5 days of pairing (cohabitation) for all females. The mean duration of mating was 2.83, 3.55, 3.08 and 3.25 days in control, low-, mid-, and high-dose groups, respectively.

No treatment-related effect was observed during the gestation, parturition and postpartum period. The mean length of gestation was 22.30, 22.10, 22.09 and 22.22 days in control, low-, mid-, and high-dose groups, respectively. All of these values were within the normal range and there were no statistically significant differences, indicating no effect of treatment on this parameter. As far as it could be observed during the study, parturition was normal for all animals.

The number of implantation sites was comparable to the control mean in all dose groups and no statistically significant differences were noted. There were no statistically significant differences or effects that could be ascribed to treatment on pre-natal, post-natal or total mortality values (litter mean and %) in any dose group.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs or abnormalities were recorded for any pups through the study.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No treatment-related mortality or effect on the survival of pups observed through the study period. There were no significant differences or effects that could be attributed to treatment on the pre-natal, post-natal, or total mortality values (litter mean and %) in any of the dose groups. The number of viable pups on PND0, 4 and 13 as well as the survival index of the pups at given time points were comparable to control value in each dose group. Thus, there was no treatment-related effect on the viability of pups at those time points.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related differences in the offspring body weights or weight gains in any treated group when compared to the control animals.

When evaluated per litter basis, the mean litter body weights on PND 0, PND 4 and PND 13 and body weight gain in the relevant periods showed no statistically significant differences compared to controls in the F1 generation (the difference was less than 8.5% in every case). In summary, there were no effects of treatment on pup weights or weight gains.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No effect of treatment on T4 (thyroxin) hormone concentration was recorded in PND13 pups. The measurement of the thyroid hormone levels in the PND4 pups was not performed as it was not deemed necessary by the Study Director / Sponsor based on the observed results of the thyroid hormone analysis or the additional histopathology results.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the anogenital distance between the treated groups (males/females) and the control animals on PND0.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There was no nipples/areolae presence observed in any of the male pups on PND13.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No statistically significant increase compared to control was detected in the absolute or relative thyroid weights in the F1 generation. Some observed minor differences (without statistical significance and dose response) were considered as animal variability, not being a treatment-related effect.

Gross pathological findings:

no effects observed

Description (incidence and severity):

F1 Generation (Found Dead)
During the lactation period no pups found dead remained intact, but some cannibalized pups were recorded. Therefore, no necropsy with macroscopic examination and no histopathological examination was performed on pups (F1 generation).

F1 Generation (Terminal - PND13)
No treatment-related macroscopic changes were seen in the F1 offspring euthanized and examined externally at scheduled termination on PND13.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No treatment-related effect on the sex ratio was observed in the study.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Table 7. Analytical Results
Nominal concentration (mg/mL)	Measured concentration (mg/mL)	Percentage of the nominal Concentration (%)
Analytical sampling #1 (Sampling: 29 October 2018)
Control	<LOQ	N/A
15	15.45 ± 0.17	103.0
50	49.76 ± 2.74	99.5
200	200.91 ± 14.64	100.5
Analytical sampling #2 (Sampling: 19 November 2018)
Control	<LOQ	N/A
15	15.69 ± 0.04	104.6
50	50.99 ± 2.16	102.0
200	203.79 ± 6.27	101.9
Analytical sampling #3 (Sampling: 17 December 2018)
Control	<LOQ	N/A
15	15.70 ± 0.23	104.7
50	48.83 ± 0.49	97.7
200	210.20 ± 7.08	105.1

Notes: Formulation samples were measured within one week after samples collection.

Mean measured concentrations with the 95% confidence intervals are shown.

LOQ (Limit of Quantification) was 3 μg/mL for analytical samples, which equalled to 3.0 mg/mL for formulation samples.

N/A: Not applicable

Table 8. Summary of Selected Body Weight Parameters of Parental Animals
Parameter	Dose Groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Males
Male, Body weight on Day 27 (g) (NS)	528.5	525.3	526.3	520.0
Male, Body weight on Day 27 (g) (NS)	Difference (%)	-0.6	-0.4	-1.6
Male, Body weight gain Day 0-7 (g) (all males) (NS)	8.8	10.8	8.8	0.6
Male, Body weight gain Day 0-7 (g) (all males) (NS)	Difference (%)	22.9	0.0	-92.6
Male, Body weight gain Day 0-27 (g) (excluded High dose males^#) (NS)	8.8	10.8	8.8	6.5
	Difference (%)	22.9	0.0	-25.7
Male, Body weight gain Day 7-14 (g) (NS)	16.0	21.6	17.6	7.5
Male, Body weight gain Day 7-14 (g) (NS)	Difference (%)	34.9	9.9	-53.1
Male, Body weight gain Day 0-27 (g) (NS)	33.5	32.3	32.0	27.7
Male, Body weight gain Day 0-27 (g) (NS)	Difference (%)	-3.6	-4.3	-17.2
Females
Female, Body weight on GD20 (g) (NS)	429.3	434.1	422.3	447.1
Female, Body weight on GD20 (g) (NS)	Difference (%)	1.1	-1.6	4.1
Female, Body weight on PPD13 (g) (DN)	360.8	380.3	373.4	391.6**
Female, Body weight on PPD13 (g) (DN)	Difference (%)	5.4	3.5	8.5
Female, Body weight gain Day 0-7 (g) (NS)	-5.8	-5.2	-4.8	0.2
Female, Body weight gain Day 0-7 (g) (NS)	Difference (%)	-11.2	-18.6	-102.9
Female, Body weight gain Day 0-PPD13 (g) (D)	93.1	106.4	105.5	119.8**
Female, Body weight gain Day 0-PPD13 (g) (D)	Difference (%)	14.3	13.3	28.6

Notes: Data (group mean values, n=9-12) were rounded to one decimal place.

#: Values of found dead High dose males (#4003 on Day 6 and #4005 on Day 12) excluded from evaluation. In case of the third High dose male showing severe clinical sign (#4001) excluded, then the resulted body weight gain for the first week of treatment is 10.1 g for the High dose group (15.6% higher than control), for the second week of treatment it is 20.7 g (29.2% higher than Control). Non-pregnant females or females with total intrauterine mortality (#1501, #1512, #2503, #4502, #4505 and #4510); female not delivering pups (#3505) and pre-terminally euthanized animals (#1507) were excluded from the evaluation of gestation and lactation periods.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s Test

D: Duncan’s Multiple Range test

NS: Statistically not significant when compared to the control.

Table 9. Results of Daily Food Consumption (Parental Generation)
Parameter	Dose groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Males
Male, Food consumption Day 0-7 (g) (DU)	28.05	27.70	27.64	23.63**
Male, Food consumption Day 0-7 (g) (DU)	difference (%)	-1.2	-1.4	-15.8
Male, Food consumption Day 0-27 (g) (NS)	27.42	28.07	27.64	26.62
Male, Food consumption Day 0-27 (g) (NS)	difference (%)	2.3	0.8	-2.9
Females
Female, Food consumption Day 0-7 (g) (NS)	18.54	18.16	17.85	19.24
Female, Food consumption Day 0-7 (g) (NS)	difference (%)	-2.0	-3.7	3.8
Female, Food consumption GD 0-20 (g) (NS)	27.06	27.10	24.67	27.51
Female, Food consumption GD 0-20 (g) (NS)	difference (%)	0.1	-8.8	1.7
Female, Food consumption PPD 0-13 (g) (NS)	52.90	56.54	57.21	58.03
Female, Food consumption PPD 0-13 (g) (NS)	difference (%)	6.9	8.2	9.7

Notes: Data (group mean values, n=9-12) were rounded to two decimal places.

Non-pregnant females or females with total intrauterine mortality (#1501, #1512, #2503, #4502, #4505 and #4510); female not delivering pups (#3505); and pre-terminally euthanized animal (#1507) were excluded from the evaluation.

GD: Gestation Day, PPD: Post-partum Day.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DU: Dunn test

NS: Statistically not significant when compared to the control.

Table 10. Results of selected FOB and SMART parameters (Parental Generation)
Parameter	Dose groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Males
Landing foot splay, mm (hind paws) (NS) HC range: 26-129	78.4	74.8	57.7	72.1
Landing foot splay, mm (hind paws) (NS) HC range: 26-129	difference (%)	-4.6	-26.4	-8.1
Grip-strength, g (forelimbs) (NS) HC range: 1100-2333	2070.5	2089.3	1878.7	2039.1
Grip-strength, g (forelimbs) (NS) HC range: 1100-2333	difference (%)	0.9	-9.3	-1.5
Grip-strength, g (hind limbs) (NS) HC range: 483-1378	707.3	832.8	775.2	820.7
Grip-strength, g (hind limbs) (NS) HC range: 483-1378	difference (%)	17.7	9.6	16.0
Total travelled distance (cm) (NS) HC data: 2307-10078	6651.8	7154.0	6011.8	7764.1
Total travelled distance (cm) (NS) HC data: 2307-10078	difference (%)	7.6	-9.6	16.7
Females
Landing foot splay, mm (NS) (hind paws) HC range: 35-108	61.9	77.7	65.8	68.7
Landing foot splay, mm (NS) (hind paws) HC range: 35-108	difference (%)	25.6	6.4	11.0
Grip-strength, g (forelimbs) (NS) HC range: 795-1935	1519.9	1619.0	1589.9	1530.1
Grip-strength, g (forelimbs) (NS) HC range: 795-1935	difference (%)	6.5	4.6	0.7
Grip-strength, g (hind limbs) (NS) HC range: 392-1265	633.5	591.3	599.9	607.9
Grip-strength, g (hind limbs) (NS) HC range: 392-1265	difference (%)	-6.7	-5.3	-4.0
Total travelled distance (cm) (NS) HC data: 2749-11632	5698.9	7095.8	6319.2	4983.6
Total travelled distance (cm) (NS) HC data: 2749-11632	difference (%)	24.5	10.9	-12.6

Notes: Data (group mean values, n=5) are rounded to one digit, HC data are rounded to whole number.

Total travelled distance of 0-60 minutes was calculated.

HC: Historical control

NS: Statistically not significant when compared to the control.

Table 11. Results of Selected Haematology Parameters (Parental Generation)
Parameter	Dose groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Males
Red Blood Cell Count (x 10⁶/μL) (DN) HC range: 7.52-9.85	8.86	8.80	9.01	8.23*
Red Blood Cell Count (x 10⁶/μL) (DN) HC range: 7.52-9.85	difference (%)	-0.6	1.7	-7.1
Mean Cell Volume (fL) (DN) HC range: 49.10-59.10	52.28	53.38	51.80	56.36**
Mean Cell Volume (fL) (DN) HC range: 49.10-59.10	difference (%)	2.1	-0.9	7.8
MCHC (g/dL) (DU) HC range:31.90-36.80	32.56	32.40	32.44	31.16*
MCHC (g/dL) (DU) HC range:31.90-36.80	difference (%)	-0.5	-0.4	-4.3
Relative amount of Eosinophils (%) (NS) HC range: 0.00-3.90	1.34	2.16	2.36	1.40
Relative amount of Eosinophils (%) (NS) HC range: 0.00-3.90	difference (%)	61.2	76.1	4.5
Females
Red Blood Cell Count (x 10⁶/μL) (NS) HC range: 5.46-8.64	7.69	7.24	7.36	7.36
Red Blood Cell Count (x 10⁶/μL) (NS) HC range: 5.46-8.64	difference (%)	-5.8	-4.3	-4.2
Mean Cell Volume (fL) (NS) HC range: 51.70-72.40	59.60	61.82	59.40	60.22
Mean Cell Volume (fL) (NS) HC range: 51.70-72.40	difference (%)	3.7	-0.3	1.0
MCHC (g/dL) (NS) HC range:30.30-36.40	31.94	31.68	32.20	32.02
MCHC (g/dL) (NS) HC range:30.30-36.40	difference (%)	-0.8	0.8	0.3
Relative amount of Eosinophils (%) (DN) HC range: 0.30-9.30	2.00	1.26	1.26	1.04*
Relative amount of Eosinophils (%) (DN) HC range: 0.30-9.30	difference (%)	-37.0	-37.0	-48.0

Notes: Data (group mean values, n=5) were rounded to two decimal places. HC: Historical control.

MCHC: Mean Cell Haemoglobin Concentration

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test

DU: Dunn test

NS: Statistically not significant when compared to control

Table 12. Results of Selected Clinical Chemistry Parameters (Parental Generation)
Parameter	Dose groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Males
Glucose (mmol/L) DU) HC range: 4.49-12.11	10.60	9.20	9.13	8.35*
Glucose (mmol/L) DU) HC range: 4.49-12.11	difference (%)	-13.2	-13.9	-21.2
Phosphorus (mmol/L) (DN) HC range:1.96-3.16	2.49	2.62	2.57	3.37**
Phosphorus (mmol/L) (DN) HC range:1.96-3.16	difference (%)	5.2	3.1	35.2
Chloride (mmol/L) (DN) HC range: 94.90-107.10	99.34	100.46	101.02	104.40**
Chloride (mmol/L) (DN) HC range: 94.90-107.10	difference (%)	1.1	1.7	5.1
Albumin (g/L) (DN) HC range: 23.10-35.60	29.80	30.30	30.04	32.90*
Albumin (g/L) (DN) HC range: 23.10-35.60	difference (%)	1.7	0.8	10.4
Urea (mmol/L) (NS) HC range: 4.01-9.89	8.27	7.76	7.68	8.39
Urea (mmol/L) (NS) HC range: 4.01-9.89	difference (%)	-6.1	-7.2	1.5
Females
Glucose (mmol/L) (NS) HC range: 5.17-11.84	7.46	8.80	7.87	7.71
Glucose (mmol/L) (NS) HC range: 5.17-11.84	difference (%)	18.0	5.5	3.4
Phosphorus (mmol/L) (NS) HC range: 1.60-3.78	3.03	3.14	2.69	2.92
Phosphorus (mmol/L) (NS) HC range: 1.60-3.78	difference (%)	3.6	-11.3	-3.9
Chloride (mmol/L) (NS) HC range:90.70-106.60	99.88	100.76	103.74	103.00
Chloride (mmol/L) (NS) HC range:90.70-106.60	difference (%)	0.9	3.9	3.1
Albumin (g/L) (NS) HC range: 25.70-42.70	30.34	31.34	30.08	32.02
Albumin (g/L) (NS) HC range: 25.70-42.70	difference (%)	3.3	-0.9	5.5
Urea (mmol/L) (DN) HC range: 4.44-11.18	17.76	14.10*	13.57**	13.38**
Urea (mmol/L) (DN) HC range: 4.44-11.18	difference (%)	-20.6	-23.6	-24.7

Notes: Data (group mean values, n=5) were rounded to two decimal places.

HC: Historical control.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test

DU: Dunn test

NS: Statistically not significant when compared to control

Table 13. Results of Selected Urinalysis Parameters (Parental Generation)
Parameter	Dose groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Males
pH (DU) HC range: 6.0-8.0	7.80	7.60	7.60	6.40*
pH (DU) HC range: 6.0-8.0	difference (%)	-2.6	-2.6	-17.9
Urinary leukocytes (DU)	0.2	0.2	0.6	1.4*
Females
pH (NS) HC range: 6.0-8.0	6.80	6.60	6.40	6.60
pH (NS) HC range: 6.0-8.0	difference (%)	-2.9	-5.9	-2.9
Urinary leukocytes (NS)	0.0	0.2	0.2	0.2

Notes: Data (group mean values, n=5) were rounded to one or two decimal places.

HC: Historical control.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DU: Dunn test

NS: Statistically not significant when compared to control

Table 14. Results of Reproductive Parameters (Parental Generation)
Parameter	Dose groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Males
Number of treated animals	12	12	12	12
Number of pre-terminal death	0^§	0	0	2^§
Number of males used for mating	11^§	11^§§	12	10
Number of successful mating	11	11	12	10
Number of fertile males	10	10	12	9^§
Male mating index (%)	100	100	100	100
Male fertility index (%)	91	91	100	90
Females
Number of treated animals	12	12	12	12
Number of pre-terminal death (before mating)	0	1^§	0	0
Number of females used for mating	12^§§	11	12	12^§§
Number of sperm-positive females	12	11	12	12
Number of females with no implantation sites	2	1	0	3
Number of pregnant females	10	10	12	9
Pregnant females with live born(s)	10	10	11	9
Pregnant females not delivered living pups	0¥	0	1¥	0
Female mating index (%)	100	100	100	100
Female fertility index (%)	83	91	100	75
Female gestation index (%)	100	100	92	100

§Note: One Control Male (#1012) died on the second day of the mating period, this animal was

excluded from evaluation.

§§Note: One female in the High dose group (#2512) was found dead before the mating started. Therefore, its male pair was not used for mating.

#: Due to pre-terminal deaths before the mating period (#4003 and #4005), two High dose males (#4002 and #4011) were used for mating with two different females. They were counted as fertile even though one mating did not result pregnant female, but the second mating was successful.

§Note: One female in the Low dose group (#2512) was found dead before the mating started.

§§Note: Although its mating pair (#1012) died on the second day of mating, the mating period of the #1512 female was continued using another male (#1001). Similarly, in the High dose group where males (#4003 and #4005) died before the mating started, the mating of their mating female pairs was conducted with different males (#4503 was mated with #4002, and #4505 was mated with #4011). No corpora lutea and/or no implantation sites were recorded in two Control (#1501, #1512), one Low dose (#2503) and three High dose (#4502, #4505, #4510) females.

¥Note: One Control (#1507) female were pre-terminally euthanized on the day of delivery after giving birth to 10 living pups, one Mid dose (#3505) female was euthanized on GD 26.

Table 15. Results of Pregnancy Evaluation (P Generation)
Parameter	Dose groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Number of evaluated females	12	11	12	12
Number of pregnant females	10	10	12	9
Duration of pregnancy (days) (NS)	22.30	22.10	22.09	22.2
Number of implantations, mean (NS)	12.50	13.55	14.75	11.67
Number of pups born, mean (NS)	13.70	13.50	12.83	14.44
Number of live born pups, mean (NS)	12.80	13.40	12.75	14.33
Pre-natal mortality, mean (NS)	2.20	1.50	2.00	1.29
Pre-natal mortality (%), mean (NS)	19.55	9.58	13.76	8.27
Post-natal mortality, mean (NS)	0.44	0.10	0.18	0.11
Post-natal mortality (%), mean (NS)	14.14	0.71	1.14	0.65
Total mortality, mean (NS)	2.33	1.60	2.17	1.29
Total mortality (%), mean (NS)	23.66	10.30	14.69	8.27

Notes: Data (group mean values) were rounded to two decimal places. Pre-natal mortality includes intrauterine mortality and the loss at delivery. Post-natal mortality PND0-13 and total mortality on PND13 are shown in the table (the number of pups culled for blood sampling on PND4 were

excluded).

NS: Statistically not significant when compared to the control

Table 16. Summary of Survival (Offspring – F1)
Parameter	Dose groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Number of evaluated litters	10	10	11	9
Number of pups born, mean (NS)	13.70	13.50	12.83	14.44
Number of live born pups, mean (NS)	12.80	13.40	12.75	14.33
Number of living pups on PND13, mean (NS)	10.89	11.30	11.73	12.22
Pre-natal mortality, mean (NS)	2.20	1.50	2.00	1.29
Pre-natal mortality (%), mean (NS)	19.55	9.58	13.76	8.27
Post-natal mortality on PND0-4, mean (NS)	0.44	0.10	0.18	0.00
Post-natal mortality on PND0-4 (%), mean (NS)	14.14	0.71	1.14	0.00
Total mortality on PND4, mean (NS)	2.33	1.60	2.17	1.29
Total mortality on PND4 (%), mean (NS)	23.66	10.30	14.69	8.27
Pups culled for blood sampling, mean (NS)	1.78	2.00	2.00	2.00
Post-natal mortality on PND0-13, mean (NS)	0.44	0.10	0.18	0.11
Post-natal mortality on PND0-13 (%), mean (NS)	14.14	0.71	1.14	0.65
Total mortality on PND13, mean (NS)	2.33	1.60	2.17	1.29
Total mortality on PND13 (%), mean (NS)	23.66	10.30	14.69	8.27
Survival index on PND0 (NS)	89.17	99.17	99.39	99.38
Sex ratio (%) on PND0 (NS)	45.04	50.79	42.48	49.88
Survival index on PND4 (NS)	85.86	99.29	98.86	100.00
Sex ratio (%) on PND4 (NS)	54.84	51.17	42.95	49.88
Survival index on PND13 (NS)	100.0	100.0	100.0	99.26
Sex ratio (%) on PND13 (NS)	53.94	51.70	41.85	50.16

Notes: Data (group mean values) were rounded to two decimal places. Sex ratio means the percentage of females per litter. Culling for blood sampling was made on PND4. Survival index was calculated in comparison with the end of previous period (on PND0 it was compared to the number of pups born, on PND4 it was compared to the number of live born pups, on PND13 it was compared to the number of pups after culling on PND4). One Control female (#1507) was pre-terminally euthanized due to animal welfare reason on the day of delivery, her litter (10 pups) was also euthanized on the same day, thus this litter is not included in the calculation after PND0.

NS: Statistically not significant when compared to control

Table 17. Summary of Mortality (Offspring – F1)
Parameter	Dose groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Number of evaluated litters	10	10	11	9
Number of pups born (NS)	137	135	154	130
Number of cannibalized pups (CH^¥)	7 / 3	1* / 1	0** / 0	1* / 1
Number of autolyzed pups (NS)	2 / 2	0 / 0	1 / 1	0 / 0
Number of stillborn pups (NS)	0 / 0	0 / 0	0 / 0	0 / 0
Number of live born pups (CH^¥)	128	134*	153**	129*
Number of found dead pups (born alive) (NA)	0 / 0	0 / 0	0 / 0	0 / 0
Number of pups euthanized^#(NA)	10 / 1	0 / 0	0 / 0	0 / 0
Number of living pups on PND0 (NA)	118	134	153	129
Number of cannibalized pups (PND0-13)	4 / 3	1 / 1	2 / 2	1/ 1
Number of autolyzed pups (PND0-13) (NA)	0 / 0	0 / 0	0 / 0	0 / 0
Number of found dead, intact pups (PND0-13) (NA)	0 / 0	0 / 0	0 / 0	0 / 0
Total number of pups died (born alive) (NA)	0 / 0	0 / 0	0 / 0	0 / 0
Culled for blood sampling on PND4 (NA)	16 / 8	20 / 10	22 / 11	18 / 9
Number of viable pups on PND13 (NS)	98	113	129	110

Notes: Mortality numbers mean number of pups / number of affected litters. PND0-13 means the lactation period, counted after the delivery was ended.

#: One Control female (#1507) was pre-terminally euthanized due to animal welfare reason on the day of delivery, her litter (10 pups) was also euthanized on the same day.

Statistical significance compared to control: * = p<0.05, ** = p<0.01.

(¥: Due to the negative trend it has no biological relevance)

NA: Not applicable

CH: Chi square test

NS: Statistically not significant compared to control

Table 18. Summary of Selected Body Weight Parameters of Offspring (F1)
Parameter	Dose Groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Number of evaluated litters	10	10	11	9
Mean litter body weight (PND0), g (NS)	6.230	6.310	6.507	6.427
Mean litter body weight (PND4), g (NS)	10.103	10.485	10.426	10.213
Mean litter body weight gain (PND0-4), g (NS)	3.913	4.164	3.919	3.787
Mean litter body weight (PND13), g (NS)	28.192	29.751	29.051	26.785
Mean litter body weight gain (PND4-13), g (NS)	18.101	19.320	18.646	16.595
Mean litter body weight gain (PND0-13), g (NS)	22.019	23.474	22.549	20.388

Notes: Body weight / body weight gain data (litter mean values) were rounded to two decimal places.

NS: Statistically not significant when compared to control

Table 19. Anogenital Distance (Offspring (F1))
Parameter	Dose Groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Male Pups
Number of evaluated litters	10	10	11	9
Number of evaluated male pups (NS)	65	68	89	65
Anogenital distance, litter mean of males (mm) (NS)	3.36	3.30	3.27	3.25
Anogenital distance, all male pups (mm) (NS)	3.34	3.31	3.27	3.27
Minimum / Maximum value, litter mean (mm) (NS)	2.55 / 4.06	2.39 / 3.86	2.64 / 3.93	2.72 / 3.71
Female Pups
Number of evaluated litters	9	10	11	9
Number of evaluated female pups (NS)	63	66	64	64
Anogenital distance, litter mean of females (mm) (NS)	1.32	1.32	1.32	1.29
Anogenital distance, mean of all females (mm) (NS)	1.32	1.32	1.33	1.29
Minimum / Maximum value, litter mean (mm) (NS)	1.03 / 1.69	1.12 / 1.62	1.04 / 1.66	1.00 / 1.52

Notes: Data (group mean or litter mean values) were rounded to one or two decimal places. In litter #1504 only one male pup was born.

NS: Statistically not significant when compared to control

Table 20. Results of Selected Organ Weights (Parental Generation)
Parameter	Dose groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Males
Number of Evaluated Males	11	11	12	10
Terminal body weight, g (NS)	506.4	502.8	500.7	498.8
Terminal body weight, g (NS)	difference (%)	-0.7	-1.1	-3.3
Liver (absolute), g (DU)	14.068	14.353	16.042*	17.718**
Liver (absolute), g (DU)	difference (%)	2.0	14.0	25.9
Liver (relative to body), % (DU)	2.782	2.851	3.200**	3.620**
Liver (relative to body), % (DU)	difference (%)	2.5	15.0	30.1
Kidney (absolute), g (NS)	3.385	3.247	3.429	3.650
Kidney (absolute), g (NS)	difference (%)	-4.1	1.3	7.8
Kidney (relative to body), % (DN)	0.669	0.647	0.685	0.746**
Kidney (relative to body), % (DN)	difference (%)	-3.4	2.4	11.4
Females
Number of Evaluated Females	9	10	11	9
Terminal body weight, g (NS)	333.1	348.5	345.0	347.8
Terminal body weight, g (NS)	difference (%)	4.6	3.6	4.4
Liver (absolute), g (DN)	12.893	14.318	14.497*	16.803**
Liver (absolute), g (DN)	difference (%)	11.0	12.4	30.0
Liver (relative to body), % (DU)	3.868	4.113	4.199	4.826**
Liver (relative to body), % (DU)	difference (%)	6.3	8.6	24.8
Kidney (absolute), g (DU)	2.200	2.193	2.241	2.470*
Kidney (absolute), g (DU)	difference (%)	-0.3	1.9	12.3
Kidney (relative to body), % (NS)	0.662	0.630	0.651	0.712
Kidney (relative to body), % (NS)	difference (%)	-4.8	-1.7	7.6

Notes: Data (group mean values) were rounded to one or three decimal places.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s Multiple Range Test

DU: Dunn test

NS: Statistically not significant when compared to the control

Table 21. Summary of Histopathological Findings in the Kidney
Parameter	Dose groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Parental Males
Tubular basophilia, bilateral, multifocal/focal or unilateralmultifocal, minimal	1 / 11	3 / 12	3 / 12	6 / 10
Tubular basophilia, bilateral, multifocal/focal or unilateral multifocal, mild	0 / 11	0 / 12	0 / 12	3 / 10
Parental Females
Tubular basophilia, bilateral, multifocal/focal, minimal	0 / 5	-	-	0 / 5
Tubular basophilia, bilateral, multifocal/focal, mild	0 / 5	-	-	0 / 5
Tubular basophilia, unilateral, focal, minimal	0 / 5	-	-	0 / 5

Notes: Incidences and total number of analysed samples are shown. Minimal unilateral focal basophilic tubules observed in Control (2/11), Low dose (1/12) and Mid dose (4/12) males were considered as within the common background and excluded from the table.

Table 22. Selected Parameters related to Thyroid Hormone Levels
Parameter	Dose groups
Parameter	Control (0 mg/Kg/day)	75 mg/Kg/day	250 mg/Kg/day	1000 mg/Kg/day
Parental Males
Number of evaluated males	11	12	12	10
T4 concentration (ng/mL) (DN) HC range: 34.3-60.7	42.42	40.63	35.78**	33.00**
T4 concentration (ng/mL) (DN) HC range: 34.3-60.7	difference (%)	-4.2	-15.7	-22.2
Thyroid gland weights (g) (NS)	0.0261	0.0263	0.0269	0.0270
Thyroid gland weights (g) (NS)	difference (%)	0.9	3.2	3.5
Thyroid gland / body weight (%) (NS)	0.0052	0.0053	0.0054	0.0055
Thyroid gland / body weight (%) (NS)	difference (%)	2.2	4.1	7.2
Parental Females
Number of evaluated Females	9	10	11	9
Thyroid gland weights (g) (NS)	0.0259	0.0220	0.0194	0.0311
Thyroid gland weights (g) (NS)	difference (%)	-15.0	-25.2	20.2
Thyroid gland / body weight (%) (NS)	0.0078	0.0063	0.0057	0.0089
Thyroid gland / body weight (%) (NS)	difference (%)	-18.7	-27.2	14.8
PND13 Pups
Number of evaluated litters	8	10	11	9
T4 concentration (ng/mL) (NS) HC range: 26.3-61.6	40.45	42.76	40.78	42.04
T4 concentration (ng/mL) (NS) HC range: 26.3-61.6	difference (%)	5.7	0.8	3.9
Thyroid gland weights (g) (NS)	0.0057	0.0057	0.0051	0.0051
Thyroid gland weights (g) (NS)	difference (%)	0.2	-10.5	-10.1
Thyroid gland / body weight (%) (NS)	2.0087	1.8811	1.7471	1.8868
Thyroid gland / body weight (%) (NS)	difference (%)	-6.4	-13.0	-6.1

Notes: Data (group mean values) were rounded to two or four decimal places. Thyroid and parathyroid weights were measured together. Thyroid gland weight for one male and one female pup per litter were determined except of litters #1504 and #1507 where there was as no living pup on PND13, furthermore eight females (#1501, #1512, #2503, #2512, #3505, #4502, #4505 and #4510) died or had no litters. Pups blood were pooled for T4 (thyroxin) determination. HC: Historical control.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s Multiple Range Test

NS: Statistically not significant when compared to the control

Conclusions:

Based on the results observed in this combined repeated dose, reproductive/developmental toxicity screening study, the No Observed Adverse Effect Level (NOAEL) for Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile was determined to be 250 mg/Kg bw/day for systemic toxicity of the male parental (adult) animals (based on effects on the kidney at the highest dose tested) and 1000 mg/Kg bw/day for systemic toxicity of the female parental (adult) animals. The NOAEL for reproductive and developmental toxicity in rats was determined to be 1000 mg/Kg bw/day, based on lack of adverse treatment-related effects observed at the highest dose tested.

Executive summary:

In a key Guideline OECD 422 combined repeated dose, reproductive/developmental toxicity screening study, the toxicity potential of the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile; EC# 915-371-2) was evaluated in rats following repeated exposure via oral gavage. Crl:WI (Wistar) rats (12/sex/dose) were exposed to the test material via gavage once daily at doses of 0, 75, 250, or 1000 mg/Kg bw/day for 2 weeks before mating, during mating, up to and including the day before the necropsy. The total exposure was 28 days in total for males while females were treated throughout gestation up to and including postpartum/lactation day (PPD) 13. The control group received the vehicle Polyethylene glycol 400 (PEG 400) only.

Parameters measured during the study included signs of morbidity and mortality, observation of clinical signs daily (general) or weekly (detailed), neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment of functional observation battery (including the measurements of the landing foot splay and grip strength) and measurement of locomotor activity was performed during the last week of treatment. Additionally, reproductive performance, pregnancy, parturition, and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND 13.

At termination, gross necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues / organs sampled and preserved in appropriate fixatives from the adult animals as well as offspring. Thyroxine (T4) levels in the adult males and PND 13 pups were also assessed. For the adult animals, a detailed histological examination was performed on a select list ofretained organs of 5 control and high dose animals per sex, and all those male / female mating pairs where no conceptus and/or no live born pups was achieved. Track down exanimation of liver samples of all remaining animals as well as kidneys and thyroids of all remaining males were also conducted.

Two male rats in the high-dose group died during the study. These two males and one surviving male in the same dose group exhibited adverse clinical signs (decreased activity, piloerection, and hunched back) which were attributed to treatment. The cause of death of the two male rats found dead during the study could not be determined at necropsy or during the histopathological examination.

No clear treatment-related effect on mean body weight, body weight gain was observed in the study (although reduced body weight gain was seen in 3/12 high dose males which died or showed severe clinical signs). No treatment-related effects were noted in the food consumption calculated for the whole treatment period in any of the treated groups when compared to the control animals.

Haematology, coagulation, and clinical chemistry parameters evaluated were unaffected by treatment and there was no treatment-related effect detected during the neurotoxicity assessment.

Treatment-related effects were observed in the liver weights of male and female rats in the mid- and high-dose groups, as well as in the kidney weights of the high dose male rats. These liver weight changes correlated with histopathological changes in the high dose animals of both sex. The kidney weight changes also correlated with histopathological findings in the high dose male rats. Liver weight changes in mid dose male rats also correlated with histopathological changes. No clear treatment-related macroscopic findings were observed in any of the dose groups at terminal necropsy, but treatment-related microscopic findings were observed during the histopathology assessment in the liver of the mid-dose group males and in both sexes of the high dose group as well as in the kidney of high-dose group male rats. The hepatic changes appeared to be non-adverse adaptive changes (centrilobular hepatocellular hypertrophy) but the changes in male kidneys in the high-dose group represented an adverse tubular degeneration/regeneration.

Based on the results of the thyroid hormone analysis, thyroid gland weights, thyroid histopathology and anogenital distance, no treatment-related endocrine disruptor effect was observed in the study.

Oestrus cycle of parental females remained unaffected by treatment and exposure to the test material had no adverse impact on the reproductive parameters evaluated, on gestation, parturition or on lactation.

Exposure to the test material had no adverse effect on F1 offspring viability or on physical or sexual development of the pups. No adverse clinical signs of toxicity were observed in the pups and gross necropsy of F1 pups also did not reveal any remarkable findings.

Based on the results observed in this combined repeated dose, reproductive/developmental toxicity screening study, the No Observed Adverse Effect Level (NOAEL) for Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile was determined to be 250 mg/Kg bw/day for systemic toxicity of the male parental (adult) animals (based on effects on the kidney at the highest dose tested) and 1000 mg/Kg bw/day for systemic toxicity of the female parental (adult) animals. The NOAEL for reproductive and developmental toxicity in rats was determined to be 1000 mg/Kg bw/day, based on lack of adverse treatment-related effects observed at the highest dose tested.