Octadecene, reaction products with hexadecene, hydrogenated

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Two studies are available on the substance as follows:

OECD 422: SynNova Base Oil: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Administration in Wistar Rats

OECD 443: Extended one-generation reproductive toxicity – with F2 generation (Cohorts 1A and 1B with extension of Cohort 1B to F2 generation

 

The results of the OECD 422 study on the substance itself demonstrated the following results:

 

The NOAEL for systemic toxicity of the parental generation was considered to be 1000 mg/kg bw/day.

The NOAEL for reproductive effects of the parental generation was considered to be 1000 mg/kg bw/day.

The NOAEL for Pup development and survival was considered to be 1000 mg/kg bw/day.

 

For the OECD 443, daily administration of SynNova® Base Oil by dietary route to male and female Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day, under the conditions of this study, did not cause test item-related adverse effects in any of the examined parameters in any of the dose groups, neither in Parental nor in the Cohort 1A, 1B, Group 1X, F1 and F2 offspring. The increased food consumption observed, is a normal physiological response to the physicochemical changes of the food caused by the inclusion of the test item in the diet.  The following results were obtained:

Parental generation:

NOAEL (systemic toxicity):      1000 mg/kg bw/day

NOAEL (reproductive effects): 1000 mg/kg bw/day

F1 generation:

NOAEL (developmental toxicity):         1000 mg/kg bw/day

NOAEL (systemic toxicity):      1000 mg/kg bw/day

NOAEL (reproductive effects): 1000 mg/kg bw/day

F2 generation:

NOAEL (developmental toxicity):         1000 mg/kg bw/day

The substance showed no effects for reproduction toxicity in either of the studies conducted.

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 September 2021 to 04 May 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Required study for Annex X. Note that this study was conducted to enable notification in Asia-Pacific regions.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 443, Extended One-Generation Reproductive Toxicity Study, Organisation for Economic Co-Operation and Development, Paris, 25 June 2018

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Justification for study design:

In accordance with the test guideline.

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar rat as a rodent is one of the standard strains for repeated dose and reproductive toxicity studies. Wistar rat was selected due to experience with this strain in toxicity and reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Han:WIST rats
Source: Toxi-Coop Zrt., H-1122 Budapest, Magyar Jakobinusok tere 4B
Hygienic level at supplier: SPF
Hygienic level during the study: Standard housing conditions
Number of groups: 4 groups (3 dose groups plus one control group)
Number of P animals: Parental (P) generation: 96 male and 96 female rats, 24 animals/sex/group for treatment.
Sex of P animals: Male and female. The females were nulliparous and non-pregnant at the start of the study.
Age of P animals: Young adult rats, approximately 11 to 12 weeks old at start of dosing and 13 to 14 weeks old at mating.
Body weight range of P animals: Males: 330 – 419 g, females: 202 – 239 g; did not exceed ± 20 % of the mean weight for each sex at onset of dosing.
Acclimation period: at least 13 days

Husbandry
Animal health: Only healthy animals were used for the test. The health status was certified by the clinical Veterinarian.
Housing: Parental animals were be group-housed until mating and were housed individually after mating until necropsy. During the lactation period, Parental dams were housed with their pups. F1 pups were group-housed after weaning until necropsy (Cohort 1A and Cohort 2A) or until the mating period (Cohort 1B). Cohort 1B animals were housed individually after mating, until necropsy. During the lactation period, Cohort 1B dams were housed with their pups.
Cage type: T3H polycarbonate
Bedding and nesting: “SAFE 3/4-S-FASERN” certified wooden chips (batch number: 03027210315 / 03027210601 / 03027210927 / 03027211209, expiry date: 15 March 2024/ 01 June 2024 / 27 September 2024 / 09 December 2024) produced by J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) and “Sizzle pet” / “Sizzle nest” nest material batch number: 201016/02 / SIZNEST00022W, expiry date: 01 December 2023 / 10 May 2024) produced by LBS (Serving Biotechnology) Ltd. (Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH, United Kingdom) were available to animals during the study.
Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19 – 24 °C (target range 22 ± 3 °C)
Relative humidity: 30 – 70 % (target range 30-70 %)
Ventilation: 15-20 air exchanges/hour
Enrichment: Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities. Nest building material allowed normal nesting behaviour. Fresh bedding was provided for the animals as frequently as appropriate/practical, but at least twice weekly.

Food and water supply
During acclimatisation, the animals received standard laboratory rat diet, ad libitum. The food is considered not to contain any contaminants that could affect the purpose or integrity of the study.

Details of the diets used during the acclimatisation were as follows:
Name: SM Rat/Mouse, Breeding & Maintenance, 10 mm, autoclavable
Manufacturer: ssniff Spezialdiäten GmbH (D-59494 Soest, Germany)
Batch number: 233 77046 / 431 81989
Expiry date: 31 September 2021 / 28 February 2022

Drinking water
Animals received tap water from the municipal supply, as for human consumption, from drinking bottles, ad libitum. Water quality control analysis and microbiological assessment are performed once per year by the laboratory of Veszprém County Government Office, Department of Public Health (Veszprém Megyei Kormányhivatal Népegészségügyi Főosztály, H-8200 Veszprém, József A. u. 36., Hungary).

Randomisation
P animals: On the day of the start of dosing, parental animals were randomly allocated to the control and dose groups by randomisation based on their most recent body weight. All animals were within 20% of the overall mean at the start of the study and group means were similar. Males and females were randomised separately.
F1 animals: At weaning (on PND 21) three males and three females from 20 litter of each dose group were selected for further examinations in Cohort 1A, Cohort 1B and Group 1X. Pups were selected and assigned randomly. In one case, due to the insufficient number of male pups in a litter, one pup from another litter with the same age has been used to complete the pups chosen for the Cohorts: the male pup 2501/4 was assigned to the litter 2510 (later animal number 6046 in Group 1X).

Animal identification
Each adult/parental animal (P Generation) and selected F1 animals (from PND 21) was individually identified by unique numbers written on the tail with an indelible pen. In case of parental (P) animals, this number was cross-referred to the Animal Master File of the Test Facility.
The animal number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental Design section.
The boxes were marked by identity cards, with information about study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery.
Identification of the new-borns (Offspring) from the day of birth to weaning was performed by permanently marking of the tips of the digits on the day of birth. The pup ID consisted of the dam’s ID, then a sequential number unique within the litter separated by a slash (e.g. 2506/3).

Route of administration:

oral: feed

Vehicle:

corn oil

Details on exposure:

Carrier item
To ensure the proper distribution of the test item in the diet, it was added with carrier oil. For this purpose, corn oil was chosen, as it was considered as proper technically for supporting the test item’s distribution.

Name: Corn Oil
Batch/Lot number: 21-0172
CAS number: 8001-30-7
Appearance: Light/Faint yellow oil
Purity: Ph.Eur.10, Supplement 10.1
Expiry date: 30 November 2022
Storage conditions: Room temperature (15-25 ºC, below 70 RH%)

Vehicle item
Ssniff® SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” was used as a vehicle for SynNova® Base oil.

Details of the diets used in the study were as follows:
Name: SM Rat/Mouse, Breeding & Maintenance, 10 mm, autoclavable
Manufacturer: ssniff Spezialdiäten GmbH (D-59494 Soest, Germany)
Batch number: Control (0 mg/kg test item):4721235 / 8311543 / 3241250 / 4602102
Low dose (1500 mg/kg test item): 4741235 / 8471244 / 3251250 / 4402501
Mid dose (5000 mg/kg test item): 4761235 / 8481244 / 3261250 / 4412501
High dose (15000 mg/kg test item): 4781335 / 84911244 / 3271250 / 4612102
Expiry date: 31 January 2022 / 31 March 2022 / 30 April 2022 / 31 May 2022

Diet
The test item was incorporated into ssniff® SM R/M-Z “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” by ssniff Spezialdiäten GmbH, D-59494 Soest Germany. Ssniff Spezialdiäten GmbH was chosen as manufacturer of the diet because of their extensive experience in the production of experimental diets for regulatory purposes.
In the first step, a premix was prepared from the proper amount of test item and corn oil (carrier). In the second step, this premix was mixed with the ingredients of the basal diet, and pelleted.
The prepared diets were stored at room temperature under dry conditions in sealed bags pending and during transport to the Test Facility. At the Test Facility, the prepared diet bags were stored in the diet storage room (19-25°C, humidity: 30-70%), pending transfer to animal room at 22 ± 3 °C for animal feeding.

Details on mating procedure:

Parental animals: Mating began 2 weeks after the initiation of dosing, with one male and one female (1:1 mating) in a single cage. Males remained with the female until copulation occurred. The presence of sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy).
Due to failing to achieve the sufficient number of pregnant animals in the low and mid dose groups during the initial mating period (day 14 to day 21), the non-pregnant animals were re mated with their original pair in order to fulfill the requirements of the OECD 443 guideline (at least 20 pregnant females per dose group).
F1 Cohort 1B animals: Animals of Cohort 1B were mated to obtain an F2 generation. Males and females of the same dose group were cohabited (avoiding the pairing of siblings) beginning on or after PND 90, but not exceeding PND 120. Procedures were similar to those for the P animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration of each diet batch delivered to the Test Facility was determined after arrival and near at the end of use.

For homogeneity determination, 3x at least 10 g pelleted diet samples were taken from the top, middle and bottom of the diet container. In total, 9 samples/diet container were taken and analysed in the laboratory of the Test Facility following proper sample preparation by the validated GC-FID method [10] (N21001-917). As for the control diet, at least 50 g pelleted diet sample were collected and analysed.

The diet formulation analyses were conducted under the control of the Analyst. According to the analytical method validation, acceptance criteria of the concentration analysis were set to be at 100 ± 25 % of the nominal value. Acceptance criteria of the homogeneity were that the CV of replicates must be less than 10%.

Duration of treatment / exposure:

The dosing of both sexes of the Parental and F1 generation was performed as detailed below:
- Parental males: were dosed from Day 0 until Day 69 days; gross necropsy on Day 70.
- Parental females were dosed from Day 0 until PPD21; gross necropsy on PPD 22.
- F1 Cohort 1A males and females: were dosed from PND21 until PND89; gross necropsy on PND90.
- F1 Cohort 1B males: were dosed from PND21 until the day before necropsy; gross necropsy after termination of Cohort 1B dams, at 21 weeks of age.
- F1 Cohort 1B females: were dosed from PND21 until PPD21; gross necropsy on PPD22.
- Group 1X animals: were dosed from PND21 until necropsy (after confirmed sexual maturation)

Frequency of treatment:

The test item was administered to the animals by dietary route on a 7 days/week basis.

Details on study schedule:

P generation: 96 male and 96 female animals (Parental generation; 24 male and female animals/dose group) were mated after two weeks treatment period. The female animals were treated during pregnancy and during the lactation period, and were necropsied after the weaning of their litters (on PPD 22). The male animals were treated totally for 10 weeks and then they were necropsied.

F1 generation: On PND 21, 20 litters of each dose group were chosen at random for continuation of treatment, out of which, each one male and one female was assigned to Cohort 1A, Cohort 1B and Group 1X.

F1 Cohort 1A (Reproductive/developmental toxicity testing; 20 males and 20 females): All animals were treated after weaning until PND89, and were necropsied on PND 90.

F1 Cohort 1B (Reproductive/developmental toxicity testing; 20 males and 20 females): Animals of Cohort 1B were mated to obtain a F2 generation. Males and females of the same dose group were cohabited (avoiding the pairing of siblings) beginning on or after PND90, but not exceeding PND120. Parental F1 males were terminated at 21 weeks of age, dams and litters (F2 generation) were sacrificed on PPD22/PND21.

Group 1X (Extra group for evaluation of sexual maturation; 20 males and 20 females): All animals were treated for at least until sexual maturation, and then were euthanized followed by the examination of their stomach.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Dietary Concentration (ppm): 0

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Dietary Concentration (ppm): 1500

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Dietary Concentration (ppm): 5000

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Dietary Concentration (ppm): 15000

No. of animals per sex per dose:

P Generation: 96 males/96 females (24 animals per dose group)
F1 Generation (Cohort 1A): 80 male/80 females (20 per dose group)
F1 Generation (Cohort 1B): 80 males/80 females (20 per dose group)
F1 Generation (Group 1X): 80 males/80 females (20 per dose group)

Control animals:

yes, plain diet

Details on study design:

The dose levels were selected by the Sponsor in consultation with the Study Director, based on available data and information from previous experimental work; including a reproduction toxicity study according to OECD 422, a teratology study according to OECD 414, a 90 day toxicity study, according to OECD 408 and a Palatability study performed at the Test Facility.

The oral (dietary) route was selected as it is one of the possible routes of human exposure.

Positive control:

Not required for the study.

Parental animals: Observations and examinations:

Clinical observations
Parental animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
General clinical observations were made once a day.

Detailed clinical observations were made on P animals outside the home cage in a standard arena in the morning hours. Observation was performed on the skin, fur, eyes and mucous membranes, occurrence of secretions and excretions, autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behavioural pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagy/self-mutilation, backward motion, abnormal vocalization, aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (hunchback posture, etc.), gait, posture and response to handling and to environmental stimulation. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
On GD 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).

Body weight measurements
P animals: Body weights of all parental animals were recorded with a precision of 1 g on the first day of dosing (Day 0, prior to start of dosing), thereafter twice weekly until pairing.
After mating, parental males were weighed at least weekly and on the day of scheduled necropsy (fasted).
After mating, parental females were weighed on GD 0, 3, 7, 10, 14, 17 and 20 and on PPD 0 (within 24 hours after parturition), 4, 7, 10, 14, 17, 21 and on the day of scheduled necropsy (fasted).

Food consumption and dose intake
The schedule of food consumption determination (by weighing of the remaining, non-consumed food and the newly provided food, with a precision of 1g) was identical to the schedule of body weight determination (without weighing the food on the day of scheduled necropsy).

During periods of pairing (cohabitation), individual food consumption was not determined.

The mean absolute daily food consumption per animal was calculated (g/animal/day) by calculating the difference in the weight of the food at the beginning and at the end of a given period for one animal. Food consumption per body weight (g/kg bw) was calculated as a quotient of the absolute food consumption and the body weight of the animal(s). Dose intake (mg/kg bw) was calculated on the basis of the food consumption per body weight and the nominal test item concentration of the food. In addition, food conversion efficiency was calculated as percentage of body weight gain per food consumed (%BWG/gFI). No food conversion efficiency was calculated for the post-partum period, as requested by the OECD 443 guideline.

Observation of the delivery process, offspring and nursing instinct
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. Any evidence of abnormal deliveries was recorded. The duration of gestation was recorded and calculated from Day 0 of pregnancy.

Dams were observed to record whether they formed a nest from the bedding material and covered their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach.

CLINICAL PATHOLOGY
Parental: prior to scheduled necropsy, clinical pathology investigations (haematology, blood clotting times, clinical chemistry and urinalysis) were conducted in 10 randomly selected male and female animals / group.
After an overnight period of food deprivation of animals, blood samples were collected by heart puncture under pentobarbital anaesthesia.

Haematology
An IDEXX ProCyte Dx Hematology Analyser was used. The blood was collected to tubes containing K2-EDTA as anticoagulant. The following parameters were determined from whole blood of selected animals, euthanized at termination:

RBC –Total number of erythrocytes; HCT –Hematocrit value: erythrocyte ratio of total blood volume; HGB – Hemoglobin concentration; MCV – Mean erythrocyte volume in total sample; MCH – Mean hemoglobin volume per red blood cell (RBC) count; MCHC – Mean hemoglobin concentration of erythrocytes; RDW – The degree of variation in size of the erythrocyte population; %RETIC – Reticulocyte percent; RETIC – Reticulocyte count; WBC – Total number of leukocytes; %NEU – Neutrophil percent; %LYM – Lymphocyte percent; %MONO – Monocyte percent; %EOS – Eosinophil percent; %BASO – Basophil percent; NEU – Neutrophil count; LYM – Lymphocyte count; MONO – Monocyte count; EOS – Eosinophil count; BASO – Basophil count; PLT – Total number of platelets; MPV – Mean platelet volume; PDW – Platelet distribution width; the degree of variation in size of the platelet population; PCT – Plateletcrit value

Blood clotting times
IDEXX Coag Dx Coagulation Analyzer was used. The blood was collected to tubes containing 3.2% sodium citrate as anticoagulant. The following parameters were evaluated in the selected animals, euthanized at termination from whole blood:

aPTT –Activated Partial Thromboplastin Time
PT – Prothrombin Time

Clinical chemistry
An IDEXX Catalyst One Chemistry Analyzer was used for clinical chemistry evaluation. The blood was collected to tubes containing lithium heparin as anticoagulant, and then processed for plasma. The following parameters evaluated in the selected animals, euthanized at termination from plasma:

ALB – Albumin; ALKP – Alkaline Phosphatase; ALT – Alanine Aminotransferase; AST – Aspartate aminotransferase; BUN/CREA – Blood Urea Nitrogen; Urea – Urea concentration; Ca – Calcium; CHOL – Cholesterol (total); CREA – Creatinine; Cl – Chloride; GGT – Gamma-glutamyltransferase; GLU – Glucosel K –Potassium; Na –Sodium; PHOS – Inorganic Phosphate; TBIL – Total Bilirubin; TP –Total Protein; TBA –Total bile acids


Urinalysis
Urine collection was conducted over approximately 16 hours in metabolic cages, during an overnight period of food deprivation in selected animals.

The urine samples were evaluated by observation and with Siemens CLINITEK Status Plus urinalyser, using Multistix® 10 SG reagent test strips. The sediment was examined under a light microscope. The following parameters were evaluated:

Visual assessment; Clarity; Colour; Volume

Urinalyser
LEU – Leukocyte; NIT – Nitrite; URO – Urobilinogen; PRO – Protein; pH; BLO – Blood (occult); SG – Specific Gravity; KET – Ketones; BIL – Bilirubin; GLU – Glucose

Sediment microscopic examination
White Blood Cell; Red Blood Cell; Epithelial Cell; Crystals; Bacteria; Amorphous globlets

THYROID HORMONE ANALYSIS

Parental animals: Blood samples were taken by cardiac puncture under pentobarbital anaesthesia from 10 randomly selected male and female animals / group into two tubes, one containing lithium heparin as anticoagulant (to be processed for plasma, for T4 and TSH measurement) and one containing no anticoagulant (to be processed for serum for potential T3 measurement).

The T4 (Thyroxine) levels were determined from the plasma samples by IDEXX Catalyst One Chemistry Analyzer (Catalyst Total T4 Test slide, batch: 708107 / 708115 / 708117 / 708121 / 708122, exp. date: 30 September 2022 / 14 December 2022 / 06 January 2023 / 26 January 2023 / 13 February 2023; measurable range: 6.4-257.4 nmol/L) on the day of sampling.

The processed remaining plasma and all serum samples were divided into at two aliquots (if applicable) and stored in a freezer.
From the first aliquots of frozen plasma samples TSH (Thyroid-stimulating hormone) level determination was performed, used Rat Thyroid Stimulating Hormone (TSH) ELISA kit (Cat. No.: abx156194, produced by Abbexa Ltd., batch number: E2110671N / E2201248U, exp. date: 30 May 2023 / 31 July 2022, (Limit of detection (LOD): 29 pg/mL / limit of quantification (LOQ): 97 pg/mL). BMG FLUOstar Omega Microplate Reader was used for the optical density measurements.

Oestrous cyclicity (parental animals):

Parental females were monitored daily for the stage of oestrous cycle by assessment of vaginal smears for two weeks before start of pairing and during pairing until evidence of mating.

When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which could induce pseudopregnancy.

Additionally, vaginal smears were prepared and examined for each Parental female animal on the day of necropsy to determine the stage of oestrous cycle and allow correlation with histopathology of the reproductive organs.
Vaginal smears were stained with 1 % aqueous methylene blue solution and were examined under a light microscope.

Sperm parameters (parental animals):

Sperm parameters were measured in all Parental males. At termination, testis and epididymis weights were recorded. One testis and one epididymis was reserved for histopathological examination. The remaining epididymis was used for enumeration of cauda epididymis sperm reserves, furthermore evaluation of sperm motility and morphology. Sperm motility was performed by preparing video records, morphology was examined by dispensing sperm suspension onto microscopical slides, followed by air drying.

Collection method:
Immediately after sacrifice, one cauda epididymis was measured, cautiously cut-in on a few sites with scissors, and placed into PE tubes containing 2.5 mL of PBS buffer with 20% fetal bovine serum, pre-warmed to 37°C and incubated for 15 minutes at 37°C to allow the sperm cells to diffuse into the medium. After 15 minutes, the tissues were taken out from the suspension.

Motility analysis:
5 µL of freshly prepared sperm-cell suspension was transferred onto a 37°C pre-warmed Shukratara® Sperm Counting Chamber, covered and video sequences of a few seconds were recorded immediately for later analysis. Using computer assisted motility analysis of the video recordings, the sperm cells were classified by a user as “non-moving” or “motile” sperm cells, for at least 200 sperms for each animal.

Enumeration of cauda epididymis sperm reserves:
Following the video recording for motility analysis, previously reserved cell suspensions were vortexed for approximately 10 seconds. 5 µL of the cell suspension was taken out and placed into a Shukratara® Sperm Counting Chamber, and sperms were counted under light microscope. Results were expressed as number of sperms per organ, and as number of sperms per g of tissues.

Morphology analysis:
For the purpose of morphology analysis, approximately 200 µL of sperm suspension was pipetted onto slides, spread and excess was decanted. Thereafter the slides were let dry, fixated in methanol and stained with Giemsa stain. Photos were taken of the slides, and morphology of the sperms was analysed based on the photos prepared. At least 200 spermatozoa per sample were examined for abnormalities such as fusion, isolated heads, misshapen heads and/or tails. Sperm cells were classified as “normal” or “abnormal”.

Litter observations:

Clinical observations

Selected F1 animals (after weaning) were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
General clinical observations were made once a day.

Detailed clinical observations were made on selected F1 animals (after weaning) outside the home cage in a standard arena in the morning hours. Observation was performed on the skin, fur, eyes and mucous membranes, occurrence of secretions and excretions, autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behavioural pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagy/self-mutilation, backward motion, abnormal vocalization, aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (hunchback posture, etc.), gait, posture and response to handling and to environmental stimulation. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
On GD 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).

F1 animals: To determine the timing of sexual maturation, vaginal patency was evaluated daily for each female beginning on PND 22. The body weight of each female was recorded on the day of vaginal patency. All males were evaluated for balanopreputial separation daily beginning on PND 35 and the body weight was recorded on the day of separation.
Body weight measurements
F1 animals: Live pups were weighed with a precision of 0.01 g at birth (PND 0) and on PND 4, 7, 10, 14, 17 and 21. After weaning, the selected animals were weighed with a precision of 1 g at least weekly and on the day of sexual maturation and on the day of scheduled necropsy (fasted). Body weight determination of the mated Cohort 1B dams followed the schedule of the dams of the parental generation.

Observation of the delivery process, offspring and nursing instinct
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births and the presence of gross abnormalities.
Runts (significantly smaller than normal pups; BW< control group mean-2*SD) were determined in the report.
In addition, the first clinical examination of the neonates included a qualitative assessment of body temperature, state of activity and reaction to handling. Intact dead pups were examined macroscopically for possible defects and cause of death, if possible.
The anogenital distance (AGD) of each pup was measured (with 0.01mm accuracy) at the time of the first weighing (PND 0). The body weight-normalized anogenital distance (the cube root of body weight) was calculated.
All the litters were controlled and recorded daily for the number of viable and dead pups, any abnormal behaviour or appearance of the pups were also recorded.
Detailed clinical observations, as applicable for the age of the animals, were performed on the days when the offspring were weighed. Signs noted included external abnormalities, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity. Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour, were also examined.
Number of nipples/areolae in male pups was recorded on PND 13.
On PND 4, the size of each litter was adjusted by eliminating (culling) extra pups (surplus pups) by random selection to yield, as nearly as possible, five pups per sex per litter. The surplus pups were subjected to gross necropsy and blood samples were taken into one tube containing lithium heparin as anticoagulant by decapitation for potential thyroid hormone (T4 and TSH) analysis. The blood was pooled by litters, processed for plasma and stored in freezer.
No pups were eliminated when litter size dropped below the culling target (10 pups/litter).
F1 pups not selected for further treatment (surplus pups) and all F2 pups were terminated on PND 21.

CLINICAL PATHOLOGY
Cohort 1A: prior to scheduled necropsy, clinical pathology investigations (haematology, blood clotting times, clinical chemistry and urinalysis) were conducted in 10 randomly selected male and female animals / group.

After an overnight period of food deprivation of animals, blood samples were collected by heart puncture under pentobarbital anaesthesia.

Haematology
An IDEXX ProCyte Dx Hematology Analyser was used. The blood was collected to tubes containing K2-EDTA as anticoagulant. The following parameters were determined from whole blood of selected animals, euthanized at termination:

RBC –Total number of erythrocytes; HCT –Hematocrit value: erythrocyte ratio of total blood volume; HGB – Hemoglobin concentration; MCV – Mean erythrocyte volume in total sample; MCH – Mean hemoglobin volume per red blood cell (RBC) count; MCHC – Mean hemoglobin concentration of erythrocytes; RDW – The degree of variation in size of the erythrocyte population; %RETIC – Reticulocyte percent; RETIC – Reticulocyte count; WBC – Total number of leukocytes; %NEU – Neutrophil percent; %LYM – Lymphocyte percent; %MONO – Monocyte percent; %EOS – Eosinophil percent; %BASO – Basophil percent; NEU – Neutrophil count; LYM – Lymphocyte count; MONO – Monocyte count; EOS – Eosinophil count; BASO – Basophil count; PLT – Total number of platelets; MPV – Mean platelet volume; PDW – Platelet distribution width; the degree of variation in size of the platelet population; PCT – Plateletcrit value

Blood clotting times
IDEXX Coag Dx Coagulation Analyzer was used. The blood was collected to tubes containing 3.2% sodium citrate as anticoagulant. The following parameters were evaluated in the selected animals, euthanized at termination from whole blood:

aPTT –Activated Partial Thromboplastin Time; PT – Prothrombin Time


Clinical chemistry
An IDEXX Catalyst One Chemistry Analyzer was used for clinical chemistry evaluation. The blood was collected to tubes containing lithium heparin as anticoagulant, and then processed for plasma. The following parameters evaluated in the selected animals, euthanized at termination from plasma:

ALB – Albumin; ALKP – Alkaline Phosphatase; ALT – Alanine Aminotransferase; AST – Aspartate aminotransferase; BUN/CREA – Blood Urea Nitrogen; Urea – Urea concentration; Ca – Calcium; CHOL – Cholesterol (total); CREA – Creatinine; Cl – Chloride; GGT – Gamma-glutamyltransferase; GLU – Glucose; K –Potassium; Na –Sodium; PHOS – Inorganic Phosphate; TBIL – Total Bilirubin; TP –Total Protein; TBA –Total bile acids

Urinalysis
Urine collection was conducted over approximately 16 hours in metabolic cages, during an overnight period of food deprivation in selected animals.
The urine samples were evaluated by observation and with Siemens CLINITEK Status Plus urinalyser, using Multistix® 10 SG reagent test strips. The sediment was examined under a light microscope. The following parameters were evaluated:

Visual assessment: Clarity; Colour; Volume

Urinalyser: LEU – Leukocyte; NIT – Nitrite; URO – Urobilinogen; PRO – Protein; pH; BLO – Blood (occult); SG – Specific Gravity; KET – Ketones; BIL – Bilirubin; GLU – Glucose
Sediment microscopic examination: White Blood Cell; Red Blood Cell; Epithelial Cell; Crystals; Bacteria; Amorphous globlets

Thyroid hormone analysis
Cohort 1A animals: Blood samples were taken by cardiac puncture under pentobarbital anaesthesia from 10 randomly selected male and female animals / group into two tubes, one containing lithium heparin as anticoagulant (to be processed for plasma, for T4 and TSH measurement) and one containing no anticoagulant (to be processed for serum for potential T3 measurement).
F1 and F2 PND21 pups: Blood samples were taken sublingually from 1 male and 1 female pups per litter (as applicable) into one tube containing lithium heparin as anticoagulant for thyroid hormone (T4 and TSH) analysis.

The T4 (Thyroxine) levels were determined from the plasma samples by IDEXX Catalyst One Chemistry Analyzer (Catalyst Total T4 Test slide, batch: 708107 / 708115 / 708117 / 708121 / 708122, exp. date: 30 September 2022 / 14 December 2022 / 06 January 2023 / 26 January 2023 / 13 February 2023; measurable range: 6.4-257.4 nmol/L) on the day of sampling.
The processed remaining plasma and all serum samples were divided into at two aliquots (if applicable) and stored in a freezer.
From the first aliquots of frozen plasma samples TSH (Thyroid-stimulating hormone) level determination was performed, used Rat Thyroid Stimulating Hormone (TSH) ELISA kit (Cat. No.: abx156194, produced by Abbexa Ltd., batch number: E2110671N / E2201248U, exp. date: 30 May 2023 / 31 July 2022, (Limit of detection (LOD): 29 pg/mL / limit of quantification (LOQ): 97 pg/mL). BMG FLUOstar Omega Microplate Reader was used for the optical density measurements.
In addition, blood samples were taken by decapitation from F1 and F2 PND4 surplus pups into one tube containing lithium heparin as anticoagulant for potential thyroid hormone (T4 and TSH) analysis. The blood was pooled by litters, processed for plasma and stored in freezer.

Based on the study results, no further thyroid hormone analysis was needed, therefore all frozen plasma and serum samples will be discarded following finalization of the report.

Postmortem examinations (parental animals):

Terminal procedures
Necropsy and macroscopic examination were performed on all Parental, animals, at the end of the dosing period. The animals were euthanized by exsanguination under pentobarbital anaesthesia (Euthanimal 40% (400 mg/mL pentobarbital sodium); Manufacturer: Alfasan Nederland BV; Batch number: 2104103-03; Expiry date: 31 May 2024). Unscheduled deaths did not occur in the study.

Macroscopic examination
After exsanguinations, the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were 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.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrous cycle and allow correlation with histopathology of the reproductive organs.

The number of implantation sites were recorded in the Parental females as applicable. The number of corpora lutea were recorded in the Parental female animals.

Intact dead pups and pups euthanized on PND 4 or PND 21 were carefully examined for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

Organ weight measurements
The following organs were trimmed of fat and weighed:
Parental animals:
With precision of 0.01g
Brain; Testes; Liver; Epididymides (total / cauda for sperm analysis); Spleen; Seminal vesicles with coagulating glands; Heart; Prostate (dorsolateral and ventral part combined); Kidneys; Uterus (including oviducts and cervix); Thymus
With precision of 0.001g
Adrenal glands; Thyroid with parathyroid glands; Pituitary; Ovaries
Testes, epididymides and the other paired organs were weighed together. If any significant difference in weight of paired organs were noted, than the individual weights were also determined. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.
 
Histopathology
On completion of the macroscopic examination, the following tissues and organs were retained from all animals:
Gross findings; Lungs with bronchi; Skeletal muscle (quadriceps); Adrenals; Lymph node; Small intestine; Animal identification; Ovary; Spinal cord; Aorta; Oviduct; Spleen; Brain; Pancreas; Sternum with marrow; Epididymis; Pituitary; Stomach; Eye with the optic nerve; Prostate; Testis; Oesophagus; Salivary gland (including mandibular, sublingual and parotid glands); Thymus; Femur with marrow; Thyroid with parathyroid gland; Heart; Tongue; Kidney; Sciatic nerve; Trachea; Large intestine; Seminal vesicle with coagulating gland; Urinary bladder; Extraorbital lachrymal gland; Uterus; Harderian gland; Skin, subcutis with mammary gland (inguinal); Vagina; Liver

The eyes with the optic nerve, testes and epidymides were preserved in modified Davidson’s fixative, all other organs in 10 % buffered formalin solution.

When microscopic examination was needed for a tissue or organ, the retained tissues and organs required for histopathology (below) were embedded in paraffin wax; sections were cut at 4-6 μm by microtome and transferred to microscopy slides. Tissue sections were stained with haematoxylin-eosin and examined by light microscope.

For the adult animals, detailed histological examination was performed as follows:

-retained tissues and organs in the Control and High dose groups of Parental animals,
-all macroscopic findings (abnormalities)
-retained reproductive organs of all animals of the low-, and mid-dose animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire, or deliver healthy offspring.
Detailed testicular histopathological examination were conducted in order to identify treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cell, or sloughing of cells into the lumen. Examination of the epididymis included evaluation of the caput, corpus and cauda using longitudinal sectioning.

Enumeration of primary follicles in ovaries
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. For randomly selected 10 females/dose group, a quantitative depletion of the primordial and small growing follicles as well as corpora lutea in post-lactational ovary were evaluated. For the enumeration of small follicles (primordial and primary follicles according to Pedersen’s follicular classification) ovary were fixed in formaldehyde, dehydrated in alcohols, clarified in xylene and embedded in paraffin. Embedded tissues were serially sliced into 4 µm sections. Five sections per animal were assessed so that a distance of 150 µm was kept between the assessed sections in order to avoid double-counting of follicles. For immunohistochemical staining, tissue sections mounted on microscope slides were deparaffinised, and cross bindings formed during formalin fixation were eliminated. With this, the epitopes became accessible for binding by the proliferating cell nuclear antigen (PCNA) antibodies. As the PCNA antibodies were biotin-labelled, they were detected with horse radish peroxidase (HRP) conjugated streptavidin. For the staining of the PCNA positive cells, 3,3’-Diaminobenzidine (DAB; a chromogenic substrate of HRP) was added, which forms a brown precipitate upon oxidization by HRP. Sections were additionally counter-stained with haematoxylin and the PCNA-positive (brown) follicles were visualized by light microscopy and counted.

Bone marrow analysis
Bone marrow smears from the femur were prepared from all Parental animals. Detailed cytological examination of hematopoietic cells was performed under oil immersion magnification (1000x). 300 nucleated cells were evaluated per animal. All the major lineages were assessed:
Granulopoiesis: Myeloblasts; Promyelocytes; Metamyelocytes; Neutrophil granulocytes (stab); Neutrophil granulocytes (band); Neutrophil granulocytes (segment, mature); Eosinophil granulocytes (premature); Eosinophil granulocytes (mature); Basophil granulocytes
Monopoiesis: Monocytes; Macrophages
Lymphopoiesis: Lymphocytes; Plasmacells
Erythropoiesis: Rubriblasts; Normoblasts (Basophilic, polychromatophilic and ortochromatophilic erythroblasts)
Thrombopoiesis: Megakaryocytes
Quantitative evaluation was done in case of myeloid and erythroid lines. Data were given both in absolute count and in percentage. Myeloid/Erythroid ratio was calculated. Semi-quantitative method was used for megakaryocytes, macrophages and plasma cells.

Postmortem examinations (offspring):

Terminal procedures
Necropsy and macroscopic examination were performed on all Cohort 1A and 1B, F1 PND21 surplus and F2 PND21 pups, at the end of the dosing period. The animals were euthanized by exsanguination under pentobarbital anaesthesia (Euthanimal 40% (400 mg/mL pentobarbital sodium); Manufacturer: Alfasan Nederland BV; Batch number: 2104103-03; Expiry date: 31 May 2024). Unscheduled deaths did not occur in the study. Based on consultation with the sponsor, from the Group 1X animals only stomachs were examined macroscopically.

Macroscopic examination
After exsanguinations, the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were 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.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrous cycle and allow correlation with histopathology of the reproductive organs.

The number of implantation sites were recorded in the Cohort 1B females as applicable. The number of corpora lutea were recorded in the Cohort 1A and Cohort 1B female animals.

Intact dead pups and pups euthanized on PND 4 or PND 21 were carefully examined for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

Organ weight measurements
The following organs were trimmed of fat and weighed:
Cohort 1A animals:
With precision of 0.01g
Brain; Testes; Liver; Epididymides (total / cauda for sperm analysis); Spleen; Seminal vesicles with coagulating glands; Heart; Prostate (dorsolateral and ventral part combined); Kidneys; Uterus (including oviducts and cervix); Thymus
With precision of 0.001g
Adrenal glands; Thyroid with parathyroid glands; Pituitary; Ovaries; Mandibular lymph node; Mesenteric lymph node

Cohort 1B animals:
With precision of 0.01g
Brain; Seminal vesicles with coagulating glands; Thymus; Prostate (dorsolateral and ventral part combined); Testes; Uterus (including oviducts and cervix); Epididymides
With precision of 0.001g
Adrenal glands; Thyroid with parathyroid glands; Pituitary; Ovaries; Mandibular lymph node; Mesenteric lymph node

F1 PND21 surplus pups:
With precision of 0.001g
All surplus pups: Epididymides; Prostate (dorsolateral and ventral part combined); Testes; Uterus (including oviducts and cervix); Ovaries
1 male and 1 female pups per litter (that ones which were used for serum thyroid hormone (T4 and TSH) analysis: Thyroid with parathyroid glands
10 pups per sex per group (in addition, mammary tissues for these pups were preserved for possible histopathological evaluation): Brain; Spleen; Thymus
F2 PND21 pups:
With precision of 0.001g
2 male and 2 female pups per litter: Epididymides; Prostate (dorsolateral and ventral part combined); Testes; Uterus (including oviducts and cervix); Ovaries
1 male and 1 female pups per litter (that ones which were used for serum thyroid hormone (T4 and TSH) analysis: Thyroid with parathyroid glands
10 pups per sex per group (in addition, mammary tissues for these pups were preserved for possible histopathological evaluation): Brain; Spleen; Thymus

Testes, epididymides and the other paired organs were weighed together. If any significant difference in weight of paired organs were noted, than the individual weights were also determined. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.

Histopathology
On completion of the macroscopic examination, the following tissues and organs were retained from all Cohort 1A and Cohort 1B animals:
Gross findings; Lungs with bronchi; Skeletal muscle (quadriceps); Adrenals; Lymph node; Small intestine; Animal identification; Ovary; Spinal cord; Aorta; Oviduct; Spleen; Brain; Pancreas; Sternum with marrow; Epididymis; Pituitary; Stomach; Eye with the optic nerve; Prostate; Testis; Oesophagus; Salivary gland (including mandibular, sublingual and parotid glands); Thymus; Femur with marrow; Thyroid with parathyroid gland; Heart; Tongue; Kidney; Sciatic nerve; Trachea; Large intestine; Seminal vesicle with coagulating gland; Urinary bladder; Extraorbital lachrymal gland; Uterus; Harderian gland; Skin, subcutis with mammary gland (inguinal); Vagina; Liver

The eyes with the optic nerve, testes and epidymides were preserved in modified Davidson’s fixative, all other organs in 10 % buffered formalin solution.

When microscopic examination was needed for a tissue or organ, the retained tissues and organs required for histopathology (below) were embedded in paraffin wax; sections were cut at 4-6 μm by microtome and transferred to microscopy slides. Tissue sections were stained with haematoxylin-eosin and examined by light microscope.

For the adult animals, detailed histological examination was performed as follows:

-retained tissues and organs in the Control and High dose groups of Cohort 1A animals,
-pituitary, testes, epididymides, seminal vesicles with coagulating glands, prostate, uterus, ovaries and vagina in the Control and High dose groups of Cohort 1B animals
-all macroscopic findings (abnormalities)
-retained reproductive organs of all animals of the low-, and mid-dose animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire, or deliver healthy offspring.
Detailed testicular histopathological examination were conducted in order to identify treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cell, or sloughing of cells into the lumen. Examination of the epididymis included evaluation of the caput, corpus and cauda using longitudinal sectioning.

Enumeration of primary follicles in ovaries
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. For randomly selected 10 females/dose group, a quantitative depletion of the primordial and small growing follicles as well as corpora lutea in post-lactational ovary were evaluated. For the enumeration of small follicles (primordial and primary follicles according to Pedersen’s follicular classification) ovary were fixed in formaldehyde, dehydrated in alcohols, clarified in xylene and embedded in paraffin. Embedded tissues were serially sliced into 4 µm sections. Five sections per animal were assessed so that a distance of 150 µm was kept between the assessed sections in order to avoid double-counting of follicles. For immunohistochemical staining, tissue sections mounted on microscope slides were deparaffinised, and cross bindings formed during formalin fixation were eliminated. With this, the epitopes became accessible for binding by the proliferating cell nuclear antigen (PCNA) antibodies. As the PCNA antibodies were biotin-labelled, they were detected with horse radish peroxidase (HRP) conjugated streptavidin. For the staining of the PCNA positive cells, 3,3’-Diaminobenzidine (DAB; a chromogenic substrate of HRP) was added, which forms a brown precipitate upon oxidization by HRP. Sections were additionally counter-stained with haematoxylin and the PCNA-positive (brown) follicles were visualized by light microscopy and counted.

Bone marrow analysis
Bone marrow smears from the femur were prepared from all Cohort 1A and 1B animals. Detailed cytological examination of hematopoietic cells was performed under oil immersion magnification (1000x). 300 nucleated cells were evaluated per animal. All the major lineages were assessed:
Granulopoiesis: Myeloblasts; Promyelocytes; Metamyelocytes; Neutrophil granulocytes (stab); Neutrophil granulocytes (band); Neutrophil granulocytes (segment, mature); Eosinophil granulocytes (premature); Eosinophil granulocytes (mature); Basophil granulocytes
Monopoiesis: Monocytes; Macrophages
Lymphopoiesis: Lymphocytes; Plasmacells
Erythropoiesis: Rubriblasts; Normoblasts (Basophilic, polychromatophilic and ortochromatophilic erythroblasts)
Thrombopoiesis: Megakaryocytes
Quantitative evaluation was done in case of myeloid and erythroid lines. Data were given both in absolute count and in percentage. Myeloid/Erythroid ratio was calculated. Semi-quantitative method was used for megakaryocytes, macrophages and plasma cells.

Statistics:

Statistical analysis was performed for the continuous variables using an automated decision tree within the R software. The following decision tree was applied:

The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Reproductive indices:

The reproductive parameters were counted by the following formulas:

Male mating index=(Number of males with confirmed mating)/(Total number of males cohabited) x 100

Male fertility index=(Number of males impregnating a female)/(Total number of males cohabited) x 100

Female mating index=(Number of sperm positive females)/(Total number of females cohabited) x 100

Female fertility index=(Number of pregnant females)/(Number of sperm positive females) x 100

Gestation index=(Number of females with liveborn pups)/(Number of pregnant females) x 100

Survival index=(Number of live pups (at designated time))/(Number of pups born) x 100

Offspring viability indices:

Survival index on PND 21 was calculated from number of pups after culling on PND 4 instead of number of pups born.

Intrauterine mortality=(Number of implantations-number of liveborns)/(Number of implantations) x 100

Total mortality=(Number of implantations-number of viable pups (at designated time))/(Number of implantations) x 100

Sex ratio=(Number of female pups (at designated time))/(Number of pups (at designated time)) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

During the general-, and detailed clinical observations, no clinical signs were observed in the current the study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was observed during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Administration of the test item incorporated into diet, did not cause any test item related effect on the body weight, or body weight gain of Parental males and females.
P generation males
Administration of the test item did not cause any effect in the body weight or body gain of the parental male animals, when compared to the control. As a sporadic event, the +52.3% elevation with a statistical significance of p<0.01 in body weight gain in the high dose-treated group restricted to the period between Day0 until Day 3 was considered incidental, rather than a test item related effect. The overall body weight and body weight gain was as expected in the animals of similar age and strain.
P generation females
There was no change in the body weight or body gain of the parental females that could be ascribed to the administration of the test item, when compared to the control. The statistically significantly lower body weight gain (-62.0% p<0.05) in the low dose group, noted in the period between PPD 17-21, was considered incidental in the absence of a dose dependency.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

P generation males
During the thorough observation period, a dose dependent elevation of food consumption was noted, attaining statistical significance at the majority of the measuring periods in the mid and high dose groups, and occasionally even in the low dose group, when compared to the control. The elevation was within the range of +2.3% to +9.9% in the low dose group, +4.8% to +8.8% in the mid dose group, and +6.8% to +12.3% in the high dose group. Statistical significance (p<0.05 or p<0.01 was observed at 3 out of 12 occasions in the low-, 9 out of 12 occasions in the mid-, and 12 out of 12 occasions in the high dose group. The overall means were higher than control in all dose groups in a dose dependent manner (+4.8%, +6.7% and +8.5% in the low, mid and high dose groups respectively) with a statistical significance of p<0.05 in the low dose group, and p<0.01 in the mid and high dose groups.
The similar trend was noted when the body weight normalized food consumption was calculated. The increase in comparison with the control was within the range of +1.3% to +6.7% in the low dose group, +3.6% to +7.9% in the mid dose group, and +6.0% to +8.4% in the high dose group. Statistical significance (p<0.05 or p<0.01 was observed at 4 out of 12 occasions in the low-, 11 out of 12 occasions in the mid-, and 12 out of 12 occasions in the high dose group. The overall means were higher than control in all dose groups in a dose dependent manner (+3.7%, +5.5% and +6.9% in the low, mid and high dose groups respectively) with a statistical significance of p<0.01 in all dose groups.
Food utilization of parental males was similar to the control in all dose groups except a single period from Day0 until Day 3, when a statistically significant +41.4%, p<0.01 elevation was noted. As this is a solitary event, without dose dependency, this observation can be regarded as incidental, and without toxicological relevance.

P generation females
Statistical significant differences in the absolute food consumption in comparison to the control were observed in the high dose-treated parental females on the pre-mating Days 3-7 (+11.4%, p<0.01) on Days 10-14 (+4.8%, p<0.05) and during the entire pre-mating period of Days 0-14 (+5.0%, p<0.05). While during the gestational days this group showed no statistical significant difference to control, statistical significant differences were noted during the post-partum days PPD 10-14(+7.1%, p<0.05), PPD 14-17 (+9.3% p<0.01) and PPD 17-21 (+6.7%, p<0.01).
Statistical significant increase of food consumption per body weight of the high dose-treated females was observed on the pre-mating days 3-7 (+11.3%, p<0.01) and on pre-mating days 10-14 (+4.6%, p<0.05) and during the entire pre-mating period (days 0 to 14: +4.7%, p<0.05). Even though statistical not significant, in the pre-mating period as of day 3, and throughout the gestational period, the test item-treated parental females a dose dependently increasing food consumption was noted. Statistical significant increased food intake was observed in the high dose-treated group, if the time weighted average of the entire gestation period (GD 0-20) was analyses (+3.9%, p<0.05). During the postnatal period, statistical significant difference in the body weight-normalized food intake to the control was noted in the high dose-treated parental female group on PPD14-17 (+7.5%, p<0.05).
Food utilization of the parental female animals showed no statistical significant difference to the control at any of the observation times.

Dose Intake of P generation
P generation males
The time-weighted average daily dose intakes of the low-, mid-, and high dose-treated males for the entire treatment period (Days 0-69) were 94.7, 321.2 and 976.6 mg/kg bw of the test item, which were 94.7%, 107.1% and 97.7% of the nominal dose.
P generation females
The time weighted mean daily dose intake of the low-, mid-, and high dose-treated females from Day 0 to PPD21 was 199.12, 601.3 and 2092.8 mg/kg bw, corresponding to 199.2%, 200.4% and 209.3% of the nominal dose.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

In the high dose-treated parental males, the relative (RETIC %) and absolute number of reticulocytes (RETIC) were increased by +15.7% (p<0.05) and +14.5% (p<0.05) compared to the control. An increased reticulocyte number (reticulocytosis) is an adaptive response to potential blood loss, but in lack of any significant change in the red blood cell parameters (erythrocytes (RBC), heamatocrit (HTC) and heamoglobin (HGB)) and in the lack of similar effect in the female animals the increased RETIC [%] and RETIC [K/L] values can be regarded as of no toxicological relevance.

Other variations were noted in a few parameters, attaining occasional statistical significance, such as higher absolute number of monocyte (MONO) in low dose females (p<0.05) and higher eosinophil (EOS) percentage in low and high dose females (p<0.05, respectively). In the lack of dose dependency, these changes are regarded as toxicologically not relevant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease of the globulin level (GLOB; -5.1%; p<0.05) and consequently an increased albumin/globulin ratio (ALB/GLOB) without a clear dose dependence was noted in the high dose-treated male group (+7.3%; p<0.05) compared to the control. Decreased synthesis of albumin and globulin generally caused by loss of hepatic functional mass, however the liver has a very large reserve capacity, therefore these laboratory abnormalities typically are not detectable until approximately 70% of hepatic functional mass has been lost . With this in mind, in the lack of any macroscopic or histopathological alteration, the observed decrease in globulin group mean and the subsequently higher ALB/GLOB ratio is considered as a normal biological variance rather than a test item related effect.

Statistically significant increase of total cholesterol (CHOL tot) group mean was noted in the mid dose-treated females (+21.2%; p<0.05). As the difference attained statistical significance only in the mid dose group and no dose dependency was observed, this change can be regarded as incidental and not related to the test item.

Endocrine findings:

no effects observed

Description (incidence and severity):

No effect of test item was observed in the thyroid hormone analysis.

Compared to the control values, there were no statistically significant differences in thyroid hormone concentrations (T4 and TSH) for Parental male and female animals.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item related changes were observed in the urinalysis parameters in Parental males and females at any dose level compared to the control group.

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related microscopic findings in evaluated tissues and organs of Parental male and female animals.

Multifocal/diffuse red/dark red discoloration and/or diffuse thickness of the mucous membrane of the stomach (glandular/non glandular region) were observed in the following animals:
P Generation: Males: No.: 3001, 4008; Females: No.: 1502, 1504, 1507, 1508, 1510, 1511, 1513, 1516, 1520, 1521, 1523, 2501, 2502, 2504, 2506, 2509, 2510, 2513, 2515, 2517, 2520, 2521, 3501, 3502, 3504, 3506, 3508, 3509, 3510, 3515, 3518, 3524, 4501, 4502, 4505, 4506, 4507, 4508, 4512, 4515, 4522 and 4524

Histological examination revealed focal congestion in all animals in these regions of mucous membrane. In addition, erosion (focal necrosis in the upper region of mucous membrane) and edema was also occurred. No ulceration or inflammatory lesions were seen.

Pyelectasia in the kidneys (unilateral or bilateral) (P Generation: No.: 4019, 3522) without degenerative, inflammatory or other histological (fibrotic etc.) lesion could be considered as a common finding in laboratory rats without toxicological significance.
Histological examination revealed slight focal lymphocytic and histiocytic infiltration in five male animals (P Generation: No.: 1003, 1010, 1012, 1021, 4019).

These findings could possibly indicate the initial phase of chronic progressive nephropathy (CPN) of laboratory rats, but in the absence of dose dependency, it is concluded the test item was not a predisposing factor in the development and pathogenesis of CPN.

The cyst in the liver (P Generation: No.: 4519) accompanied with focal purulent cholangitis in one case (P Generation: No.: 4519) and the depressed area in the cortex of the right kidney in one Parental control female animal (P Generation: No.: 1523) associated microscopically with multifocal interstitial nephritis could be considered as individual diseases.

Hemorrhage in the thymus, in the mandibular lymph node, in the mesenteric lymph node and in the seminal vesicle (P Generation: No.: 2012, 1522) without inflammatory or degenerative lesions could be connected with the euthanasia and agony.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Assessment of ovarian toxicity
In the Parental female animals belonging to the control and high-dose groups, the ovaries, uterus, cervix, vagina had a normal structure characteristic of the species, age and phase of the sexual cycle. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well.
The number of corpora lutea was comparable with the control in each of the treated dose groups, without any statistically significant difference.
Enumeration of small follicles in the control and high dose cohort 1A animals did not reveal any test item-related effect indicating ovarian toxicity. Number of small follicles was similar in the control and high dose groups.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No effect of the test item on oestrous cycles was noted. The mean cycle length was 3.9-4.1 days for each group after starting of the treatment, during the 14 days pre-mating period.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

In the Parental generation, statistically significantly higher than control group mean was noted in the percent of sperms with normal appearance in the low and mid dose groups (+1.2% and 1.3%, both p˂0.01), however no dose response was observed, therefore it was considered rather incidental than test item related change, without toxicological relevance.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the control and test item-dosed groups with regard to reproductive ability, mating or gestation indices that could be ascribed to test item administration.

Although all animals mated and showed successful coitus (sperm positive vaginal smears) in the initial period, due to failing to achieve the sufficient number of pregnant animals in the low and mid dose groups, the non-pregnant animals (No.: 2507, 2511, 2512, 2519, 2522, 3505, 3507, 3512, 3517, 3519, 3521, 3522) were re-mated with their original pair in order to fulfill the requirements of the OECD 443 guideline (at least 20 pregnant females per dose group).
All re-mated animals became pregnant in the low and mid dose groups, which confirmed that the elevated number of the non-pregnant animals (5/24 and 7/24) in the initial period in these groups were not related to the test item. Therefore, for the evaluation of the reproductive parameters the data of all females have been used.

Successful coitus (sperm positive vaginal smears) occurred within 5 days of pairing (cohabitation) in all groups. The mean duration of mating was 2.5, 2.5, 2.5 and 2.3 days in the control, low, mid and high dose groups, respectively.

The mating index was 100% in all groups (males and females). Two females in the control (No.: 1506, 1517) and three females in the high dose group (No.: 4509, 4517, 4520) were non-pregnant. The male and female fertility index was 92%, 100%, 100% and 88 % in the control, low, mid and high dose groups, respectively. The gestation index was 100% in all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Clinical signs:

no effects observed

Description (incidence and severity):

During the general-, and detailed clinical observations, no clinical signs were observed in the current the study.

OFFSPRINGS PND0-PND21 (F1, F2)
Evidence of suckling on PND 0 was recorded for all viable pups of all dose groups and control group.
All surviving pups were externally normal and were symptom free from PND 0 up to and including on PND 21 in all dose groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No mortality was observed during the study.

Viability of F1 pups
There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values in any of the dose groups.
The mean number of implantation sites was comparable to the control mean in all dose groups.
The number of viable pups on PND0 and PND4 as well as pup survival indices on PND0 and PND4 were comparable to control values in each dose group. In the mid dose group, totally 10 pups died between PND5 and 21, which resulted statistically higher mean number and percentage of died pups (0.42 vs. 0.00; 4.29% vs. 0.00%; p˂0.05 respectively), and decreased survival index (-4.3%; p˂0.05) during this period. As no mortality was observed in the low dose group and only one pup died in the high dose group between PND5-21, the elevated number of dead pups in the mid dose group can be regarded as incidental and not a test item related effect.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

OFFSPRINGS PND0-PND21 (F1, F2)
At PND 0, runts (pups with body weights smaller than the mean-2xSD of the body weights of controls) were observed with a frequency of 11/243, 6/266, 6/268 and 14/235 in control, low, mid and high dose-treated groups, respectively.

F1 generation males (PND 21-89)
At the beginning of the observation period on PND21, a lower body weight was noted in all dose groups (-2.8%, -3.8% and -3.6% in the low, mid and high dose groups respectively), attaining statistical significance in the mid and high dose groups (p<0.01 and p<0.05 respectively), in comparison with the control. Later, until PND89, there were also some sporadic occasions, where statistically significant lower body weight was observed in the mid dose group, such as on PND35 (-2.9%, p<0.05), on PND49 (-3.1%, p<0.05) and on PND63 (-3.1%, p<0.05). The difference at PND 21 is the result of the reduced body weights of the pups at weaning (see relevant section), which was quickly compensated in the low and high dose groups, and finally, on PND89 the body weight of the low, mid and high dose groups were similar to the control. With this in mind, and in the lack of clear dose response, the differences in body weight are considered as incidental, without toxicological relevance.
The body weight gain of the F1 generation was very similar to the control in all dose groups with the exception of a limited number of isolated occasions, such as a statistically significant lower body weight gain (-4.5%, p<0.01) in the mid dose group between
PND 28-35, and a statistically significant elevation in the high dose-treated males between PND 84-89 (+13.8%, p<0.05). The overall body weight gain of the test item-treated animals from PND21 to PND89 was comparable to the control.

F1 generation females (PND 21-89)
In the F1 generation females, statistical significant difference to the control was observed on the first day only in the mid-, (-2.9%, p<0.05) and high dose-treated groups (-3.6%, p<0.01) and no statistical significant difference could be seen on none of the later treatment days and in none of the treatment groups. The reduced body weight of these groups is likely a result of the reduced mean litter-mean values of the female F1 pups at the time of weaning, which were -3.7% (p<0.05), -3.1% (NS) and -5.4% (p<0.01). As the body weights of all test item-treated groups approached and even exceeded the body weight of the control group, and as the initial difference was <4%, these observations can be regarded as without toxicological relevance.
Statistical significant differences to the control was noted in the body weight gain on some occasions, such as in the high dose-treated group between PND 21-28 (+3.3%, p<0.05) and PND 84-89 (+40.2%, p<0.05), in the mid dose-treated group between PND 21-28 and PND 28-35 (+5.7% and -4.3%, both with p<0.05) and in the low dose-treated group between PND 63-70 (+20.9%, p<0.05). The overall body weight gain of the high dose-treated group was also significantly higher than control (+4.7%, p˂0.05). As these observations did not show any dose dependency and because of the body weight values on PND 89 did not show any significant difference to the control, these observations can be regarded as minor variances without toxicological relevance.

Cohort 1B males
Administration of the test item incorporated into diet, did not cause any test item related effect on the body weight, or body weight gain of cohort 1B males. A few transient and isolated, but statistically significant changes including -4.4% less than control body weight mean on week 16 in the mid dose group (p<0.05), and lower than control body weight gain in the low and mid dose groups (-21.3%, p<0.05 and -29.5% p<0.01 respectively) on week 16, furthermore -20.4% less than control body weight gain in the high dose group with a statistical significance of p<0.05 on week 17, were considered incidental and toxicologically irrelevant in the lack of dose response and/or their impermanence.

Cohort 1B females
Similarly to the males, the body weight and body weight gain of cohort 1B females was also unaffected in the observation period from GD 0 until PPD 21. The statistically significantly higher than control body weight in the low dose at all occasions, and in the high dose group on PPD 17 and PPD 21 were considered incidental and toxicologically irrelevant in the lack of dose response. Likewise, minor but statistically significant (all p˂0.05) changes in body weight gain such as lower than control body weight gain between GD0 and GD3 in the mid dose group, and higher than control body weight gain between GD14-17 in the high dose group, between PPD 7-10 in the low dose group, between PPD 10-14 in the high dose group and between PPD 14-17 in the mid dose group were considered not test item related, but incidental, without toxicological relevance in the lack of dose response and/or their transience.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation males (PND 21-89)
During the thorough observation period, an apparent elevation of food consumption was noted in the high dose group, attaining statistical significance at the majority of the measuring periods, when compared to the control. The elevation was within the range of +4.9% to +8.7%. Statistical significance (p<0.05 or p<0.01) was observed at 9 out of 10 occasions. Between Days 21-89, the overall mean of the high dose group was 6.8% higher than control, with a statistical significance of p<0.01. The statistically significant 4.9% higher than control mean between Days 21-28 in the mid dose group was restricted to only to the first observation period.
The similar trend was noted when the body weight normalized food consumption was calculated, except that the mid- and at a lower magnitude the low dose was affected as well. The increase in comparison with the control was within the range of +1.5% to +5.6% in the low dose group, +0.8% to +7.7% in the mid dose group, and +6.1% to +12.5% in the high dose group. Statistical significance (p<0.05 or p<0.01 was observed at 2 out of 10 occasions in the low-, 6 out of 10 occasions in the mid-, and 10 out of 10 occasions in the high dose group. The overall means were higher than control in all dose groups in a dose dependent manner (+3.3%, +4.4% and +8.6% in the low, mid and high dose groups respectively) with a statistical significance of p<0.01 in all dose groups.
Food utilization group means of F1 males showed a dose dependent decrease at the vast majority of the measuring periods, attaining statistical significance at some occasions in the mid and the high dose group, and at two occasions also in the low dose group. The overall food utilization mean showed a dose dependent decrease (-2.2%, -4.0%, -6.2%), attaining statistical significance in the mid and high dose groups (p<0.01).

F1 generation females (PND 21-89)
At the first observation period between Days 21-28, a dose dependent increase in food consumption was noted in all test item treated dose groups (+4.2%. +6.9%, +7.8% in the low, mid and high dose groups respectively), attaining statistical significance in all dose groups (p<0.05 in the low dose group, and p<0.01 in the mid-, and high dose groups). In the low- and mid dose groups, the initial elevation was transient, no effect was noted later in the observation period, between Days 28-89). On the other hand, an apparent elevation of food consumption was noted in the high dose group, attaining statistical significance at the majority of the measuring periods, when compared to the control. The elevation was within the range of +1.4% to +10.5%. Statistical significance (p<0.05 or p<0.01 was observed at 7 out of 10 occasions. The overall mean of the high dose group was 6.8% higher than control with a statistical significance of p<0.01.
Similarly, when the body weight normalized food consumption was calculated, an initial, but temporary, statistically significant (p<0.05 in the low dose group, and p<0.01 in the mid-, and high dose groups) elevation was noted in the food consumption in all dose groups (+4.8%, +7.8% and +9.8% in the low, mid and high dose groups respectively), restricted to the first observation period between Days 21-28, which eliminated later in the study. In contrast, the high dose group was affected nearly during the whole period between Day 21 -89. An increase in comparison with the control was observed at 9 out of ten occasions and was within the range of +2.3% to +9.8%. Statistical significance (p<0.05 or p<0.01 was observed at 7 out of 10 occasions. The overall mean in the high dose group was 6.1% higher than control, and showed statistical significance (p<0.01).
Food utilization of F1 females was somewhat lower than control (within the range of
-6.8% - -13.9%) in the high dose group during the whole observation period between Day 21 and 89, with a very limited number of exceptions such as between Day 42-49 and Day 84-89. The overall mean in the high dose group was -4.2% less than control, and a statistical significance was observed also (p<0.01). On an isolated occasion only between Days 28-35, a statistically significantly lower than control utilization was noted in the mid dose group as well (-5.2%, p<0.01).

Cohort 1B males
No statistically significant effect was noted in cohort 1B males in absolute food consumption, however both the interim and overall means were higher than control. On the other hand, when body weight normalized food consumption was calculated, an apparent and consequent effect was noted in the high dose group, namely that the amount of consumed food given in the ratio of the animals’ body weight, was statistically significantly (p˂0.05 or p˂0.01) higher than control in nearly each (7 out of 8) of the examined periods. The elevation was between the range of +2.9% - +6.8%. A similar, but somewhat less intensive effect was noted also in the mid dose group, where higher than control mean values were noted almost at each of the measuring periods, within the range of +3% - +6.5% with a statistical significance (p˂0.05 or p˂0.01) at 2 out of 8 occasions. The overall mean was significantly higher than control in the mid and high dose groups showing dose dependence (+3.5% and +5.6% respectively). The overall food utilization was somewhat less in all dose groups in comparison with the control, but without statistical significance. However, at some measuring points such as week 16 in the low and mid dose group, furthermore on weeks 17 and 19 in the high dose group, the differences in comparison with the control were significant statistically (p˂0.05 or p˂0.01).

Cohort 1B females
When compared to the control, an apparent elevation in absolute food consumption was noted in the high dose group both in the gestation, and in the post-partum periods. The interim mean values were higher than control at 6 out of 6 occasions during the gestational period, and also 6 out of 6 occasions in the post partum period. The elevation was within the range of +7.4%-+13.9% between GD0 and GD20, while +4.5%-+10.7% between PPD0-PPD21, and showed statistical significance (p<0.05 or p<0.01) at 4 out of 6 occasions in the gestational, and 2 out of 6 occasions in the post-partum period. The overall mean in the high dose group was 10.4% higher than control, with a statistical significance (p<0.01), between GD0-20 and 8.3% higher than control, with a statistical significance (p<0.05), between PPD0-21.
Other changes showing statistical significance (p<0.05 or p<0.01) including -7.1% less than control group mean in the mid dose group between GD0-GD 3, +10.5% and +11.2% higher than control group mean in the low dose group between GD10-14 and GD17-20 respectively, +9.6% and +10.7% higher than control group mean in the mid dose group between GD17-20 and between PPD 17-21 were considered incidental in the lack of a clear dose response.
An apparent elevation of body weight normalized food consumption was observed in high dose group thorough the gestational period. The elevation was within the range of +1.8% and 9.3% in comparison with the control. Statistical significance (p<0.05 or p<0.01) at 4 out of 6 measuring occasions were observed. The overall mean in the gestation period in the high dose group, was 6.0% higher than control, showing statistical significance of p<0.05. A somewhat less intensive effect was noted in the post-partum period, where the interim group means in the high dose groups were generally higher than control, but no statistical significance was observed.
Other minor changes were observed in the low dose-treated group in the period of GD 0-3 (-10.0%) and PPD 17-21 (-11.2%) and in the mid dose-treated group in the period of GD 0-3 (-10.8%), all with statistical significance of p<0.01. As these changes were without dose response, can be considered as incidental, without toxicological relevance.
Food utilization of the Cohort 1B female animals showed no statistical significant difference to the control in any of the treatment groups at any of the observation times.

Dose Intake of weaned F1 generation (PND21-89)
F1 generation males (PND 21-89)
The time-weighted average daily dose intakes of the low-, mid-, and high dose-treated males for the entire treatment period (Days 21-89) were 156.3, 526.7 and 1643.4 mg/kg bw of the test item, which were 156.3%, 175.6% and 164.3% of the nominal dose.

F1 generation females (PND 21-89)
The time-weighted average daily dose intakes of the low-, mid-, and high dose-treated females for the entire treatment period (Days 21-89) were 161.9, 547.5 and 1717.2 mg/kg bw of the test item, which were 161.9%, 182.5% and 171.7% of the nominal dose.

Dose Intake of Cohort 1B
Cohort 1B males
The time-weighted average daily dose intakes of the low-, mid-, and high dose-treated males between week 14 and 21 were 86.0, 297.9 and 912.8 mg/kg bw of the test item, which were 86.0%, 99.3% and 91.3% of the nominal dose.

Cohort 1B females
The time weighted mean daily dose intake of the low-, mid-, and high dose-treated females from GD 0 to PPD21 was 255.6, 943.7 and 2884.2 mg/kg bw, corresponding to 155.6%, 214.6% and 188.4% of the nominal dose.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology of Cohort 1A
In male animals, a dose dependent increase of group mean monocyte percent (MONO%) was noted in all test item treated groups (group means 5.07%, 5.99%, 6.15% and 7.15% in the control, low, mid and high dose groups respectively), attaining statistical significance in low and high dose groups (p<0.05 and p<0.01, respectively). Due to the nature and magnitude of the change, no substantial or noteworthy effect on the general state of health or well-being of the animals is expected, therefore it is considered as biologically non-significant effect without toxicological relevance. Also in high dose-treated male animals, a +69.3% elevation of group mean monocyte count (MONO) was observed in comparison with the control, showing statistical significance (p<0.05), though the mean value (0.232x109/L) is within the historical control mean ±2SD (0.00 - 0.323), therefore no toxicological meaningful is ascertained.

A -11% lower than control lymphocyte ratio (LYM [%]) without association with a similar shift in the absolute count value is considered to have no toxicological relevance, but being the consequence of the relatively higher number of monocytes in leukocyte population.

Some minor, but statistically significant (p<0.05) changes were noted in parameters characterizing platelet count and size, such as elevated mean plateletcrit value (PCT, +9.7%) in high dose-treated males, decreased mean platelet volume (MPV, -3.5%) in high dose-treated females, and lower than control platelet distribution width (PDW, -4.7%) in low dose-treated females, were not associated with any significant effect in the measured blood clotting parameters, therefore were considered as biologically insignificant variance with no toxicological relevance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical chemistry of Cohort 1A
Statistically significant increase of the urea group mean concentration (UREA; +12.5%; p<0.05) and subsequently, increased blood urea nitrogen/creatinine ratio (BUN/CREA) was noted in the high dose-treated male group (+15.8%; p<0.01). Urea is the principal nitrogenous waste product of metabolism, and generated from protein breakdown. It is eliminated from the body almost exclusively by the kidneys in urine, therefore the measurement of its concentration is part of the assessment of kidney function. The causes of elevated urea concentration can be renal disease/failure, dehydration, heart failure, gastrointestinal bleed, high-protein diet, ageing, trauma, severe infection, or starvation. Each of these causative factors can be excluded based on the result of the clinical observation, body weight and food consumption measurements, other clinical pathology measurements (unaffected creatinine concentration, normal ion-concentrations and calculated plasma osmolality), necropsy, organ weight measurements and histopathology examination, therefore this change is considered a normal biological variation rather than a test item related toxic effect.

In comparison with the control, an increase was noted in high dose females in alkaline phosphatase (ALKP) level, showing statistical significance (+34.6%; p<0.05). Elevation in ALKP plasma concentration may reflect to changes of hepatic origin, bone- and endocrine diseases, however in this case it is not reasoned by other observations (clinical pathology, organ weight, and histopathology). On the other hand, the high dose group mean (51.4 U/L) is within the historical control mean ±2SD (0.00 – 109.1) . With this in mind, the increase of ALKP activity is considered as a normal biological variance and the statistical significance is resulted by the relatively low control group mean.

Statistically significant decrease of alkaline phosphatase (ALKP) levels were noted in the low dose-treated male (-19.6%; p<0.05), which is considered incidental in the lack of dose-response.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item related changes were observed in the urinalysis parameters in F1 Cohort 1A males and females at any dose level compared to the control group.

Sexual maturation:

no effects observed

Description (incidence and severity):

Compared to the control, there was no statistical significant difference in the sexual maturation neither of the male nor of the female animals in any of the dose groups.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distances of the males and females of the various treatment groups were statistically indistinguishable for the corresponding control groups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No test item-related effect was observed in male pups on nipple retention in any of the treated groups on PND 13.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A
Variations were observed, occasionally attaining statistical significance such as statistically lower absolute thyroid and parathyroid gland weight in mid dose-treated males (-6.9%; p˂0.05), lower absolute and brain weight relative thyroid and parathyroid gland weight and in low and mid dose-treated females (abs.:-9.3% and -9.0%, relBrW: -7.8% and -7.4%, all p˂0.05), lower absolute pituitary weight in mid dose females (-7.8; p<0.05) and lower body weight normalized pituitary weight in high dose-treated females (-8.9%; p<0.05). The differences were without dose dependency and/or showed no consistent response between genders, and/or were not correlated with histopathological findings therefore considered incidental and not related to treatment.

Cohort 1B
No biologically or statistically significant changes were noted compared to the control in any of the test item-treated male groups.
Statistically significant higher absolute, body and brain weight normalized uterus weight was noted in mid dose females (+24.4%, +22.6% and +26.5%; all p<0.05). These changes could be attributed to the higher number of females observed with dilatation of uterine body and horn (4 in the mid dose group and only one in the control) which is not test item related but the consequence of the normal oestrous cycle of the animals.
Other variations attained statistical significance (all p˂0.05) such as lower body weight normalized brain (-4.4%) and ovaries (-12.6%) weight in the low dose-treated females, higher brain weight normalized body weight (+4.7%) in low dose females and higher brain weight normalized adrenal glands weight (+11.5%) in mid dose-treated females without any consistent dose dependency considered incidental and as of no toxicological relevance.

Organ weights of lymph nodes of Cohort 1A and 1B

In male animals of cohort 1A, an elevation of group means was noted in absolute mesenterial lymph node weight in all treated groups (+16.6%, +9.6%, +18.5% in low, mid and high dose groups respectively) when compared to the control, attaining statistical significance in the low and high dose groups (p˂0.01 and p˂0.05 respectively). A similar trend was observed in the low, mid and high dose-treated groups when the mesenteric lymph node weights were normalized either to the body weight (+16.4%, p<0.01; +10.9%, p<0.05; and +16.6%, p< 0.05) or to the brain weight (+16.7%, p<0.01; +11.1%, NS; and +17.7%, p<0.05). All these changes detailed above were without dose dependence and no changes were observed in cohort 1B animals, therefore in the lack of histopathological findings in the high dose treated animals it was considered incidental rather than a test item related effect.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related findings were noted in Cohort 1A, Cohort 1B and Group 1X animals of any dose group as well as control group during the macroscopic examination.

Description (incidence and severity):

No test item-related microscopic findings in evaluated tissues and organs of Cohort 1A and Cohort 1B male and female animals.

Multifocal/diffuse red/dark red discoloration and/or diffuse thickness of the mucous membrane of the stomach (glandular/non glandular region) were observed in the following animals:
- Cohort 1A: Males: No.: 5018, 7014, 8013, 8014; Females: No.: 7503, 7520, and 8517
- Cohort 1B: Males: No.: 5034; Females: No.: 5524, 5525, 5529, 5530, 5533, 5536, 5539, 6521, 6522, 6524, 6526, 6528, 6530, 6531, 6532, 6534, 6535, 7521, 7522, 7524, 7526, 7534, 7535, 7538, 7540, 8522, 8524, 8525, 8528, 8529, 8530, 8533, 8535, 8536 and 8539.

Histological examination revealed focal congestion in all animals in these regions of mucous membrane. In addition, erosion (focal necrosis in the upper region of mucous membrane) and edema was also occurred. No ulceration or inflammatory lesions were seen.

For the purpose of clarification, the stomach of the euthanized F1 Group 1X animals were inspected macroscopically in order to examine if the necropsy findings in the stomach of the dams (in all groups, also including control) is specific for the lactating dams or if it is detectable also in the F1 Group 1X animals, which similarly to the dams, also had an increased intake of the formulated diet. As there gastric mucosa of the Group 1X animals was symptom free, it is very likely that the changes observed in the dams is not test item related, but the increased sensitivity of the mucosa can be ascribed to their endocrine status Furthermore, this assumption can be supported with observations in a similar study performed in the Test Facility, where hypersensitivity of the gastric mucosa was also noted in 6/9 dams in the control group with the same endocrine status.

Pyelectasia in the kidneys (unilateral or bilateral) (Cohort 1A: No.: 7015, 7520, 8001, 8011; Cohort 1B: No.: 5023, 5034, 5038, 5039, 7029, 8021, 8025, 8029, 8036) without degenerative, inflammatory or other histological (fibrotic etc.) lesion could be considered as a common finding in laboratory rats without toxicological significance.
Histological examination revealed slight focal lymphocytic infiltration and tubular basophilia in two cases (Cohort 1A: No.: 5019, 8012) in the kidneys. Furthermore, mild focal lymphocytic infiltration and tubular basophilia accompanied with tubular atrophy and tubular dilatation was noted in one control Cohort 1B male (Cohort 1B: No.: 5034) animal.
Bilateral, light brown multifocal discoloration and multifocal depressed area in the kidneys was noted during necropsy in one high dose-treated Cohort 1B male animal (Cohort 1B: No.: 8034). Moderate focal lymphocytic infiltration and tubular basophilia accompanied with tubular atrophy and tubular dilatation could be observed microscopically.

These findings could possibly indicate the initial phase of chronic progressive nephropathy (CPN) of laboratory rats, but in the absence of dose dependency, it is concluded the test item was not a predisposing factor in the development and pathogenesis of CPN.

Enlargement left mandibular lymph node associated microscopically with hyperplasia (Cohort 1A: No.: 5012) is an immunomorphological phenomenon without pathological significance.

Involution of the thymus (recorded as small thymus macroscopically) in one control, one low and one high dose Cohort 1B female animal (Cohort 1B: No.: 5537, 6537, 8533) could be in connection with the age of animal and considered as individual disorders.
Hemorrhage in the thymus, in the mandibular lymph node, in the mesenteric lymph node and in the seminal vesicle (Cohort 1A: No.: 8520; Cohort 1B: No.: 5026, 5033, 8032, 8527) without inflammatory or degenerative lesions could be connected with the euthanasia and agony.

Assessment of ovarian toxicity
In the F1 (Cohort 1A and 1B) female animals belonging to the control and high-dose groups, the ovaries, uterus, cervix, vagina had a normal structure characteristic of the species, age and phase of the sexual cycle. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well.
The number of corpora lutea was comparable with the control in each of the treated dose groups, without any statistically significant difference.
Enumeration of small follicles in the control and high dose cohort 1A animals did not reveal any test item-related effect indicating ovarian toxicity. Number of small follicles was similar in the control and high dose groups.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

Immunophenotyping of lymphocytes in the spleen performed with the combination of six specific antibodies directed against membrane markers such as CD45, CD3, CD4, CD8, CD45 RA and CD161 did not reveal any potential impact of the test item on the developing immune system. The ratio of T and B lymphocytes, NT and NKT subpopulations within the population of splenocytes was comparable with the control in each of the treated dose groups. The percentage and ratio of the helper (CD4+) and cytotocix (CD8+) T cells did not show any difference in the treated dose groups in comparison with the control. In the lack of dose response, the statistically significantly less than control group mean in cytotoxic T-cell percent CTC [% TC] (-19.5%, p˂0.05) in mid dose females was considered incidental without toxicological relevance.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive effects

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Variations were observed, occasionally attaining statistical significance (all p˂0.05) such as
statistically lower absolute seminal vesicle weight in low dose males (-14.9%), lower body
weight adjusted epididymides and thyroid and parathyroid gland weight (-6.6% and -11.2%
respectively) in mid dose-treated males, lower absolute and body weight relative uterus
weight in low dose females (-10.7% and -8.3% respectively) and higher brain weight relative
ovaries weight in high dose treated female pups (+17.6%). The differences were without dose
dependence and/or were significant only in either one of absolute or body/brain weight normalized organ weight and were without associating necropsy findings. Therefore these
were considered incidental and not related to treatment.

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No effect of test item was observed in the thyroid hormone analysis.
Compared to the control values, there were no statistically significant differences in thyroid
hormone concentrations (T4 and TSH) for F1 and F2 PND21 pups.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects noted.

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Haematology of Parental Generation

Selected haematology data of parental males

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (300)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
RETIC [%]	2.99	3.03	3.17	3.46	1.3	6.0	15.7 DN*
RETIC [K/µL]	257.23	266.46	273.33	294.54	3.6	6.3	14.5 DN*

Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test

Selected haematology data of parental females

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (300)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
EOS [%]	0.21	0.40	0.24	0.50	90.5 DN*	14.3	138.1 DU*
MONO [x10^9/L]	0.19	0.35	0.28	0.26	82.4 DU*	45.1	32.1

Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test

 

Haematology of Cohort 1A

Selected haematology data of Cohort 1A males

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (300)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
LYM [%]	66.87	67.17	66.42	59.47	0.5	-0.7	-11.1 DU*
MONO [%]	5.07	5.99	6.15	7.15	18.1 DN*	21.3	41.0 DN**
MONO [x10^9/L]	0.14	0.20	0.17	0.23	47.4	21.9	69.3 DN*
PCT [%]	0.55	0.59	0.57	0.60	8.0	3.5	9.7 DN*

Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test

Selected haematology data of Cohort 1A females

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (300)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
MPV [fL]	8.65	8.59	8.54	8.35	-0.7	-1.3	-3.5 DU*
PDW [fL]	8.36	7.97	8.17	7.94	-4.7 DU*	-2.3	-5.0

Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test

 

Clinical Chemistry of Parental Generation

Selected clinical chemistry data of parental males

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (300)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
GLOB [g/L]	27.2	26.4	26.8	25.8	-2.9	-1.5	-5.1 DU*
ALB/ GLOB	1.1	1.1	1.1	1.2	0.9	-1.8	7.3 DU**

Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test

Selected clinical chemistry data of parental females

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (300)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
CHOL Tot. [mmol/L]	1.89	2.18	2.29	2.20	15.6	21.2 DN*	16.6

Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test

 

Clinical chemistry of Cohort 1A

Selected clinical chemistry data of Cohort 1A males

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (300)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
UREA [mmol/L]	5.53	6.00	5.87	6.22	8.5	6.1	12.5 DN*
BUN/ CREA	57.5	56.6	55.2	66.6	-1.6	-4.0	15.8 DN**
ALKP [U/L]	94.9	76.3	91.8	91.4	-19.6 DN*	-3.3	-3.7

Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test

Selected clinical chemistry data of Cohort 1A females

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (300)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
ALKP [U/L]	38.2	49.7	44.4	51.4	30.1	16.2	34.6 DN*

Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test

 

Organ weights of Parental Generation

Selected organ weight data of parental males

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (300)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
TESTES [g]	3.88	3.72	3.68	3.72	-4.1	-5.0 DN*	-4.1 DU*
TESTES [%BW]	0.839	0.800	0.790	0.795	-4.7	-5.8 DN*	-5.2
TESTES [%BrW]	174.86	167.89	167.98	167.711	-4.0	-3.9	-4.1 DN*
SPLEEN [g]	0.754	0.847	0.772	0.854	12.3 DU*	2.4	13.3 DN**
SPLEEN [%BW]	0.163	0.183	0.165	0.183	12.0 DU*	1.1	12.0 DN*
SPLEEN [%BrW]	34.00	38.24	35.17	368.47	12.4 DU**	3.4	13.1 DN**
THYROID PARATHY [g]	0.024	0.025	0.024	0.026	3.8	-0.2	6.1 DN*
THYROID PARATHY [%BW]	0.0052	0.0053	0.0080	0.0054	1.6	-4.0	3.2
THYROID PARATHY [%BrW]	1.0854	1.1269	1.0930	1.1518	3.8	0.7	6.1 DN*

Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test

 

Organ weights of lymph nodes of Cohort 1A and 1B

Mesenteric and mandibular lymph node weight data of Cohort 1A and 1B male animals

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (300)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
COHORT 1A
LYMPH N. MESENTERIC [g]	0.187	0.218	0.205	0.221	16.6 DN**	9.6	18.5 DN*
LYMPH N. MESENTERIC [%BW]	0.0474	0.0551	0.0525	0.0552	16.4 DN**	10.9 DN*	16.6 DN*
LYMPH N. MESENTERIC [%BrW]	8.620	10.057	9.577	10.146	16.7 DN**	11.1	17.7 DN*
Cohort 1B
LYMPH N. MESENTERIC [g]	0.268	0.249	0.265	0.264	-7.0	-0.8	-1.4
LYMPH N. MESENTERIC [%BW]	0.0527	0.0500	0.0542	0.0526	-5.1	2.9	-0.2
LYMPH N. MESENTERIC [%BrW]	11.836	10.994	11.921	11.676	-7.1	0.7	-1.4

 

Reproductive ability assessment and indices of P Generation

Summary of reproductive parameters (P Generation, males)

Parameters	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Number of dosed animals	24	24	24	24
Number of males used for mating	24	24	24	24
Number of successful matings	24	24	24	24
Number of infertile animals	2	0	0	3
Male mating index (%)	100	100	100	100
Male fertility index (%)	92	100	100	88

Summary of reproductive parameters (P Generation, females)

Parameters	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Number of dosed animals	24	24	24	24
Number of females used for mating	24	24	24	24
Number of sperm positive females	24	24	24	24
No of females with no implantation sites	2	0	0	3
Number of pregnant females	22	24	24	21
Number of pregnant females with liveborn	22	24	24	21
Female mating index (%)	100	100	100	100
Female fertility index (%)	92	100	100	88
Female gestation index (%)	100	100	100	100

 

Reproductive ability assessment and indices of Cohort 1B

Summary of reproductive parameters (Cohort 1B, males)

Parameters	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Number of dosed animals	20	20	20	20
Number of males used for mating	20	20	20	20
Number of successful matings	20	20	20	20
Number of infertile animals	0	2	2	2
Male mating index (%)	100	100	100	100
Male fertility index (%)	100	90	90	90

Summary of reproductive parameters (Cohort 1B, females)

Parameters	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Number of dosed animals	20	20	20	20
Number of females used for mating	20	20	20	20
Number of sperm positive females	20	20	20	20
No of females with no implantation sites	0	2	2	2
Number of pregnant females	20	18	18	18
Number of pregnant females with liveborn	19	18	18	18
Female mating index (%)	100	100	100	100
Female fertility index (%)	100	90	90	90
Female gestation index (%)	95	100	100	100

 

Gestation of P and F1 generation

Summary of the pre-natal and gestation periods (P Generation, Cohort 1B)

Parameters	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
P GENERATION
Gestation, length, mean (days)	22.1	22.0	22.1	22.0
Parturition, normal	22/22	24/24	24/24	21/21
COHORT 1B
Gestation, length, mean (days)	22.2	22.3	22.2	22.2
Parturition, normal	19/19	18/18	18/18	18/18

 

OFFSPRING PND0-PND21 (F1, F2)

Viability of F1 pups

Selected mean offspring viability data by litter (P Generation)

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (300)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
Implantation sites [n]	11.59	11.50	12.13	12.10	-0.8	4.6	4.4
Pups born [n]	11.14	11.13	11.21	11.29	-0.1	0.6	1.3
Live born [n]	11.05	11.08	11.17	11.19	0.3	1.1	1.3
Prenatal mortality [%]	4.99	3.27	8.49	7.43	-34.4	70.2	48.9
Viable pups PND 0 [n]	11.05	11.08	11.17	11.19	0.3	1.1	1.3
Pups dies on PND 0 [n]	0.00	0.00	0.00	0.00	NA	NA	NA
Pups died on PND 0 [%]	0.00	0.00	0.00	0.00	NA	NA	NA
Survival index PND 0 [%]	98.99	99.68	99.62	99.13	0.7	0.6	0.1
Total mortality PND 0 [%]	4.99	3.27	8.49	7.43	-34.4	70.2	48.9
Viable pups PND 4 [n] (before culling)	10.91	10.92	10.96	10.95	0.1	0.5	0.4
Pups died PND 1-4 [n]	0.14	0.17	0.21	0.24	22.2	52.8	74.6
Pups died PND 1-4 [%]	1.15	1.55	2.07	1.93	34.5	79.8	67.5
Survival index PND 4 [%] (before culling)	97.84	98.13	97.55	97.21	0.3	-0.3	-0.6
Total mortality PND 4 [%] (before culling)	6.14	4.82	10.28	9.24	-21.5	67.5	50.5
Viable pups PND 21 [n]	9.36	9.42	9.00	9.86	0.6	-3.9	5.3
Pups died PND 5-21 [n]	0.00	0.00	0.42	0.05	NA	NA DU*	NA
Pups died PND 5-21 [%]	0.00	0.00	4.29	0.48	NA	NA DU*	NA
Survival index PND 4-21 [%]	100.00	100.00	95.69	99.52	0.0	-4.3 DU*	-0.5
Total mortality PND 21 [%] (incl. culled pups)	6.14	4.82	13.20	9.61	-21.5	115.0	56.5

REMARKS: ±% = Percent Deviation Versus Control / NS = Not Significant / * = p<0.05 / ** = p<0.01 / DU = Dunn’s Test / DN = Dunnett’s Test / NA = Not applicable / PND = postnatal day

 

Viability of F2 pups

Selected mean offspring viability data by litter (Cohort 1B)

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (300)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
Implantation sites [n]	12.37	12.83	13.56	13.78	3.8	9.6	11.4
Pups born [n]	11.74	12.17	13.06	12.50	3.7	11.2	6.5
Live born [n]	11.58	11.94	12.83	12.44	3.2	10.8	7.5
Prenatal mortality [%]	7.12	6.70	4.98	10.14	-5.0	-30.1	42.5
Viable pups PND 0 [n]	11.58	11.94	12.83	12.44	3.2	10.8	7.5
Pups dies on PND 0 [n]	0.00	0.00	0.00	0.00	NA	NA	NA
Pups died on PND 0 [%]	0.00	0.00	0.00	0.00	NA	NA	NA
Survival index PND 0 [%]	98.87	98.24	98.32	99.57	-0.6	-0.6	0.7
Total mortality PND 0 [%]	7.12	6.70	4.98	10.14	-5.8	-30.1	42.5
Viable pups PND 4 [n] (before culling)	11.32	11.44	12.56	11.39	1.1	11.0	0.6
Pups died PND 1-4 [n]	0.26	0.50	0.28	1.06	90.0	5.6	301.1
Pups died PND 1-4 [%]	2.05	3.84	2.24	8.09	87.5	9.5	294.8
Survival index PND 4 [%] (before culling)	96.82	94.63	96.14	91.48	-2.3	-0.7	-5.5
Total mortality PND 4 [%] (before culling)	9.15	10.17	7.16	17.68	11.2	-21.7	93.3
Viable pups PND 21 [n]	8.95	9.22	9.68	9.24	3.1	8.0	3.2
Pups died PND 5-21 [n]	0.21	0.11	0.28	0.35	-47.2	31.9	67.6
Pups died PND 5-21 [%]	2.21	1.11	2.78	3.53	-49.7	25.7	59.7
Survival index PND 4-21 [%]	97.78	98.89	97.22	96.47	1.1	-0.6	-1.3
Total mortality PND 21 [%] (incl. culled pups)	11.29	10.85	9.44	16.16	-3.9	-16.4	43.1

REMARKS: ±% = Percent Deviation Versus Control / NS = Not Significant / * = p<0.05 / ** = p<0.01 / DU = Dunn’s Test / DN = Dunnett’s Test / NA = Not applicable / PND = postnatal day

 

Body weight, body weight gain and anogenital distance F1 pups

Mean BW and BWG values of male and female F1 offspring by litter

Parameters	Historical control (mean±2*SD)	Group / Concentration (mg/kg bw/day)				Difference to control [%]
		CONTR. (0)	LOW (100)	MID (100)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
BW PND 0	5.103-7.395	6.021	6.146	6.1616	5.995	2.1	2.3	-0.4
BW PND 4	7.993-13.246	10.129	9.930	9.984	9.735	-2.0	-1.4	-3.9
BW PND 7	13.949-18.699	15.592	15.081	14.910	14.822	-3.3	-4.4	-4.9
BW PND 10	18.469-26.324	21.810	20.905	20.727	20.783	-4.2 DN*	-5.0	-4.7
BW PND 14	24.685-36.709	30.317	29.205	29.086	28.949	-3.7	-4.1	-47.5 DN*
BW PND 17	28.618-44.412	36.358	35.137	35.456	34.622	-3.4	-2.5	-4.8 DN*
BW PND 21	42.184-59.471	50.465	48.471	48.896	47.836	-4.0 DN*	-3.1	-5.2 DN**
BWG PND 0-4	2.633-6.109	4.111	3.788	3.825	3.730	-7.9	-7.0	-9.3
BWG PND 4-7	5.013-6.811	5.399	5.134	4.917	5.082	-4.9	-8.9 DN*	-5.9
BWG PND 7-10	4.274-7.855	6.218	5.824	5.781	5.961	-6.3 DN*	-7.0	-4.1
BWG PND 10-14	5.989-10.612	8.507	8.301	8.188	8.166	-2.4	-3.7	-4.0
BWG PND 14-17	3.552-8.084	6.041	5.932	6.370	5.674	-1.8	5.4	-6.1
BWG PND 17-21	12.617-16.007	14.107	13.334	13.440	13.214	-5.5 DN*	-4.7	-6.3 DN*
BWG PND 0-21	36.119-53.410	44.428	42.321	42.723	41.834	-4.7 DN*	-3.8	-5.8 DN**

REMARKS: Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test, PND = postnatal day

 

Mean BW, BWG and AGD values of male F1 offspring by litter

Parameters	Historical control (mean±2*SD)	Group / Concentration (mg/kg bw/day)				Difference to control [%]
		CONTR. (0)	LOW (100)	MID (100)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
BW PND 0	5.140-7.721	6.226	6.334	6.374	6.128	1.7	2.4	-1.6
BW PND 4	8.080-13.622	10.290	10.172	10.253	9.845	-1.1	-0.4	-4.3
BW PND 7	14.071-19.325	15.799	15.386	15.316	15.061	-2.6	-3.1	-4.7
BW PND 10	18.913-26.895	22.051	21.304	21.198	21.076	-3.4	-3.9	-4.4
BW PND 14	25.356-37.544	30.708	29.668	29.496	29.635	-3.4	-3.9	-4.4
BW PND 17	29.429-45.360	36.860	35.670	36.115	35.116	-3.2	-2.0	-4.7 DN*
BW PND 21	43.862-60.681	51.412	59.540	50.124	48.779	-3.6	-2.5	-5.1 DN*
BW PND 28#	76.1-101.4	88.4	87.8	86.6	85.6	-0.68	-2.02	-3.19
BWG PND 0-4	2.626-6.214	4.066	3.842	3.869	3.709	-5.5	-4.9	-8.8
BWG PND 4-7	5.088-6.963	5.478	5.200	5.060	5.196	-5.1	-7.6	-5.1
BWG PND 7-10	4.526-7.886	6.252	5.918	5.845	6.015	-5.3	-6.5	-3.8
BWG PND 10-14	6.126-10.966	8.657	8.365	8.226	8.289	-3.4	-5.0	-4.3
BWG PND 14-17	3.763-8.126	6.151	6.002	6.619	5.751	-2.4	7.6	-6.5
BWG PND 17-21	13.539-16.215	14.552	13.869	14.009	13.663	-4.7	-3.7	-6.1 DN*
BWG PND 0-21	37.601-54.414	45.184	43.207	43.731	42.638	-4.4 DN*	-3.2	-5.6 DN*
BWG PND 21-28#	30.2-44.0	36.9	37.8	37.0	35.9	2.35	0.41	-2.67
AGD PND 0	1.994-3.524	2.887	2.813	2.933	2.887	-2.5	1.6	0.0
nAGD PND 0	1.078-1.893	1.570	1.521	1.582	1.579	-3.1	0.8	0.6

REMARKS: Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test, PND = postnatal day / #=Data for “BW PND 28” and “PWG PND 21-28” were taken from the analysis of the F1 cohorts

Mean BW, BWG and AGD values of female F1 offspring by litter

Parameters	Historical control (mean±2*SD)	Group / Concentration (mg/kg bw/day)				Difference to control [%]
		CONTR. (0)	LOW (100)	MID (100)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
BW PND 0	4.909-7.220	5.845	6.004	6.005	5.837	2.7	2.7	-0.1
BW PND 4	7.721-13.013	9.987	9.749	9.818	9.608	-2.4	-1.7	-3.8
BW PND 7	13.738-18.233	15.372	14.813	14.640	14.567	-3.6	-4.8	-5.2
BW PND 10	17.923-25.921	21.544	20.565	20.404	20.500	-4.5 DN*	-5.3	-4.8 DN*
BW PND 14	23.884-36.126	29.917	28.831	28.764	28.536	-3.6 DN*	-3.9	-4.6 DN*
BW PND 17	27.673-43.729	35.826	34.731	34.980	34.149	-3.1	-2.4	-4.7 DN*
BW PND 21	40.174-58.779	49.557	47.746	48.007	46.887	-3.7 DN*	-3.1	-5.4 DN**
BW PND 28#	68.3-92.3	79.9	79.9	80.2	79.2	-0.06	0.35	-0.94
BWG PND 0-4	2.571-6.034	4.146	3.745	3.813	3.760	-9.7	-8.0	-9.3
BWG PND 4-7	4.885-6.738	5.315	5.064	4.831	4.963	-4.7	-9.1 DN*	-6.6
BWG PND 7-10	4.004-7.857	6.172	5.752	5.736	5.933	-6.8 DN*	-7.1	-3.9
BWG PND 10-14	5.769-10.398	8.373	8.266	8.161	8.036	-1.3	-2.5	-4.0
BWG PND 14-17	3.312-8.080	8.373	8.266	8.161	8.036	-1.3	-2.5	-4.0
BWG PND 17-21	11.389-16.162	13.731	13.015	13.027	12.738	-5.2	-5.1	-7.2 DN*
BWG PND 0-21	34.323-52.892	43.702	41.737	42.008	41.037	-4.5 DN*	-3.9	-6.1 DN**
BWG PND 21-28#	24.3-38.4	30.5	31.4	32.3	31.6	2.89	5.68	3.33
AGD PND 0	0.841-1.295	0.990	0.948	0.983	0.944	-4.2	-0.7	-4.6
nAGD PND 0	0.451-0.724	0.550	0.522	0.541	0.525	-5.0	-1.5	-4.5

REMARKS: Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test, PND = postnatal day / #=Data for “BW PND 28” and “PWG PND 21-28” were taken from the analysis of the F1 cohorts

 

Body weight, body weight gain and anogenital distance F2 pups

Mean BW and BWG values of male and female F2 offspring by litter

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (100)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
BW PND 0	5.890	6.117	6.072	5.853	3.9	3.1	-0.6
BW PND 4	9.479	9.688	9.767	9.494	2.2	3.0	0.2
BW PND 7	14.590	14.669	14.607	14.443	0.5	0.1	-1.0
BW PND 10	20.290	19.935	20.322	19.823	-1.8	0.2	-2.3
BW PND 14	28.727	28.247	28.575	28.468	-1.7	-0.5	-0.9
BW PND 17	34.759	34.204	34.537	34.868	-1.6	-0.6	0.3
BW PND 21	47.844	46.691	47.95	47.880	-2.4	0.1	0.1
BWG PND 0-4	3.569	3.546	3.680	3.563	-0.6	3.1	-0.2
BWG PND 4-7	5.029	4.978	4.885	4.933	-1.0	-2.9	-1.9
BWG PND 7-10	5.682	5.234	5.649	5.360	-7.9	-0.6	-5.7
BWG PND 10-14	8.399	8.268	8.196	8.471	-1.6	-2.4	0.9
BWG PND 14-17	6.033	5.957	5.962	6.325	-1.3	-1.2	4.8
BWG PND 17-21	13.085	12.487	13.367	13.012	-4.6	2.2	-0.6
BWG PND 0-21	41.892	40.553	41.830	41.941	-3.2	-0.1	0.1

REMARKS: PND = postnatal day

 

Mean BW, BWG and AGD values of male F2 offspring by litter

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (100)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
BW PND 0	6.045	6.298	6.266	6.020	4.2	3.7	-0.4
BW PND 4	9.507	9.874	9.990	9.642	3.9	5.1	1.4
BW PND 7	14.691	14.809	14.885	14.643	0.8	1.3	-0.3
BW PND 10	20.294	20.030	20.802	19.880	-1.3	2.5	-2.0
BW PND 14	28.814	28.360	29.467	28.777	-1.6	2.3	-0.1
BW PND 17	34.968	34.346	35.574	35.309	-1.8	1.7	1.0
BW PND 21	48.227	47.028	49.472	48.851	-2.4	2.6	1.3
BWG PND 0-4	3.462	3.545	3.705	3.540	2.4	7.0	2.2
BWG PND 4-7	5.075	4.943	7.913	4.968	-2.6	-3.2	-2.1
BWG PND 7-10	5.603	5.152	5.770	5.205	-8.0	3.0	-7.1
BWG PND 10-14	8.420	8.240	8.466	8.404	-2.1	0.5	-0.2
BWG PND 14-17	6.154	5.986	6.107	6.377	-2.7	-0.8	3.6
BWG PND 17-21	13.260	12.712	13.898	13.542	-4.1	4.8	2.1
BWG PND 0-21	42.125	40.724	43.187	42.703	-3.3	2.5	1.4
AGD PND 0	2.943	3.128	3.155	2.952	6.3 DU**	7.2 DU*	0.3
nAGD PND 0	1.619	1.696	1.642	1.627	4.7	1.4	0.5

REMARKS: Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test, PND = postnatal day

Mean BW, BWG and AGD values of female F2 offspring by litter

Parameters	Group / Concentration (mg/kg bw/day)				Difference to control [%]
	CONTR. (0)	LOW (100)	MID (100)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
BW PND 0	5.736	5.918	5.887	5.689	3.2	2.6	-0.8
BW PND 4	9.389	9.525	9.558	9.333	1.4	1.8	-0.6
BW PND 7	14.389	14.562	14.351	14.228	1.2	-0.3	-1.1
BW PND 10	20.099	19.908	19.888	19.677	-1.0	-1.0	-2.1
BW PND 14	28.441	28.156	27.833	28.125	-1.0	-2.1	-1.1
BW PND 17	34.397	34.028	33.680	34.381	-1.1	-2.1	0.0
BW PND 21	47.232	46.375	46.657	47.008	-1.8	-1.2	-0.5
BWG PND 0-4	3.624	3.578	3.666	3.573	-1.3	1.1	-1.4
BWG PND 4-7	4.952	5.033	4.877	4.897	1.6	-1.5	-1.1
BWG PND 7-10	5.685	5.346	5.537	5.449	-6.0	-2.6	-4.14
BWG PND 10-14	8.342	8.248	7.945	8.448	-1.1	-4.8	1.3
BWG PND 14-17	5.956	5.872	5.847	6.256	-1.4	-1.8	5.0
BWG PND 17-21	12.836	12.348	12.977	12.627	-3.8	-1.6	-0.5
BWG PND 0-21	41.446	40.436	40.782	41.259	-2.5	-1.6	-0.5
AGD PND 0	1.102	1.096	1.102	1.059	-0.6	0.0	-3.9
nAGD PND 0	0.618	0.606	0.611	0.595	-1.8	-1.1	-3.7

REMARKS: Statistical significance compared to control: * = p<0.05, ** = p<0.01, DN: Dunnett’s Multiple Range Test, DU: Dunn Test, PND = postnatal day

 

Organ weight of F1 PND21 Pups

Selected absolute and relative organ weights of male, PND 21, F1 pups

Parameters	Historical control (mean± 2*SD)	Group / Concentration (mg/kg bw/day)				Difference to control [%]
		CONTR. (0)	LOW (100)	MID (100)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
BODY WEIGHT [g]	42.6-60.1	50.3	48.4	47.0	47.4	-3.9	-6.7 DN**	-5.8 DN**
BODY WEIGHT [%BrW]	2899.7-3740.3	3189.0	3177.4	3183.3	3080.7	-0.4	-0.2	-3.4
BRAIN [g]	1.472-1.620	1.563	1.518	1.548	1.518	-2.8	-1.0	-2.9 DU*
BRAIN [%BW]	2.665-3.379	3.156	3.178	3.150	3.2690	0.7	-0.2	3.3
SPLEEN [g]	0.157-0.379	0.260	0.245	0.219	0.236	-5.9	-15.7 DN*	-9.2
SPLEEN [%BW]	0.326-0.718	0.522	0.502	0.444	0.503	-3.9	-15.0 DN**	-3.7
SPLEEN [%BrW]	10.374-24.320	16.647	16.047	14.161	15.512	-3.6	-14.9 DN*	-6.8
PROSTATE [g]	0.0322-0.0746	0.0518	0.0493	0.0481	0.0477	-4.7	-7.1 DN*	-7.8 DN*
PROSTATE [%BW]	0.068-0.140	0.103	0.102	0.103	0.101	-1.2	-0.2	-1.9
PROSTATE [%BrW]	2.114-4.788	3.341	3.160	3.268	3.215	-5.4	-2.2	-3.8
TESTES [g]	0.176-0.299	0.243	0.230	0.220	0.226	-5.3	-9.5 DN**	-6.8 DN*
TESTES [%BW]	0.390-0.533	0.481	0.473	0.467	0.477	-1.5	-2.8	-0.8
TESTES [%BrW]	11.814-18.887	15.291	15.049	14.965	14.784	-1.6	-2.1	-3.3

 

Selected absolute and relative organ weights of female, PND 21, F1 pups

Parameters	Historical control (mean± 2*SD)	Group / Concentration (mg/kg bw/day)				Difference to control [%]
		CONTR. (0)	LOW (100)	MID (100)	HIGH (1000)	LOW (100)	MID (300)	HIGH (1000)
BODY WEIGHT [g]	39.9-58.2	49.1	46.4	46.1	45.1	-5.5 DN**	-6.2 DN**	-8.1 DN**
BODY WEIGHT [%BrW]	2678.9-3849-.0	3321.2	3070.8	3111.0	3129.9	-7.5 DN**	-6.3 DN*	-5.8 DN*
BRAIN [g]	1.452-1.554	1.526	1.451	1.480	1.506	-4.9 DN**	-3.0 DN*	-1.3
BRAIN [%BW]	2.509-3.664	3.021	3.262	3.230	3.202	8.0 DN**	6.9 DN*	6.0 DN*
THYMUS [g]	0.143-0.305	0.240	0.200	0.206	0.229	-16.5 DN*	-14.1	-4.5
THYMUS [%BW]	0.329-0.582	0.473	0.449	0.446	0.488	-5.1	-5.6	3.1
THYMUS [%BrW]	9.621-20.172	15.709	13.777	13.865	15.234	-12.3 DN*	-11.7	-3.0
OVARIES [g]	0.0092-0.0248	0.0185	0.0160	0.0167	0.0170	-13.3 DN**	-9.6 DN*	-8.3 DN*
OVARIES [%BW]	0.0207-0.0495	0.0378	0.0344	0.0363	0.0377	-8.8 DN*	-4.0	-0.2
OVARIES [%BrW]	0.625-1.644	1.312	1.018	1.168	1.162	-22.4 DN**	-11.0	-11.5 DN*
UTERUS [g]	0.026-0.079	0.058	0.051	0.052	0.052	-12.0 DN**	-9.1 DN**	-9.2 DN**
UTERUS [%BW]	0.069-0.143	0.117	0.109	0.114	0.116	-7.1 DN*	-2.7	-0.9
UTERUS [%BrW]	1.751-5.252	3.653	3.317	3.323	3.437	-9.2	-9.0	-5.9

 

Conclusions:

In summary, daily administration of SynNova® Base Oil by dietary route to male and female Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day, under the conditions of this study, did not cause test item-related adverse effects in any of the examined parameters in any of the dose groups, neither in Parental nor in the Cohort 1A, 1B, Group 1X, F1 and F2 offspring. The increased food consumption observed, is a normal physiological response to the physicochemical changes of the food caused by the inclusion of the test item in the diet.

Parental generation:
NOAEL (systemic toxicity): 1000 mg/kg bw/day
NOAEL (reproductive effects): 1000 mg/kg bw/day

F1 generation:
NOAEL (developmental toxicity): 1000 mg/kg bw/day
NOAEL (systemic toxicity): 1000 mg/kg bw/day
NOAEL (reproductive effects): 1000 mg/kg bw/day

F2 generation:
NOAEL (developmental toxicity): 1000 mg/kg bw/day

Executive summary:

The purpose of this study was to conduct an Extended One-Generation Reproductive Toxicity Study (EOGRTS), according to OECD No. 443 guideline and OECD No. 151 guidance document. The main objective of this Extended One-Generation Reproductive Toxicity Study was to evaluate the pre- and postnatal effects of the test substance on development as well as a thorough evaluation of the systemic toxicity in pregnant and lactating females and young and adult offspring, thereby investigating effects of exposure on specific life stages not covered by other types of toxicity studies.

 

One control and 3 test item-treated groups of Hannover Wistar rats were treated daily by oral (dietary) administration.

 

The study design followed the requirements of the OECD No.443 guideline and the relevant OECD guidance documents.

 

P generation: 96 male and 96 female animals (Parental generation; 24 male and female animals/dose group) were mated after two weeks treatment period. The female animals were treated during pregnancy and during the lactation period, and were necropsied after the weaning of their litters (on PPD 22). The male animals were treated totally for 10 weeks and then they were necropsied.

 

F1 generation: On PND 21, 20 litters of each dose group were chosen at random for continuation of treatment, out of which, each one male and one female was assigned to Cohort 1A, Cohort 1B and Group 1X.

 

F1 Cohort 1A (Reproductive/developmental toxicity testing; 20 males and 20 females): All animals were treated after weaning until PND89, and were necropsied on PND 90.

 

F1 Cohort 1B (Reproductive/developmental toxicity testing; 20 males and 20 females): Animals of Cohort 1B were mated to obtain a F2 generation. Males and females of the same dose group were cohabited (avoiding the pairing of siblings) beginning on or after PND90, but not exceeding PND120. Parental F1 males were terminated at 21 weeks of age, dams and litters (F2 generation) were sacrificed on PPD22/PND21.

 

Group 1X (Extra group for evaluation of sexual maturation; 20 males and 20 females): All animals were treated for at least until sexual maturation, and then were euthanized followed by the examination of their stomach.

 

The experimental design was as follows:

 

Group No.	Group Designation	Target Dose level (mg/kg bw/day)	Dietary Concentration (ppm)	Number of animals
				Males	Females
PARENTAL GENERATION
1	Control	0	0	24	24
2	Low Dose	100	1500	24	24
3	Mid Dose	300	5000	24	24
4	High Dose	1000	15000	24	24
COHORT 1A (F1 GENERATION)
1	Control	0	0	20	20
2	Low Dose	100	1500	20	20
3	Mid Dose	300	5000	20	20
4	High Dose	1000	15000	20	20
COHORT 1B (F1 GENERATION)
1	Control	0	0	20	20
2	Low Dose	100	1500	20	20
3	Mid Dose	300	5000	20	20
4	High Dose	1000	15000	20	20
GROUP 1X (F1 GENERATION)
1	Control	0	0	20	20
2	Low Dose	100	1500	20	20
3	Mid Dose	300	5000	20	20
4	High Dose	1000	15000	20	20

 

Parameters measured during the study included signs of morbidity and mortality twice daily, daily general observation and weekly detailed observation of clinical signs, at least weekly body weight and food consumption in Parental generation and in Cohort 1A, 1B and also in Group 1X animals. Sexual maturation examination was performed in all Cohort 1A, 1B and Group 1X animals.

 

Oestrous cycles were monitored in Parental generation females before mating and during mating period and for a period of two weeks from PND75 in Cohort 1A and 1B females. In Cohort 1B females, observation of cycles was continued until evidence of mating.

 

Clinical pathology evaluation, including haematology, coagulation, clinical chemistry, T4 and TSH concentration measurement and urinalysis, was performed in 10 randomly selected Parental and Cohort 1A male and female animals from each dose group. In addition, bone marrow smear evaluation was performed in the selected Cohort 1A male and female control and high dose-treated animals.

 

Reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult Parental and Cohort 1B animals, and viability, clinical signs and development were evaluated in their F1 and F2 offspring until PND21. In addition, T4 and TSH concentration measurement was performed in F1 and F2 offspring on PND21 form 1 male and 1 female pup from each litter, where applicable.

 

At termination, necropsy with macroscopic examination was performed. Selected organs were weighed and/or preserved in appropriate fixatives from the Parental, Cohort 1A and 1B animals, and from the F1 surplus pups (remaining after assignment of Cohort animals) and F2 PND21 pups as well. A detailed histological examination was performed on all retained organs in the control and high dose-treated Parental and Cohort 1A animals and on the selected organs in the Cohort 1B animals from the control and high dose groups.

Additional histopathological investigations were made of all macroscopic findings and of all males that failed to sire and of all females that failed to deliver healthy pups.

 

For the investigation of possible pre- and post-natal-induced immunotoxic effects, 10 male and 10 female from each treatment group of Cohort 1A (1 male or 1 female per litter) were subjected to splenic lymphocyte subpopulation analysis at termination.

 

Sperm parameters (motility and morphology) and enumeration of cauda epididymis sperm reserves were evaluated in all Parental and Cohort 1A males and enumeration of small follicles of Cohort 1A females (10 females each, of the control and high dose-treated groups) were performed.

 

In summary, daily administration of SynNova^® Base Oil by dietary route to male and female Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day, under the conditions of this study, did not cause test item-related adverse effects in any of the examined parameters in any of the dose groups, neither in Parental nor in the Cohort 1A, 1B, Group 1X, F1 and F2 offspring. The increased food consumption observed, is a normal physiological response to the physicochemical changes of the food caused by the inclusion of the test item in the diet.

 

Parental generation:

NOAEL (systemic toxicity):             1000 mg/kg bw/day

NOAEL (reproductive effects):        1000 mg/kg bw/day

 

F1 generation:

NOAEL (developmental toxicity):   1000 mg/kg bw/day

NOAEL (systemic toxicity):             1000 mg/kg bw/day

NOAEL (reproductive effects):        1000 mg/kg bw/day

 

F2 generation:

NOAEL (developmental toxicity):   1000 mg/kg bw/day

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 November 2019 to 21 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 422 (2016): Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Justification for study design:

The Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in rats was conducted according to OECD 422 guideline and OECD No. 43 guidance document. The guideline was designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing.
The purpose of this study was to obtain information on the possible toxic effects of the test item (SynNova Base Oil) following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels. A control group received the vehicle only (corn oil).
The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 13 post-partum. Reversibility of any treatment-related changes were evaluated following a 14-day recovery period.

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as 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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Crl:WI (Wistar) rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany), from SPF colony.
Housing conditions: Standard laboratory conditions
Number of animals: Total: 75 male and 75 female rats.
Main animals:
60 male and 60 female rats in the acclimatisation/pre-treatment period, and 12/sex/group (4 groups) for treatment, plus 5 males and 5 females to serve as a positive control. Animals originated from different units, to avoid brother/sister mating.
Recovery animals:
For the recovery groups, 5 animals/sex/group were used for Control and High dose groups.
A sufficient number of spare animals was ordered (additional use of animals was documented in the raw data and reported).
Age of animals: Young adult rats, at least 8 weeks old at starting and at least 12 weeks at mating. The age range within the study was kept to the minimum practicable; minor variations were acceptable for practical reasons.
Body weight range: Males: 366 – 429 g (main + recovery) and 366-416 g (PC group), females: 229 – 292 g (main + recovery) and 252 – 278 g (PC group); did not exceed ± 20 % of the mean weight for each sex at onset of treatment (Day 0)*.
Acclimation period: At least 5 days
*Note: In case of PC animals, as their treatment was performed one day before the necropsy, the body weight values on Day 0 of the main animals were shown to provide more informative data.

Husbandry
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian. Females were nulliparous and non-pregnant.
Cage type: Type II and III polycarbonate
Bedding and nesting: SAFE 3/4-S Hygienic Animal Bedding (Batch No.: 03027191023 / 03027190506, Expiry date: 23 October 2022 / 06 May 2022) and SAFE Crinklets Natural (Batch No.: 05072190726 / 05072190729, Expiry date: 26 July 2022 / 29 July 2022) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study.
Details of the bedding and nesting materials were documented in the raw data.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.0-24.7°C
Relative humidity: 20-67%
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 2 animals (main animals) or 4 animals (recovery and PC animals) of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed, respectively. Group
housing allowed social interaction and the deep wood sawdust bedding and cardboard tunnels (Fun tunnel; supplied by LBS (Serving Biotechnology) Ltd, Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH, United Kingdom) allowed digging and other normal rodent activities (i.e. nesting).
Environmental parameters (temperature and relative humidity) were continuously measured, minimum and maximum values were recorded twice a day during the study.

Food and water supply
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch number: 74655782 / 23258297, Expiry date: 30 April 2020 / 30 June 2020) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest Germany), ad libitum, and tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum. The diet supplier provided analytical certificates for the batches used, which are archived with the raw data.
Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8200 Veszprém, József Attila u. 36., Hungary). The quality control results are archived with the raw data at the Test Facility.
The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Animal identification
Each adult/parental animal (P generation) was identified by a unique number within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at the Test Facility.
During the pre-exposure period, animals were identified with temporary numbers only.
After this 2-week period, a randomisation was performed based on the body weights and the selected animals received their final animal numbers, as follows:
This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section. The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery.
Identification of the new-borns (Offspring, F1 generation) was performed by ink marking of the digit-tips on the day of birth.

Randomisation
All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges on Day 0. 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. Positive control animals were randomized with the main animals
Recovery groups were randomized separately from the main groups.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Based on the dose range finding (DRF) study, corn oil was selected as vehicle for this study in agreement with the Sponsor, according to the formulation and analytical trials. Relevant information of the chemical used as vehicle are shown below:
Name: Corn oil
Manufacturer/Supplier: ACROS Organics
Batch/Lot number: A0395699
Expiry date: 30 April 2020
Storage conditions: Room temperature

Details on mating procedure:

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, for up to 14 days. 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:

Formulations were prepared fresh prior to administration to animals or at the appropriate frequency to allow their use according to stability assessment results of the analytical method validation study performed at the Test Site #1 for analytical work.
Dose levels and concentrations to be employed, as well as instructions regarding the frequency of dose formulations preparation and their storage pending use was documented in the raw data and reported.
Analysis of test item formulations for concentration and homogeneity was performed using a validated GC-FID (Gas Chromatography with Flame Ionization Detector) method (validated concentration range was 60-700 mg/mL). Duplicate samples of approximately 0.5 mL (accurately weighed 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.
Acceptance criterion of the concentration analysis was set according to the analytical method validation, expected to be at 100 ± 15% of the nominal concentration.
Acceptance criterion of the homogeneity was that the CV (coefficient of variation) of replicates (top, middle and bottom of test formulations) must be less than 15%.

Duration of treatment / exposure:

Dosing of both sexes began, after 5 days of acclimatisation and a pre-exposure period of 14 days. Dosing was conducted 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.
The first day of dosing of each animal was regarded as Day 0.

Frequency of treatment:

The 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 tipped gavage needle attached to a syringe. A constant volume of 1.5 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight in each case.

Details on study schedule:

Males were dosed for 29 days (14 days pre-mating and 15 days mating/post-mating) and then euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13-day post-partum dosing). The day of birth (when parturition was complete) was defined as Post-partum Day (PPD) 0.
All F1 offspring were terminated on Day 13 post-partum (F1 offspring selected for blood sampling on PND4 were terminated on that day). In order to allow for overnight fasting of dams with urine collection on PPD14, the offspring were euthanized on PPD/PND13 and the dams on PPD/PND14.
Recovery animals (males and females) scheduled for follow-up observations were not mated, but after 28 days of daily treatment, they were kept for 14 days without treatment* to detect delayed occurrence, or persistence of, or recovery from toxic effects

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 animals/sex/groups, 4 groups.

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study, with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
In the DRF study, no test item related effect was observed up to the limit of 1000 mg/kg bw/day. Therefore, 1000 mg/kg bw/day was considered to be suitable as a top dose for the current study. Based on this information and using a factor of 3, the doses of 100, 300 and 1000 mg/kg bw/day were deemed suitable for the purpose of the study.
The oral route was selected as it is one of the possible routes of human exposure.

Positive control:

Not required for the screening study.

Parental animals: Observations and examinations:

Clinical observations and Functional observation battery (FOB)
All animals:
Animals 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 daily*, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment. The principles and criteria summarized in the Humane Endpoints Guidance Document (OECD) were taken into consideration.
*Note: No general clinical observations were made on those days when detailed clinical observations were made (except of one case on 03 December 2019 due to a technical error.

More detailed examinations were performed once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment.
These observations were performed outside the home cage in a standard arena, at similar day times as practical. The 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.
All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including their onset, degree and duration as applicable.
On Gestation day (GD) 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).

Five main males and five main females/group were selected + all recovery animals:
Assessment of potential test item related neurotoxicity was performed during the last exposure week (males on Day 23; females on PPD9-12; recovery animals on Day 41).
Selected animals were subjected to the Functional observation battery (FOB) including Irwin test (with assessment of grip strength) and measurement of landing foot splay and fore/hind limb grip strength. In order to avoid hypothermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes during FOB or 90 minutes during SMART measurement.
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, exophthalmos, 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. 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.
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 item. 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 and the appropriate grip support. The results were tabulated with individual and mean data.
Motor 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 for a 1-hour observation time, when DVD recording of 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 distance travelled 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.

Body weight measurement
All adult animals were weighed with an accuracy of 1 g at least weekly during the preexposure period, then on Day 0 (randomisation) and 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 at termination. The body weight of the female animals measured on GD 3, 10 and 17 as well as PPD 10 were only additional measurements as aid for the calculation of accurate treatment volumes, thus these data were not evaluated statistically.

Food consumption measurement
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) except on one case (measurement was made on Day 36 instead of Day 35).

Observation of the delivery process, offspring and nursing instinct
Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. All observations were recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition.
Dams were observed for signs of nest building with the bedding material and for covering their new-borns. Evidence of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded.
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. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

THYROID HORMONE ANALYSIS
For thyroid hormone analysis, blood samples were taken by venepuncture (sublingual) into tubes containing K3-EDTA as anticoagulant as follows:
-from all dams on PPD14 (females)
-from all adult males at termination.
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, 4 ºC).
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 as a backup) and stored in an ultrafreezer (-80 ± 10 °C) until shipping for analysis.

CLINICAL PATHOLOGY
All animals were fasted (overnight period of food deprivation, in case of dams: after the litter had been culled). From the selected animals blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
For urine collection and terminal blood sampling in all selected animals (5 male and 5 female main animals/group plus all recovery animals), 3 samples were taken from each animal: one for haematology (in 0.5mL tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in 1.4 mL tubes with sodium citrate as anticoagulant) and one to obtain serum (in 1.5 mL tubes with no anticoagulant) for clinical chemistry.
Haematology and blood clotting times - see "Any other information" for parameters evaluated.
Clinical chemistry - see "Any other information" for parameters evaluated.

Urinalysis
Urine sampling was performed prior to necropsy by placing the selected main animals and all recovery animals in metabolic cages for approximately 16 hours. See "Any other information" for paramters evaluated.

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments starts. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.
Additionally, vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.

Sperm parameters (parental animals):

Not specified

Litter observations:

Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND0) and on PND4 and PND13, with accuracy of 0.01g. All litters were checked and recorded daily for the number of viable and dead pups. None of the found dead pups were intact (not cannibalized or autolysed) at the time of detection, therefore they could not be subjected to necropsy for macroscopic examination.
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 (individual records were maintained).
*Note: One control pup (#1504/4) was indicated as male at birth, thus all the processes required for male pups were conducted. However, at necropsy the sex was confirmed as female based on the internal sex organs (this is in line with the number of nipples).
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.

THYROID HORMONE ANALYSIS
For thyroid hormone analysis, blood samples were taken by decapitation (in case of pups) into tubes containing K3-EDTA as anticoagulant as follows:
-from up to two pups per litter on PND4,
- at least two pups per litter on PND13 (pups),
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, 4 ºC).
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 as a backup) and stored in an ultrafreezer (-80 ± 10 °C) until shipping for analysis.

Postmortem examinations (parental animals):

Dams were sacrificed on PPD14 after fasting overnight.
At termination, the surviving adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination.
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.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.
The number of implantation sites and of corpora lutea were recorded in female animals as applicable.

Organ weight measurements
All animals
At the time of termination, body weight and the weight of the following organs from all surviving adult animals were determined:
- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights.
Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported.

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

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 main animals/sex/group plus all recovery animals),
-all organs where macroscopic findings (abnormalities) were seen.
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, bone marrow, and blood smears if examined).
Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.

Postmortem examinations (offspring):

All pups were culled on PND13.
Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities, where possible. The anaesthetic product was diluted for pups’ euthanasia as required.

Statistics:

Data were recorded on the appropriate forms from the relevant SOPs of the Test Facility and then tabulated using the Microsoft Office Word and/or Excel, or collected using the software PROVANTIS v.9, as appropriate. Group means and standard deviations were calculated from numerical data obtained in the study. The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 or with the program package SPSS PC+4.0. The following test are utilised follwing decision trees:
Bartlett's test, a one-way analysis of variance (ANOVA), Duncan's Multiple Range test, Kolmogorow-Smirnow test, Mann-Whitney U-test, Chi-squared test.
Shapiro-Wilk and Levene tests, an Anova / Ancova (oneway analysis of variance), Dunnett’s (Multiple Range) test, Kruskal-Wallis analysis of variance, Dunn test; identifying differences of <0.05 or <0.01 as appropriate. Cochran-Armitage test for trend, Chi-squared test is used for statistical differences relative to control.

Reproductive indices:

Parental Males
- Number of pairings
- Number of fertile pairings
- Number of infertile males
- Male mating index
- Male fertility index

Parental Females
- Oestrus cycle data
- Number of pairings
- Number of pregnant females
- Number of sperm positive, but non-pregnant females
- Number of non-mated females
- Female mating index
- Female fertility index
- Gestation index
- Duration of pregnancy (days)
- Number of corpora lutea / dams
- Number of implantations / dams
- Number of dams with live pups Day 0, 4 and 13
- Pre-implantation mortality
- Intrauterine mortality
- Total mortality (intra and extra uterine mortality)

Offspring viability indices:

Offspring
- Mean pup body weight (per pup within the group and per litter) on PND0, 4 and 13
- Mean pup body weight gain (per litter) between PND0-4, PND4-13 and PND0-13
- Number of live births per litter, and number of viable pups per litter on postnatal Days 0, 4 and 13
- * Survival Index of pups on postnatal Days 0, 4 and 13
- F*Sex ratio % (on postnatal Days 0, 4 and 13)
- Thyroid hormone analysis
- Anogenital distance , nipple retention

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in the study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was seen during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item related changes were observed in the body weight and body weight gain parameters of the test item treated animals (main or recovery) in any of the sexes when compared to control data.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test item related changes were observed on food consumption of the test item treated animals (main or recovery) in any of the sexes when compared to control data.
Significantly higher food consumption than control was recorded in the first week of the pre-mating period for the Low and Mid dose females and in the second week of the pre-mating period for all test item treated females. But there was no dose response and no similar trends were seen in the males during the same periods or in the females any other periods later in the study. Therefore, these occasionally increased food consumption parameters were not considered to be a test item related effect.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed in the haematology parameters.
Statistically significant differences were observed in some cases in main or recovery animals, but there was no relationship with dose and/or all recorded values were within the historical control range. These differences were considered to not reflect an effect of the test item but being incidental.

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 that could be ascribed to the test item administration.
For all statistically significant differences, either the histopathology results confirm a lack of treatment related adverse effects, or they were considered to be incidental, with no relationship with dose and/or all recorded values were near or within the historical control ranges. These statistical differences were considered to not reflect an adverse effect of the test item.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item-related changes were observed in the urinalysis parameters.

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 noted during the assessment of grip strength, landing foot splay or locomotor activity.
All dose groups of males and females 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 20-30 minutes.
There was no statistical significance between the test item treated animals and the Control when evaluating the overall total travelled distance (0-60 minutes), occasional statistical significance for an individual segment without a clear trend or dose response was not considered as test item related effect. The test item did not increase or decrease the normal locomotor activity, all treated groups (main and recovery animals) had a profile of activity the same as historical control data.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item-related findings were observed.
All observed changes were seen in control and/or treated animals, or without meaningful differences in severity and incidence, therefore were regarded as incidental, procedure related or a common background.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

RECOVERY ANIMAL EVALUATIONS
No treatment related changes were seen in Main group animals of either sex for any in-life parameter, clinical pathology, necropsy or histopathology. Similarly in the recovery animals, there were no changes observed that could indicate any delayed toxicity.

THYROID HORMONE ANALYSIS
No effect of test item was observed in the study based on the results of thyroid hormone analysis and thyroid gland weights.
Compared to the relevant control values, there were no statistically significant differences in T4 thyroid hormone concentrations for parental males and PND13 pups.
No statistically significant increase compared to control, was detected in the absolute or relative thyroid weights of adults and F1 generation. Some observed minor differences (without statistical significance and dose response) were considered as animal variability, not being a test item related effect.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

No effect of test item on oestrus cycles was noted.
Each female selected for the study showed acceptable cycles (mean cycle length of 4.00-4.04 days) before starting the treatment period.
No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods (mean cycle length was 4.00-4.04 days in the test item treated group ad 4.02 days in the control group). Prolonged oestrus was recorded for one Low dose female; this fact was considered as being an occasional finding, not being a test item related effect.

Reproductive function: sperm measures:

not examined

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 the administration of the test item. Both the mating and fertility index was 100% in all groups (males and females). The gestation index was also 100% in all groups.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred mostly within 5 days of pairing (cohabitation), only in case a Low dose female (#2504) it lasted for 12 days and in case of a High dose female (#4511) it lasted for 14 days. The mean duration of mating was 3.33, 3.42, 2.50 and 3.58 days in the Control, Low, Mid and High dose groups, respectively.
There was no effect of treatment noted during the gestation period, parturition and postpartum period in any of dose groups.
The mean duration of pregnancy was comparable in the Control and test item treated groups.
As far as it could be observed during the study, the parturition was normal for all animals, no abnormal delivery was noted.
The number of implantation sites was comparable to the control mean in all dose groups, 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.

In summary, daily administration of SynNova Base Oil by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day, under the conditions of this study, did not result in mortality or any clinical signs.
The bodyweight, body weight gain, and food consumption of the test item treated groups did not show any test item related effect.
At the functional observation battery (FOB) and locomotor activity measurement, there were no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups.
No test item-related findings were noted in the clinical pathology parameters.
No test item effect on oestrus cycle of parental females was noted.
No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14.
No test item related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs.
Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Evidence of suckling was recorded for all live born pups in the study.
The ratio of female pups was slightly lower in the High group than in the Control group, but as there was no statistical significance, thus this fact was considered as biological variability, not related to the test item.
Based on the external evaluation, no clinical signs or abnormalities were recorded for any pups except of one Low dose male pup (#2505/17) and one Mid dose pup (#3503/2), where haemorrhage on the nose / snout was recorded. This finding was considered as minor, incidental finding, not related to the test item treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item effect on mortality or survival of the pups (F1 generation).
The number of viable pups on PND0, PND4 and PND13 as well as pup survival indices on PND0, PND4 and PND13 were comparable to control values in each dose group.
There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related differences in the offspring body weights or weight gains in any test item treated group when compared to the controls. The measured values were within the range commonly recorded for this strain and age.
When evaluated per litter basis, the mean litter body weights and/or body weight gain on PND0, 4 and 13 showed no toxicologically significant differences compared to controls in the F1 generation.

Food consumption and compound intake (if feeding study):

not examined

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No test item effect was observed on anogenital distance during the study.
No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when litter mean values were compared to control.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No test item effect was observed on nipple retention during the study.
There was no nipples/areolae presence seen in any of the male pups on PND13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item related macroscopic changes were seen in F1 offspring generation euthanized and examined externally at scheduled termination on PND13.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No effect of test item was observed in the study based on the results of thyroid hormone analysis and thyroid gland weights.
Compared to the relevant control values, there were no statistically significant differences in T4 thyroid hormone concentrations for parental males and PND13 pups.
No statistically significant increase compared to control, was detected in the absolute or relative thyroid weights of adults and F1 generation. Some observed minor differences (without statistical significance and dose response) were considered as animal variability, not being a test item related effect.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic finding were recorded for F1 pups at necropsy.
Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: development and survival

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Summary of reproductive parameters (males)

Parameters	Dose group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Number of treated animals	12	12	12	12
Number of males used for mating	12	12	12	12
Number of successful mating	12	12	12	12
Number of infertile animals	0	0	0	0
Male mating index (%)	100	100	100	100
Male fertility index (%)	100	100	100	100

 

Summary of reproductive parameters (females)

Parameters	Dose group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Number of treated animals	12	12	12	12
Number of females used for mating	12	12	12	12
Number of sperm positive females	12	12	12	12
Number of females with no implantation sites	0	0	0	0
Number of pregnant females	12	12	12	12
Number of pregnant females with live born	12	12	12	12
Female mating index (%)	100	100	100	100
Female fertility index (%)	100	100	100	100
Female gestation index (%)	100	100	100	100

 

Summary of the pregnancy evaluation

Parameters	Dose group / Concentration (mg/kg bs/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Number of evaluated females	12	12	12	12
Number of pregnant females	12	12	12	12
Duration of pregnancy (days)	22.67	22.33	22.42	22.42	NS
Number of implantations, mean	17.58	16.83	17.75	16.33	NS
Number of pups born, mean	16.25	15.00	16.25	15.42	NS
Number of live born pups, mean	16.25	14.92	16.17	15.42	NS
Pre-natal mortality, mean	1.33	1.92	1.58	0.92	NS
Pre-natal mortality (%), mean	7.33	10.90	8.93	6.39	NS
Post-natal mortality, mean	1.00	0.17	0.33	1.08	NS
Post-natal mortality (%), mean	6.76	0.99	2.06	6.50	NS
Total mortality, mean	2.33	2.08	1.93	2.00	NS
Total mortality (%), mean	12.63	11.81	10.79	12.75	NS

Notes: Data (group and mean) 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

 

Summary of survival (offspring)

Parameters	Dose group / Concentration (mg/kg bs/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Number of evaluated litters	12	12	12	12
Number of pups born, mean	16.25	15.00	16.25	15.42	NS
Number of live born pups, mean	16.25	14.92	16.17	15.42	NS
Number of living pups on PND13, mean	13.25	12.75	13.83	12.33	NS
Pre-natal mortality, mean	1.33	1.92	1.58	0.92	NS
Pre-natal mortality (%), mean	7.33	10.90	8.93	6.39	NS
Post-natal mortality on PND0-4, mean	0.83	0.17	0.25	0.83	NS
Post-natal mortality on PND0-4 (%), mean	5.83	0.99	1.59	4.87	NS
Total mortality on PND4, mean	2.17	2.08	1.83	1.75	NS
Total mortality on PND4 (%), mean	11.71	11.82	10.33	11.27	NS
Pups culled for blood sampling, mean	2.00	2.00	2.00	2.00	NA
Post-natal mortality on PND0-13, mean	1.00	0.17	0.33	1.08	NS
Post-natal mortality on PND0-13 (%), mean	6.76	0.99	2.06	6.50	NS
Total mortality on PND13, mean	2.33	2.08	1.92	2.00	NS
Total mortality on PND13 (%), mean	12.63	11.81	10.79	12.75	NS
Survival index on PND0	100.00	99.51	99.40	100.00	NS
Sex ratio (%) on PND0	47.87	48.41	49.11	40.72	NS
Survival index on PDN4	94.17	99.01	98.41	95.13	NS
Sex ratio (%) on PND4	48.32	48.40	49.71	41.10	NS
Survival index on PND13	98.96	100.00	99.44	98.12	NS
Sex ratio (%) on PND13	48.24	47.98	49.19	40.72	NS

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).

NS: Statistically not significant when compared to control, NA: Not applicable

Summary of mortality (offspring)

Parameters	Dose group / Concentration (mg/kg bs/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Number of evaluated litters	12	12	12	12
Number of pups born	195 / 12	180 / 12	195 / 12	185 / 12	NS
Number of cannibalized pups	0 / 0	0 / 0	0 / 0	0 / 0	NA
Number of autolyzed pups	0 / 0	1 / 1	1 / 1	0 / 0	NA
Number of stillborn pups	0 / 0	0 / 0	0 / 0	0 / 0	NA
Number of live born pups	195 / 12	179 / 12	194 / 12	185 / 12	NS
Number of found dead pups (born alive)	0 / 0	0 / 0	0 / 0	0 / 0	NA
Number of living pups on PND0	195 / 12	179 / 12	194 / 12	185 / 12	NA
Number of cannibalized pups ((PND0-13)	11 / 3	2 / 2	4 / 3	2 / 2	NA
Number autolyzed pups (PND0-13)	1 / 1	0 / 0	0 / 0	11 / 3	NA
Number of found dead, intact pups (PND0-13)	0 / 0	0 / 0	0 / 0	0 / 0	NA
Total number of pups died (born alive)	12 / 4	2* / 2	4* / 3	13 / 5	CH
Culled for blood sampling on PND4	24 / 12	24 / 12	24 / 12	24 / 12	NA
Number of viable pups on PND13	159 / 12	153 / 12	166 / 12	148 / 12	NS

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

Statistical significance compared to control: * = p<0.05, ** = p<0.01 (negative trends has no biological relevance)

NA: Not applicable, CH: Chi square test, NS = Statistically not significant compared to control

 

Selected body weight data (offspring)

Parameters	Dose group / Concentration (mg/kg bs/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Number of evaluated litters	12	12	12	12
Mean litter body weight (PND0), g	6.55	6.41	6.52	6.33	NS
Mean litter body weight (PND4), g	10.58	10.66	10.72	11.12	NS
Mean litter body weight gain (PND0-4), g	4.03	4.25	4.19	4.34	NS
Mean litter body weight (PND13), g	29.39	30.86	30.51	31.56	NS
Mean litter body weight gain (PND4-13), g	18.81	20.20	19.82	20.46	NS
Mean litter body weight gain (PND0-13), g	22.83	24.46	23.99	24.78	NS

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

NS: Statistically not significant when compared to control.

Anogenital distance

Parameters	Dose group / Concentration (mg/kg bs/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Male pups
Number of evaluated litters	12	12	12	12
Anogenital distance, litter mean of males (mm)	3.59	3.77	3.81	3.83	NS
Minimum / Maximum value, litter mean (mm)	3.1 / 4.1	3.3 / 4.2	3.3 / 4.2	3.4 / 4.4
Anogenital distance ratio by BW cube root	0.544	0.578	0.574	0.556	NS
Female pups
Number of evaluated litters	12	12	12	12
Anogenital distance, litter mean of females (mm)	1.59	1.74	1.78	1.77	NS
Minimum / Maximum value, litter mean (mm)	1.2 / 2.0	1.4 / 2.1	1.5 / 2.1	1.4 / 2.2
Anogenital distance ratio n by BW cube root	0.254	0.282	0.282	0.274	NS

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

BW: body weight, NS: Statistically not significant when compared to control

 

Selected parameters related to thyroid hormone levels

Parameters	Dose group / Concentration (mg/kg bs/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Parental males
Number of evaluated males	12	12	12	12
T4 concentration (ng/mL)                        HC range: 26.3-61.6	16.25	14.92	16.17	15.42	NS
	difference (%)	1.5	3.0	-0.6
Thyroid gland weights (g)	0.0272	0.0276	0.0278	0.0280	NS
	difference (%)	1.5	2.5	3.1
Thyroid gland / body weight (%)	0.0060	0.0061	0.0060	0.0063	NS
	difference (%)	2.2	0.5	5.2
PND13 pups
Number of evaluated litters	12	12	12	12
T4 concentration (ng/mL)                        HC range: 34.3-60.7	48.38	46.96	45.12	47.73	NS
	difference (%)	-2.9	-6.8	-1.4
Thyroid gland weights (g)	0.0050	0.0050	0.0051	0.0051	NS
	difference (%)	0.8	2.5	1.7
Thyroid gland / body weight (x 10^-4)	1.6898	1.6258	1.7028	1.6360	NS
	difference (%)	-3.8	0.8	-3.2

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. Pups blood were pooled for T4 (thyroxin) determination. HC: Historical control. NS: Statistically not significant when compared to control.

Conclusions:

In summary, daily administration of SynNova Base Oil by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day, under the conditions of this study, did not result in mortality or any clinical signs.
The bodyweight, body weight gain, and food consumption of the test item treated groups did not show any test item related effect.
At the functional observation battery (FOB) and locomotor activity measurement, there were no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups.
No test item-related findings were noted in the clinical pathology parameters.
No test item effect on oestrus cycle of parental females was noted.
No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14.
There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic finding were recorded for F1 pups at necropsy.
No test item related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs.
Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.
The NOAEL for systemic toxicity of the parental generation was considered to be 1000 mg/kg bw/day.
The NOAEL for reproductive effects of the parental generation was considered to be 1000 mg/kg bw/day.
The NOAEL for Pup development and survival was considered to be 1000 mg/kg bw/day.

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of SynNova Base Oil test item following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels. A control group received the vehicle only (corn oil).

The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 13 post-partum. Reversibility of any treatment-related changes were also evaluated following a dedicated 14-day recovery period.

 

The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a dose range finding (DRF) study. Based on the results of the DRF study, 1000 mg/kg bw/day was selected as the High dose for this study.

 

RESULTS

In summary, daily administration of SynNova Base Oil by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day, under the conditions of this study, did not result in mortality or any clinical signs.

 

The bodyweight, body weight gain, and food consumption of the test item treated groups did not show any test item related effect.

 

At the functional observation battery (FOB) and locomotor activity measurement, there were no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups.

 

No test item-related findings were noted in the clinical pathology parameters.

 

No test item effect on oestrus cycle of parental females was noted.

 

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14.

 

There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic finding were recorded for F1 pups at necropsy.

 

No test item related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs.

 

Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.

 

The NOAEL for systemic toxicity of the parental generation was considered to be 1000 mg/kg bw/day.

The NOAEL for reproductive effects of the parental generation was considered to be 1000 mg/kg bw/day.

The NOAEL for Pup development and survival was considered to be 1000 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

This study is classified as K1, reliable without restriction as it was in accordance with OECD test guidelines and conducted in a GLP accredited laboratory.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

In summary, daily administration of SynNova® Base Oil by oral gavage to pregnant Hannover Wistar rats from gestation day 6 (GD6) to gestation day 19 (GD19) at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any mortality or clinical signs of the dams under the conditions of this study.

There was no test item-related effect on maternal body weight, body weight gain and food consumption in any of the test item-treated groups.

No remarkable internal or external observations related to the test item treatment were recorded for any of the pregnant animals during necropsy.

No remarkable abnormalities were observed on the placentas in any examined groups.

There were no test item related changes in the examined parameters of the intrauterine development up to and including 1000 mg/kg bw/day.

No test item-related effect was observed on the fetal body weight and the number of viable fetuses in any of the test item-treated groups, as well as their sex distribution were comparable with the control.

Based on the results of thyroid hormones analysis, thyroid weights, histopathological evaluation of thyroids and the measurement of anogenital distance of fetuses, no endocrine disruptor effect was observed in the study.

There were no statistically significant effects of the test item on the external, visceral and/or skeletal development of fetuses in the study.

The No Observed Effect Level (NOEL) under the conditions of this study for maternal and fetal developmental toxicity is considered to be 1000 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 May - 25 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

K1 GLP study to current guidelines. This study was conducted to meet the requirements of other jurisdictions such as China.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See "Any other information on materials and methods incl. tables" for details

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

Hannover

Details on test animals or test system and environmental conditions:

Species and strain: Han:WIST rats
Source: Toxi-Coop Zrt., H-1122 Budapest, Magyar Jakobinusok tere 4B
Hygienic level at supplier: SPF
Hygienic level during
the study: Standard housing conditions
Justification of strain: The rat is regarded as a suitable rodent species for reproduction studies and the test guideline states it is the preferred rodent species. The Hannover Wistar rat was selected due to experience with this strain in teratology studies.
Number of animals: 89 female animals; 22, 22, 22 and 23 mated female animals in the control, low, mid and high dose groups, respectively,
30 male animals for mating; no study-procedures were carried out on the male animals; untreated, proven breeders from NEXTREAT Laboratories’ spare colony were used.
Age of animals at start: Young adult rats, nulliparous and non-pregnant, 11 -12 weeks old
Body weight at the start: Did not exceed ± 20% of the mean weight at onset of treatment and were in the range of 201-246 g
Acclimatisation period: At least 20 days
Animal health: Only healthy animals were used for the test. The health status was certified by the Veterinarian.
Cage type: T3H polycarbonate
Housing / Enrichment: The animals were housed individually. “SAFE 3/4-S-FASERN” certified wooden chips (batch number: 03027210315, expiry date: 15 March 2024) produced by J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) and “Sizzle pet” nest material (batch number: 491607, expiry date: 05 August 2023) produced by LBS (Serving Biotechnology) Ltd. (Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH, United Kingdom) were available to animals during the study. Fresh bedding was provided for the animals as frequently as appropriate/practical, but at least twice weekly. Copies of the Certificates of Analyses are archived with the raw data.
Cages were arranged in such a way that possible effects due to cage placement are minimised.
Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20 – 24 °C (target: 22 ± 3 °C)
Relative humidity: 39 – 67 % (target: 30-70%)
Ventilation: 15-20 air exchanges/hour

The minimum and maximum temperature and relative humidity values were recorded daily during the study.

Food and water Supply

Animals received standard laboratory rat diet, ad libitum. The food is not considered to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate for the batch used, which is archived with the raw data.

Details of the diet used in the study were as follows:

Name: SM Rat/Mouse, Breeding & Maintenance, 10 mm, autoclavable
Manufacturer: ssniff Spezialdiäten GmbH (D-59494 Soest, Germany)
Batch number: 233 77046
Expiry date: 30 September 2021

Animals received tap water from the municipal supply, as for human consumption, from drinking bottles, ad libitum. Water quality control analysis and microbiological assessment are performed once per year by the laboratory of Veszprém County Government Office, Department of Public Health (Veszprém Megyei Kormányhivatal Népegészségügyi Főosztály, H-8200 Veszprém, József A. u. 36., Hungary). The quality control results are retained in the archives of NEXTREAT Laboratories.


Animal identification
Adult animals were identified by temporary numbers prior to mating. Once the mating has been confirmed, the dams allocated to the study were identified by definitive numbers written with indelible ink on the tail, and cross referenced with the temporary numbers. During necropsy and caesarean section procedures each evaluated dam was given additional number (evaluation number), indicating group number.

The housing cages were identified with individual identity cards, with information about study code, sex, dose group, cage number, animal number, date of mating and caesarean section/necropsy date.

The litters were identified at necropsy with litter numbers. The flasks used for fixation and all sheets used for recording data were identified only by the litter numbers up to the end of fetal examinations. The fetuses were identified during the caesarean section with individual numbers according to their implantation sites. For visceral examination they were identified by digit-clipping; for skeletal examination the fetuses were identified by means of a water-proof plastic ribbon tied around their neck.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

A constant volume of 1.5 mL/kg body weight was administered to all dose groups, including the control. The individual volume of the treatment was based on the most recent individual body weight of the animals.

The control or test item formulations were administered to mated, sperm positive assumed pregnant female rats daily by oral gavage on a 7 days/week basis, approximately at similar times with minor variations as practical, from GD6 to GD19.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the test item formulations for concentration and homogeneity was performed at the Test Facility. Top, middle and bottom duplicate samples were taken from the test item formulations two times during the study (during the first and last weeks of the treatment period), one set to analyze and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

The formulation analyses were conducted under the control of the Analyst. Acceptance criteria of the concentration analysis was 100 ± 15% of the nominal concentration. Acceptance criteria of the homogeneity was that the RSD% of replicates (top, middle and bottom of test item formulations) should be less than 10%.

Details on mating procedure:

Mating procedure, Randomization

The oestrus cycle of female animals was examined a day before start of pairing. After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2 hours (1 male : 1 female) until at least 22 sperm positive females/group were attained. The mating of siblings was avoided and the mating partners of the females were randomly chosen. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of sperm in the vaginal smear was considered as evidence of copulation (GD0). Sperm positive females were separated and caged individually.

The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that group averages of the body weight were as similar as possible. Females inseminated by the same male were evenly distributed across groups.

Duration of treatment / exposure:

Daily

Frequency of treatment:

7days/week

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

89 female animals; 22, 22, 22 and 23 mated female animals in the control, low, mid and high dose groups, respectively.

Control animals:

yes, concurrent no treatment

Details on study design:

The objective of the study was to assess the effects of the test item on pregnant female rats and on the developing conceptuses and provide general information concerning the effects of prenatal exposure on the pregnant test animals and on the developing organism [1,2]; this may include assessment of maternal effects as well as death, structural abnormalities, or altered growth in the fetus. One control and 3 test item-treated groups of Hannover Wistar rats were treated daily by oral (gavage) administration.

The test item was administered daily to pregnant animals from implantation to one day prior to the day of scheduled kill, which was as close as possible to the normal day of delivery without risking loss of data resulting from early delivery. The experiment was not intended to examine solely the period of organogenesis (e.g. gestation days 5-15 in the rodent), but also effects through the entire period of gestation to the day before caesarean section. The day of mating (when the sperm-positive vaginal smear was identified) was regarded as gestation day 0 (GD0). Treatment was performed daily by oral (gavage) administration, between GD6 to GD19. Caesarean section and necropsy with macroscopic examination were performed on GD20.


Justification of the doses and route of administration
The dose levels were selected by the Sponsor in consultation with the Study Director, based on the results from an OECD 422 reproductive toxicity study [3] with the aim of inducing toxic effects but no death or suffering at the highest dose, and to obtain a No Adverse Effect Dose Level (NOAEL) at the lowest dose.

The oral route was selected since it is one of the routes of administration requested by the regulatory authorities, and it is considered suitable to provide the exposure required for this developmental toxicology study.

ANIMAL WELFARE
Procedures involving the care and use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). During the study, the care and use of animals were conducted in accordance with the relevant principles currently used with regard to animal welfare.

This study was performed with vertebrate animals as no in vitro alternative was available. The study was designed such that the minimum number of animals was used.
 

Maternal examinations:

Clinical signs
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). The principles and criteria summarized in the OECD Humane Endpoints Guidance Document No. 19 were taken into consideration [5].

General clinical observations were made daily, preferably at the same time(s) each day after treatment and once before necropsy.

Detailed clinical observations were made on all animals at the onset of treatment (GD6) then on GD13 and at necropsy (GD20). Observation was performed on the skin, fur, eyes and mucous membranes, occurrence of secretions and excretions, autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as presence of clonic or tonic movements, stereotypes, bizarre behaviour was also observed. Special attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

On GD13 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, which is considered to confirm implantation).

Body weight
Body weight of each animal was recorded with precision of ±1 g on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Body weight gain of pregnant females was calculated for each interval, including
GD 0-6, GD 6-20 and GD 0-20.

Food consumption
Food consumption was measured with precision of ±1 g on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption of pregnant females was calculated for each interval, including GD0-6, GD6-20 and GD0-20.

Ovaries and uterine content:

Before expected delivery, on GD20, Caesarean section was performed on each treated dam. Sodium pentobarbital (Euthanimal 40% (400 mg/mL pentobarbital sodium); Manufacturer: Alfasan Nederland BV; Batch number: 2001004-06; Expiry date: 31 January 2023) was administered by intramuscular injection for euthanasia, followed by exsanguination.

The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes. All gross findings are retained in 10% buffered formalin solution.

The weight of the thyroid gland with parathyroid glands for all dams was measured with a precision of 0.001 g. As a paired organ, it was weighed individually, but reported together. Absolute organ weights were measured, and relative paired organ weights to the body weights were calculated and reported. The organs are retained in 10% buffered formalin solution.

The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. If no implantation sites were evident but corpora lutea were present, the uterus was stretched and hold in front of a light source to clearly identify the implantation sites. Uteri that appeared non-gravid were further examined to confirm the non-pregnant status by Salewski staining method.

The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percent of pre- and post-implantation losses was calculated. The degree of resorption (early, late) was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.

Animals were checked for early delivery or abortion, but no signs of these phenomena were noted.

Blood sampling:

For thyroid hormone analysis, blood samples were taken by cardiac puncture into two tubes, one containing lithium heparin as anticoagulant (to be processed for plasma) and one containing no anticoagulant (to be processed for serum) at study termination (at least 1 mL each). The samples were taken before 11:30 AM each day, to avoid the diurnal variation of the hormone concentration among animals. Blood samples from non-pregnant females were not pooled with pregnant dams.

The processed plasma was divided into two aliquots and the serum into three aliquots. The first plasma aliquots were assessed for T4, the first and second serum aliquots for T3 and TSH, respectively.

Thyroxine (T4)

The T4 levels were determined from the plasma samples by IDEXX Catalyst One Chemistry Analyzer (Catalyst Total T4 Test slide, batch: 708096, exp.date: 01 June 2022
measurable range: 6.4-257.4 nmol/L).
 

Triiodothyronine (T3)

Total Triiodothyronine (T3) ELISA kit (Cat. No.: RCD025R, produced by BioVendor – Laboratorní medicína a.s., batch number: X21-090, exp.date: 28 February 2022, (Limit of detection (LOD): 0.049 ng/mL / limit of quantification (LOQ): 0.164 ng/mL) was used for T3 level determination from the serum samples. BMG FLUOstar Omega Microplate Reader was used for the optical density measurements.

Thyroid-stimulating hormone (TSH)

Rat Thyroid Stimulating Hormone (TSH) ELISA kit (Cat. No.: abx156194, produced by Abbexa Ltd., batch number: E2108560W, exp.date: 31 March 2022, (Limit of detection (LOD):
29 pg/mL / limit of quantification (LOQ): 97 pg/mL) was used for TSH level determination from the serum samples. BMG FLUOstar Omega Microplate Reader was used for the optical density measurements.

The samples were analysed on the day of sampling or stored in a freezer until analysis.

The remaining plasma and serum aliquots are stored as backups for confirmatory analyses. Any samples not required for analysis will be discarded following finalization of the report.

Fetal examinations:

After ensuring humane death, each fetus was weighed individually (accuracy ±0.01 g) and subjected to external examination. The sex of each fetus was determined and the anogenital distance was determined of all live fetuses. Thereafter, the fetuses were individually identified; approximately half of each litter was subjected to detailed visceral examination, and the other half was processed for skeletal examination.

Particular attention was paid to the reproductive tract which was examined for signs of altered development. External fetal sex (as determined by gross examination) was compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue malformations). In addition, indication of incomplete testicular descent/cryptorchidism was noted in male fetuses. 

For the fetuses subjected to visceral examination, the abdominal and thoracic regions were opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomiya mixture, then after fixation the body was micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

For the fetuses subjected to skeletal examination, the abdominal region was opened and the viscera and skin of fetuses were removed and the cadaver was fixed in alcian-blue - acetic acid - ethanol mixture. After fixation in isopropanol, the skeletons were stained by KOH-Alizarin
red-S method and examined by means of a dissecting microscope.

All abnormalities (variations, malformations and retardations) found during the fetal examinations were recorded.

Statistics:

See "Any other information on materials and methods incl. tables"

Historical control data:

As per lab historical data.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in the study.

Mortality:

no mortality observed

Description (incidence):

There was no mortality during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effect was observed on body weight and body weight gain in the low, mid and high dose groups (100, 300 and 1000 mg/kg bw/day) when compared to the control.

The statistically significant increase in mean body weight gain in the high dose group between GD12 and GD 14 (p˂0.05) was considered not test item related, and was rather a result of the relatively higher number of females (3 out of 21 dams) with less than 6 fetuses in the control dose group in comparison with the treated animals.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no toxicologically relevant effect noted in food consumption in any of the treated groups.

Compared to the control, statistically significant higher group mean food consumption was noted at the high dose group between GD12 and GD 14 (p˂0.05). The cumulative food consumption GD6 – GD20 and GD0-20 was also statistically significantly higher (p˂0.05, respectively) in this group. This can also be explained with the lower number of fetuses in the control dose group, resulting in a less intensive appetite and body weight gain of the pregnant females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

No test item-related difference was observed in thyroid and parathyroid gland weights between the control group and treated groups (100, 300 and 1000 mg/kg bw/day).

Statistically significantly (p˂0.05) higher than control relative thyroid weight (adjusted to body weight) group mean was noted in the low dose group. As the mean value was within the historical control range, and no dose response was shown, the change was considered to be incidental, and not test item related.

The mean TSH, T4 and T3 level was comparable in the control and test item treated groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item related changes were observed in gravid uterine weight of the low, mid and high dose group animals (100, 300 and 1000 mg/kg bw/day) when compared to control data.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item related macroscopic findings were noted at any dose level (100, 300 and
1000 mg/kg bw/day) at necropsy.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No structure/morphology effect was noted in the thyroid and parathyroid glands during histopathological evaluation.

No test item-related difference was observed in thyroid and parathyroid gland weights between the control group and treated groups (100, 300 and 1000 mg/kg bw/day).

Statistically significantly (p˂0.05) higher than control relative thyroid weight (adjusted to body weight) group mean was noted in the low dose group. As the mean value was within the historical control range, and no dose response was shown, the change was considered to be incidental, and not test item related.

The mean TSH, T4 and T3 level was comparable in the control and test item treated groups.

Other effects:

no effects observed

Description (incidence and severity):

No test item related changes were observed in gravid uterine weight of the low, mid and high dose group animals (100, 300 and 1000 mg/kg bw/day) when compared to control data.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Eighty-nine sperm positive females were included in the study (22, 22, 22 and 23 in the control, low, mid and high dose group, respectively). The number of confirmed pregnant, evaluated dams in the dose groups treated at 0, 100, 300 and 1000 mg/kg/day was 21, 21, 21 and 22, respectively.

There were no test item-related differences in the number of corpora lutea, in pre-implantation loss, in number of implantations, in early embryonic loss and in late embryonic loss between the control group and the treated groups (100, 300 and 1000 mg/kg bw/day).

There was no statistically significant difference in fetal death and post-implantation loss in any test item treated group compared to the control.

The total intrauterine mortality was comparable with the control, no statistically significant difference was observed.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no test item-related differences in the number of corpora lutea, in pre-implantation loss, in number of implantations, in early embryonic loss and in late embryonic loss between the control group and the treated groups (100, 300 and 1000 mg/kg bw/day).

There was no statistically significant difference in fetal death and post-implantation loss in any test item treated group compared to the control.

The total intrauterine mortality was comparable with the control, no statistically significant difference was observed.

Other effects:

no effects observed

Description (incidence and severity):

No macroscopic abnormalities were observed in the placentas in any of the experimental groups in the study.

Details on maternal toxic effects:

No toxic effects were noted.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No effects at the highest dose tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The weight of fetuses in the test item-treated dose groups was comparable with the control.

No test item-related differences were observed in the total number of body weight retarded fetuses (evaluated as external variation) in any of the test item treated groups.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no toxicologically significant difference in the sex distribution of fetuses between of the control and treatment groups.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

No test item effect was observed on anogenital distance of the fetuses.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no fetal external, visceral or skeletal abnormalities ascribed to the test item administration to pregnant female rats from GD6 to 19, at dose levels up to and including 1000 mg/kg bw/day. The number and percentages of the abnormalities recorded in test item treated groups was comparable or lower than control.

Two fetuses showed skeletal malformations (split vertebrae and hemivertebra) in the mid dose group, with no dose response therefore are regarded as incidental.

Minor structural changes (variations) occurred, including thymic cord, 2 or less incomplete ossification of the skull bones, 3 or more unossified sternal bodies, supernumerary ribs (14th, short unilateral or bilateral) slight wavy/wavy ribs, unossified vertebrae, bipartite and/or unilateral ossification of vertebrae, less than 3 ossified metacarpal bones, or less than 4 ossified metatarsal bones observed with low incidence throughout all groups including controls, or with lower incidence in the treated groups than in the controls or without a dose response and/or without attaining statistical significance. These variations are common background changes in this strain of rats were not considered toxicologically significant, or related to test item administration.

Visceral malformations:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects at the highest dose tested

Abnormalities:

no effects observed

Developmental effects observed:

no

Overall status of the fetuses

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of evaluated litters	21	21	21	22
Examined fetuses, total	213	234	214	238
Overall intact fetuses, mean	9.14	10.38	9.14	10.00	NS
Overall intact fetuses (%), mean	88.1	93.2	89.2	92.7	NS
Overall intact fetuses, total	192	218	192	220	NS
Fetuses with overall variation, mean	1.00	0.76	0.95	0.82	NS
Fetuses with overall variation, (%), mean	11.9	6.8	10.0	7.3	NS
Fetuses with overall variation, total	21	16	20	18	NS
Fetuses with overall malformation, mean	0.0	0.0	0.1	0.0	NS
Fetuses with overall malformation (%), mean	0.0	0.0	0.9	0.0	NS
Fetuses with overall malformation, total	0	0	2	0	NS

NS: Statistically not significant compared to control

 

Summary of Body weight, litter weight and sex distribution of fetuses

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of evaluated litters	21	21	21	22
Examined fetuses, total	213	234	214	238
Mean fetal weight (g)	3.317	3.284	3.373	3.375	NS
Mean body weight of male fetuses (g)	3.407	3.395	3.461	3.469	NS
Mean body weight of female fetuses (g)	3.246	3.176	3.299	3.292	NS
Mean litter weight (g)	33.69	36.49	34.38	36.60	NS
Fetuses with retarded body weight (%), (runts)	7.05	2.90	3.24	0.86	NS
Number of fetuses with retarded body weight	10	7	7	2	NS
Anogenital distance of male fetuses (mm), mean	2.238	2.253	2.250	2.264	NS
Anogenital distance of female fetuses (mm), mean	1.259	1.271	1.263	1.267	NS
Ratio of male fetuses (%)	39.7	46.0	48.9	46.3	NS
Ratio of female fetuses (%)	60.3	54.0	51.1	53.7	NS

NS: Statistically not significant compared to control

 

Summary of Body weight, litter weight and sex distribution of fetuses

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of evaluated litters	21	21	21	22
Examined fetuses, total	213	234	214	238
Mean fetal weight (g)	3.317	3.284	3.373	3.375	NS
Mean body weight of male fetuses (g)	3.407	3.395	3.461	3.469	NS
Mean body weight of female fetuses (g)	3.246	3.176	3.299	3.292	NS
Mean litter weight (g)	33.69	36.49	34.38	36.60	NS
Fetuses with retarded body weight (%), (runts)	7.05	2.90	3.24	0.86	NS
Number of fetuses with retarded body weight	10	7	7	2	NS
Anogenital distance of male fetuses (mm), mean	2.238	2.253	2.250	2.264	NS
Anogenital distance of female fetuses (mm), mean	1.259	1.271	1.263	1.267	NS
Ratio of male fetuses (%)	39.7	46.0	48.9	46.3	NS
Ratio of female fetuses (%)	60.3	54.0	51.1	53.7	NS

NS: Statistically not significant compared to control

 

Summary of pregnancy data

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of mated females	22	22	22	23
Number of non-pregnant females	1	1	1	1
Number of evaluated females on GD20 (Caesarean section)	21	21	21	22

 

DOSE FORMULATION ANALYSIS

 

Concentration and homogeneity of the dose formulations were determined twice during the study (03 and 22 June 2021).

 

Based on the results, all test item formulations were shown to be homogeneous and were found to be in the range of 94 to 106% of nominal concentrations, as detailed in the table below. No test item was detected in the negative (vehicle) control sample. Based on these results, formulations were considered suitable for the study purposes.

 

Analytical results

Nominal concentration (mg/mL)	Measured concentration ± 95% confidence interval (mg/mL)	RSD (%)	Percentage of the nominal concentration (%)
1^st Analytical sampling (Sampling: 03 June 2021)
Control	not detectable	-	-
66.7	70.78±0.95	1	106
200	188.38±7.77	4	94
666.7	630.98±11.29	2	95
2^nd Analytical sampling (Sampling: 22 June 2021)
Control	not detectable	-	-
66.7	68.96±1.21	2	103
200	188.04±2.28	1	94
666.7	651.72±13.42	2	98

 

HISTOPATHOLOGICAL OBSERVATIONS

EVALUATED ANIMALS

TEST ITEM: SynNova® Base Oil					DOSE: 100 mg/kg bw/day (low dose)							SEX: Female								STUDY CODE: N21001-414
OBSERVATIONS	ANIMAL NUMBERS:		2501	2502		2503	2504	2505	2506	2507	2508		2509	2510	2511	2512	2513	2514	2515		2516	2517	2518	2519	2520	2521
THYROID GLAND (a):		lesion	-	-		-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-
THYROID GLAND (b):		lesion	-	-		-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-

 

NON PREGNANT / NON EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil

DOSE: 100 mg/kg bw/day (low dose)

SEX: Female

STUDY CODE: N21001-414

OBSERVATIONS

ANIMAL NUMBERS:

# 2522 NP

THYROID GLAND (a):

lesion

-

THYROID GLAND (b):

lesion

-

 

 

 

 

EVALUATED ANIMALS

TEST ITEM: SynNova® Base Oil					DOSE: 100 mg/kg bw/day (low dose)							SEX: Female								STUDY CODE: N21001-414
OBSERVATIONS	ANIMAL NUMBERS:		2501	2502		2503	2504	2505	2506	2507	2508		2509	2510	2511	2512	2513	2514	2515		2516	2517	2518	2519	2520	2521
THYROID GLAND (a):		lesion	-	-		-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-
THYROID GLAND (b):		lesion	-	-		-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-

 

NON PREGNANT / NON EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil

DOSE: 100 mg/kg bw/day (low dose)

SEX: Female

STUDY CODE: N21001-414

OBSERVATIONS

ANIMAL NUMBERS:

# 2522 NP

THYROID GLAND (a):

lesion

-

THYROID GLAND (b):

lesion

-

 

EVALUATED ANIMALS

TEST ITEM: SynNova® Base Oil					DOSE: 300 mg/kg bw/day (mid dose)							SEX: Female								STUDY CODE: N21001-414
OBSERVATIONS	ANIMAL NUMBERS:		3501	3502		3503	3504	3505	3506	3507	3508		3509	3510	3511	3512	3513	3514	3515		3516	3517	3518	3519	3520	3521
THYROID GLAND (a):		lesion	-	-		-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-
THYROID GLAND (b):		lesion	-	-		-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-

 

NON PREGNANT / NON EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil

DOSE: 300 mg/kg bw/day (mid dose)

SEX: Female

STUDY CODE: N21001-414

OBSERVATIONS

ANIMAL NUMBERS:

# 3522 NP

THYROID GLAND (a):

lesion

-

THYROID GLAND (b):

lesion

-

 

EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil					DOSE: 1000 mg/kg bw/day (high dose)								SEX: Female								STUDY CODE: N21001-414
OBSERVATIONS	ANIMAL NUMBERS:		4501	4502		4503	4504	4505	4506	4507	4508	4509		4510	4511	4512	4513	4514	4515	4516		4517	4518	4519	4520	4521	4522
THYROID GLAND (a):		lesion	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-
THYROID GLAND (b):		lesion	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-

 

 

 

NON PREGNANT / NON EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil				DOSE: 1000 mg/kg bw/day (high dose)		SEX: Female	STUDY CODE: N21001-414
OBSERVATIONS	ANIMAL NUMBERS:		# 4523 NP
THYROID GLAND (a):		lesion	-
THYROID GLAND (b):		lesion	-

 

EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil			TEST SYSTEM: Han:Wist rat			STUDY CODE: N21001-414
HISTOPATHOLOGICAL OBSERVATIONS		GROUPS (mg/kg bw/day)		0	100		300	1000
THYROID GLAND (a):	lesion			0/21	0/21		0/21	0/22
THYROID GLAND (b):	lesion			0/21	0/21		0/21	0/22

 

 

NON PREGNANT / NON EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil			TEST SYSTEM: Han:Wist rat			STUDY CODE: N21001-414
HISTOPATHOLOGICAL OBSERVATIONS		GROUPS (mg/kg bw/day)		0	100		300	1000
THYROID GLAND (a):	lesion			0/1	0/1		0/1	0/1
THYROID GLAND (b):	lesion			0/1	0/1		0/1	0/1

 

 

 

COMMENT:                                                                 NOT PRESENT                              =    -                                                                                                       ABBREVATIONS: M    =MORIBUND                         R= RECOVERY

                                                                                     PRESENT                                      =    +                                                                D    =DIED                                                                                     a = one side                                 b = other side

                                                                                     NO DATA                                      =    /                                                                 Æ    =MISSING ORGAN

                                                                                     GRADE OF ALTERATION:           1     =    MINIMAL CHANGE                            NAFC    =NO ABNORMAL FINDING

                                                                                                                                           2     =    MILD CHANGE                                   TO CORRELATE WITH NECROPSY OBSERVATION

                                                                                                                                           3     =    MODERATE CHANGE                        X     =NOT EXAMINED

                                                                                                                                           4     =    SEVERE (MARKED) CHANGE           NP  = NON PREGNANT

 

Conclusions:

In summary, daily administration of SynNova® Base Oil by oral gavage to pregnant Hannover Wistar rats from gestation day 6 (GD6) to gestation day 19 (GD19) at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any mortality or clinical signs of the dams under the conditions of this study.

There was no test item-related effect on maternal body weight, body weight gain and food consumption in any of the test item-treated groups.

No remarkable internal or external observations related to the test item treatment were recorded for any of the pregnant animals during necropsy.

No remarkable abnormalities were observed on the placentas in any examined groups.

There were no test item related changes in the examined parameters of the intrauterine development up to and including 1000 mg/kg bw/day.

No test item-related effect was observed on the fetal body weight and the number of viable fetuses in any of the test item-treated groups, as well as their sex distribution were comparable with the control.

Based on the results of thyroid hormones analysis, thyroid weights, histopathological evaluation of thyroids and the measurement of anogenital distance of fetuses, no endocrine disruptor effect was observed in the study.

There were no statistically significant effects of the test item on the external, visceral and/or skeletal development of fetuses in the study.

The No Observed Effect Level (NOEL) under the conditions of this study for maternal and fetal developmental toxicity is considered to be 1000 mg/kg bw/day.

Executive summary:

The purpose of this Prenatal Developmental Toxicity Study was to assess the effects of the test item on pregnant female rats and on the developing conceptuses and provide general information concerning the effects of prenatal exposure on the pregnant test animals and on the developing organism according to the OECD 414 test guideline.

 

Animals (one control and 3 test item treated groups) were treated daily by oral gavage administration, from Gestation Day 6 (GD6) up to and including GD19 (sperm positive day = day 0 of pregnancy, GD0). Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

 

The doses administered were 100, 300 and 1000 mg/kg bodyweight (bw)/day. The dose levels were selected by the Sponsor in consultation with the Study Director based on the results from an OECD 422 reproductive toxicity study [3] with the aim of inducing toxic effects (developmental and/or maternal toxicity) but no death or suffering at the highest dose.

 

Treatment solutions were analysed for test item concentration twice during the treatment period.

 

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Gross macroscopic examination was performed at necropsy. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentae were examined macroscopically. For the purpose of assessing the endocrine disrupter potential of the test item, the thyroid glands were processed for histopathological examination following the measurement of the organ weight. In addition, TSH, T3 and T4 plasma/serum concentrations were measured.

 

Eighty-nine sperm positive females were included in the study, twenty-two in the control, low dose and mid dose groups and twenty-three in the high dose group.

 

All test item formulations were within the range of 94 to 106% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control sample. Based on these results, test item formulations were considered suitable for the study purposes.

 

In summary, daily administration of SynNova® Base Oil by oral gavage to pregnant Hannover Wistar rats from gestation day 6 (GD6) to gestation day 19 (GD19) at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any mortality or clinical signs of the dams under the conditions of this study.

 

There was no test item-related effect on maternal body weight, body weight gain and food consumption in any of the test item-treated groups.

 

No remarkable internal or external observations related to the test item treatment were recorded for any of the pregnant animals during necropsy.

 

No remarkable abnormalities were observed on the placentas in any examined groups.

 

 

 

There were no test item related changes in the examined parameters of the intrauterine development up to and including 1000 mg/kg bw/day.

 

No test item-related effect was observed on the fetal body weight and the number of viable fetuses in any of the test item-treated groups, as well as their sex distribution were comparable with the control.

 

Based on the results of thyroid hormones analysis, thyroid weights, histopathological evaluation of thyroids and the measurement of anogenital distance of fetuses, no endocrine disruptor effect was observed in the study.

 

There were no statistically significant effects of the test item on the external, visceral and/or skeletal development of fetuses in the study.

 

The No Observed Effect Level (NOEL) under the conditions of this study for maternal and fetal developmental toxicity is considered to be 1000 mg/kg bw/day.

 

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

K1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

For reproduction toxicity, two studies are available on the substance as follows:

OECD 422: SynNova Base Oil: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Administration in Wistar Rats

OECD 443: Extended one-generation reproductive toxicity – with F2 generation (Cohorts 1A and 1B with extension of Cohort 1B to F2 generation

The results of the OECD 422 study on the substance itself demonstrated the following results:

The NOAEL for systemic toxicity of the parental generation was considered to be 1000 mg/kg bw/day.

The NOAEL for reproductive effects of the parental generation was considered to be 1000 mg/kg bw/day.

The NOAEL for Pup development and survival was considered to be 1000 mg/kg bw/day.

For the OECD 443, the following results were obtained:

Parental generation:

NOAEL (systemic toxicity):      1000 mg/kg bw/day

NOAEL (reproductive effects): 1000 mg/kg bw/day

F1 generation:

NOAEL (developmental toxicity):         1000 mg/kg bw/day

NOAEL (systemic toxicity):      1000 mg/kg bw/day

NOAEL (reproductive effects): 1000 mg/kg bw/day

F2 generation:

NOAEL (developmental toxicity):         1000 mg/kg bw/day

The results of the OECD 414 study on the substance itself demonstrated the following result:

The No Observed Effect Level (NOEL) under the conditions of this study for maternal and fetal developmental toxicity is considered to be 1000 mg/kg bw/day.

The substance showed no effects for reproduction toxicity in any of the studies conducted. Classification is not justified.