Octadecene, reaction products with hexadecene, hydrogenated

Toxicity to reproduction

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

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, Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test, Organisation for Economic Co-Operation and Development, Paris, 29 July 2016

Deviations:

yes

Remarks:

See "any other information" for details

GLP compliance:

yes

Limit test:

yes

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.

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

Details on route of administration:

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

Vehicle:

corn oil

Details on oral 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

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.
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.
The formulation analysis was conducted within the determined stability period under the control of the PI #1 in compliance with the Study Plan as well as the relevant SOPs of the Test Site #1 for analytical work.
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.
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.
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.

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.

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:

This 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.
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 [3], with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Based on the results, 1000 mg/kg bw/day was selected as the High dose for this study.

Positive control:

Not required for the Repeated Dose Toxicity study.

Observations and examinations performed and frequency:

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.
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.

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.
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 pre-exposure 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).

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 parameters evaluated.

Sacrifice and pathology:

All animals
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.

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.

Other examinations:

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.

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 (when using Provantis) or with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest).

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in the study.
No treatment related changes were seen in Main group animals of either sex for any in-life parameter.

Mortality:

no mortality observed

Description (incidence):

No mortality was seen during the study.
No treatment related changes were seen in Main group animals of either sex for any in-life parameter.

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.
No treatment related changes were seen in Main group animals of either sex for any in-life parameter.

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:

no effects observed

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.
No treatment related changes were seen in Main group animals of either sex for clinical pathology. Similarly in the recovery animals, there were no changes observed that could indicate any delayed toxicity.

Clinical biochemistry findings:

no effects observed

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.
No treatment related changes were seen in Main group animals of either sex for clinical pathology. Similarly in the recovery animals, there were no changes observed that could indicate any delayed toxicity.

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

Description (incidence and severity):

No treatment-related effects were observed in the experimental animals.
There were no statistically significant and biologically relevant differences among groups in the weights of organs measured when compared to Controls in any sexes.
Some occasionally observed statistically significant values were considered to be incidental and not test item-related. These observed values were within the historical control ranges in all cases, there were no clear dose responses and/or values were without any similar trend in the other sex or main/recovery animals. Furthermore, there was no histopathological correlate for the weight changes. Thus, they were considered as having no toxicological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related changes were observed.
All observed changes were considered incidental or a common background.
No treatment related changes were seen in Main group animals of either sex for necropsy. Similarly in the recovery animals, there were no changes observed that could indicate any delayed toxicity.

Neuropathological findings:

not examined

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.
No treatment related changes were seen in Main group animals of either sex for histopathology. Similarly in the recovery animals, there were no changes observed that could indicate any delayed toxicity.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

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.

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

Critical effects observed:

no

Summary of Total Incidence of Clinical Signs – Main and Recovery Animals

Observation Type: All Types From Day: -14 to 63	Male				Female
	0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day	0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Total Number of Animals:	17	12	12	17	17	12	12	17
Normal Number of Animals Affected % of Affected Animals Number of Times Recorded	17 100 765	12 100 540	12 100 540	17 100 765	17 100 1064	12 100 837	12 100 826	17 100 1063
Terminal Euthanasia Number of Animals Affected % of Affected Animals Number of Times Recorded	17 100 17	12 100 12	12 100 12	17 100 17	17 100 17	12 100 12	12 100 12	17 100 17

 

Summary of Bodyweight Main and Recovery data – Males

Bodyweight (gram)

Sex: Male Day(s) Relative to Start Date		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
-14	Mean SD Max Min N %Diff	322.1  I^1 13.5 342 289 12 -	321.0 15.1 342 294 12 -0.3	317.3 18.0 341 292 12 -1.5	321.0 13.2 337 290 12 -0.3
-7	Mean SD Max Min N %Diff	363.2  I^1 13.1 383 336 12 -	362.7 16.5 387 338 12 -0.1	360.8 16.1 385 332 12 -0.6	360.1 12.4 382 338 12 -0.8
0	Mean SD Max Min N %Diff	400.4  I^1 13.5 429 374 17 -	399.2 16.4 424 366 12 -0.3	400.1 15.4 425 375 12 -0.1	399.5 13.1 421 377 17 -0.2
7	Mean SD Max Min N %Diff	419.7  I^1 13.2 444 400 17 -	420.2 26.5 451 372 12 0.1	426.0 21.6 461 383 12 1.5	416.6 13.8 445 394 17 -0.7
14	Mean SD Max Min N %Diff	444.3  R^2 18.0 482 420 17 -	438.9 31.4 484 382 12 -1.2	451.4 26.3 492 406 12 1.6	439.9 12.2 462 417 17 -1.0
21	Mean SD Max Min N %Diff	459.5 R^1 18.9 497 433 17 -	460.3 33.6 503 395 12 0.2	471.3 24.1 505 423 12 2.6	456.4 14.9 490 436 17 -0.7
28	Mean SD Max Min N %Diff	475.4  R^1 22.4 521 435 17 -	479.4 37.6 534 406 12 0.9	490.9 28.6 533 437 12 3.3	470.7 14.4 497 450 17 -1.0
35	Mean SD Max Min N %Diff	484.0  I^2 12.6 502 471 5 -	- - - - - -	- - - - - -	493.0 17.0 519 476 5 1.9
42	Mean SD Max Min N %Diff	503.6  I^2 11.2 520 489 5 -	- - - - - -	- - - - - -	506.8 18.8 536 486 5 0.6

Statistical Test: Decision Tree-VES        Transformation: Automatic

1[R – Automatic Transformation: Rank]

2[I – Automatic Transformation: Identity (No Transformation)]

 

Summary of Bodyweight Main data – Females – From Day -14 to Mating Period

Bodyweight (gram)

Sex: Male Day(s) Relative to Start Date		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
-14	Mean SD Max Min N %Diff	226.2  I^1 12.8 250 210 12 -	225.8 9.8 242 209 12 -0.1	228.1 10.5 247 213 12 0.8	227.5 9.6 247 213 12 0.6
-7	Mean SD Max Min N %Diff	246.0  I^1 11.3 261 228 12 -	249.0 12.4 279 229 12 1.2	248.5 10.3 269 232 12 1.0	244.3 11.2 268 228 12 -0.7
0	Mean SD Max Min N %Diff	236.6  I^1 13.3 286 238 12 -	265.2 12.0 286 243 12 0.6	265.1 12.0 292 246 12 0.6	262.8 13.9 283 229 12 -0.3
7	Mean SD Max Min N %Diff	273.6  I^1 13.7 299 248 12 -	278.5 11.3 302 265 12 1.8	277.5 15.5 306 256 12 1.4	276.3 15.3 305 2351 12 1.0
14	Mean SD Max Min N %Diff	280.5  I^1 15.5 311 256 12 -	287.6 11.9 304 269 12 2.5	289.2 13.4 312 272 12 3.1	284.1 16.3 316 263 12 1.3

Statistical Test: Decision Tree-VES        Transformation: Automatic

1 [I – Automatic Transformation: Identity (No Transformation)]

 

Summary of Bodyweight Main data – Females – Gestation Period

Bodyweight (gram)

Sex: Male Day(s) Relative to Start Date		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
0	Mean SD Max Min N %Diff	285.0  I^1 17.3 315 251 12 -	292.5 15.5 317 269 12 2.6	291.9 15.1 312 268 12 2.4	291.1 21.2 325 263 12 2.1
7	Mean SD Max Min N %Diff	320.2  I^1 16.9 355 295 12 -	328.8 14.6 357 306 12 2.7	331.6 21.2 371 298 12 3.6	328.4 19.4 362 297 12 2.6
14	Mean SD Max Min N %Diff	358.2  I^1 18.8 399 334 12 -	368.6 16.9 397 343 12 2.9	370.3 26.1 419 330 12 3.4	364.0 20.3 403 333 12 1.6
20	Mean SD Max Min N %Diff	454.0  I^1 22.8 507 422 12 -	462.2 24.4 508 415 12 1.8	468.5 34.4 538 414 12 3.2	458.0 31.1 518 405 12 0.9

Statistical Test: Decision Tree-VES        Transformation: Automatic

1 [I – Automatic Transformation: Identity (No Transformation)]

 

Summary of Bodyweight Main data – Females – Lactation Period

Bodyweight (gram)

Sex: Male Day(s) Relative to Start Date		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
0	Mean SD Max Min N %Diff	349.2  I^1 18.7 381 322 12 -	365.1 23.2 403 337 12 4.6	366.4 27.8 426 323 12 4.9	352.8 25.5 406 304 12 1.1
4	Mean SD Max Min N %Diff	363.6  R^2 18.0 403 337 12 -	376.8 14.9 401 354 12 3.6	378.3 27.0 442 340 12 4.0	369.2 22.7 428 341 12 1.5
13	Mean SD Max Min N %Diff	398.3  I^1 23.4 450 357 12 -	410.6 19.1 441 388 12 3.1	404.3 28.0 464 356 12 1.5	399.2 26.1 450 353 12 0.5

Statistical Test: Decision Tree-VES        Transformation: Automatic

1 [I – Automatic Transformation: Identity (No Transformation)]

2 [R – Automatic Transformation: Rank]

 

Summary of Bodyweight Recovery data – Females

Bodyweight (gram)

Sex: Male Day(s) Relative to Start Date		0 mg/kg bw/day	1000 mg/kg bw/day
0	Mean SD Max Min N %Diff	263.2  I^1 6.8 273 256 5 -	263.4 5.9 271 257 5 0.1
7	Mean SD Max Min N %Diff	269.0  I^1 11.7 286 254 5 -	270.4 14.5 282 246 5 0.5
14	Mean SD Max Min N %Diff	284.0  I^1 13.2 305 274 5 -	279.4 20.9 297 248 5 -1.6
21	Mean SD Max Min N %Diff	288.0  I^1 16.4 313 273 5 -	290.2 19.8 316 262 5 0.8
28	Mean SD Max Min N %Diff	303.0  I^1 18.3 333 285 5 -	297.2 19.4 313 265 5 -1.9
35	Mean SD Max Min N %Diff	312.8  I^1 19.9 347 298 5 -	310.8 25.3 332 269 5 -0.6
42	Mean SD Max Min N %Diff	317.4  I^1 23.0 358 304 5 -	312.2 25.2 336 272 5 -1.6

Statistical Test: Decision Tree-VES        Transformation: Automatic

1 [I – Automatic Transformation: Identity (No Transformation)]

 

Summary of Grip Strength Main Data

Day: 23 Relative to Start Date

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Grip Str. Avg. Fore. (g)	Mean SD Max Min N %Diff	1653.5I^1 124.6 1819 1535 5 -	1521.3 166.0 1614 1226 5 -8.0	1488.9 71.5 1572 1397 5 -10.0	1574.1 146.7 1772 1368 5 -4.8
Grip Str. Avg. Hind. (g)	Mean SD Max Min N %Diff	705.9  R^2 62.8 771 633 5 -	645.0 148.0 753 393 5 -8.6	653.9 44.1 697 605 5 -7.4	676.2 148.8 806 484 5 -4.2

1 [I – Automatic Transformation: Identity (No Transformation)]

2 [R – Automatic Transformation: Rank]

 

Summary of Grip Strength Main Data

Day: 49 Relative to Start Date

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Grip Str. Avg. Fore. (g)	Mean SD Max Min N %Diff	1216.3R^1 252.3 1542 970 5 -	1166.6 131.0 1330 1023 5 -4.1	1120.1 88.9 1271 1059 5 -7.9	1066.5 65.5 1127 974 5 -12.3
Grip Str. Avg. Hind. (g)	Mean SD Max Min N %Diff	458.5  L^2 88.5 607 385 5 -	484.3 32.6 518 440 5 5.6	448.5 61.2 541 382 5 -2.2	501.8 128.8 716 385 5 9.5

1 [R – Automatic Transformation: Rank]

2 [L – Automatic Transformation: Log]

 

Summary of Grip Strength Recovery Data

Day: 41 Relative to Start Date

Sex: Male		0 mg/kg bw/day	1000 mg/kg bw/day
Grip Str. Avg. Fore. (g)	Mean SD Max Min N %Diff	1803.6I^1 145.0 1946 1610 5 -	1816.5 149.4 1997 1585 5 0.7
Grip Str. Avg. Hind. (g)	Mean SD Max Min N %Diff	740.5  I^1 125.8 886 561 5 -	734.7 96.0 860 634 5 -0.8

1 [I – Automatic Transformation: Identity (No Transformation)]

 

Summary of Grip Strength Recovery Data

Day: 41 Relative to Start Date

Sex: Female		0 mg/kg bw/day	1000 mg/kg bw/day
Grip Str. Avg. Fore. (g)	Mean SD Max Min N %Diff	1430.6I^1 129.8 1555 1275 5 -	1332.5 102.2 1502 1243 5 -6.9
Grip Str. Avg. Hind. (g)	Mean SD Max Min N %Diff	594.1  I^1 32.6 643 556 5 -	614.7 87.2 758 529 5 3.5

1 [I – Automatic Transformation: Identity (No Transformation)]

 

Summary of Landing Splay Test Main Data

Day: 23 Relative to Start Date

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Foot-Splay Hind. Mean (mm)	Mean SD Max Min N %Diff	83.8    I^1 23.9 110 51 5 -	88.3 7.7 98 77 5 5.4	83.3 17.2 104 57 5 -0.6	77.1 18.1 93 50 5 -8.0

1 [I – Automatic Transformation: Identity (No Transformation)]

 

Summary of Landing Splay Test Main Data

Day: 49 Relative to Start Date

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Foot-Splay Hind. Mean (mm)	Mean SD Max Min N %Diff	71.0    I^1 5.7 77 64 5 -	92.9 18.5 114 69 5 30.8	83.2 13.1 96 67 5 17.2	69.3 22.7 93 38 5 -2.3

1 [I – Automatic Transformation: Identity (No Transformation)]

 

Summary of Landing Splay Test Recovery Data

Day: 41 Relative to Start Date

Sex: Male		0 mg/kg bw/day	1000 mg/kg bw/day
Foot-Splay Hind. Mean (mm)	Mean SD Max Min N %Diff	81.2    I^1 20.0 100 57 5 -	98.9 14.0 109 75 5 21.8

1 [I – Automatic Transformation: Identity (No Transformation)]

 

Summary of Landing Splay Test Recovery Data

Day: 41 Relative to Start Date

Sex: Female		0 mg/kg bw/day	1000 mg/kg bw/day
Foot-Splay Hind. Mean (mm)	Mean SD Max Min N %Diff	77.8    I^1 9.4 91 66 5 -	69.2 14.1 85 54 5 -11.1

1 [I – Automatic Transformation: Identity (No Transformation)]

 

Summary of Locomotor Activity Main Data (SMART)

Day: 24 Relative to Start Date

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
LMA Dist. 0-5 min (cm)	Mean SD Max Min N %Diff	1238.8I^1 248.0 1640 1004 5 -	1092.9 454.1 1450 302 5 -11.8	1160.1 232.1 1508 899 5 -6.4	1221.8 351.1 1751 911 5 -1.4
LMA Dist. 5-10 min (cm)	Mean SD Max Min N %Diff	1117.5I^1 314.4 1626 765 5 -	936.6 365.0 1310 354 5 -16.2	992.7 217.8 1326 799 5 -11.2	1011.6 92.2 1111 887 5 -9.5
LMA Dist. 10-15 min (cm)	Mean SD Max Min N %Diff	1043.2I^1 250.9 1364 748 5 -	829.3 152.7 990 598 5 -20.5	870.4 247.8 1214 569 5 -16.6	820.8 179.1 1124 683 5 -21.3
LMA Dist. 15-20 min (cm)	Mean SD Max Min N %Diff	794.6  I^1 125.0 983 633 5 -	740.7 280.7 1142 372 5 -6.8	775.0 228 1151 562 5 -2.5	578.9 211.5 914 351 5 -27.2
LMA Dist. 20-25 min (cm)	Mean SD Max Min N %Diff	777.9  I^1 422.0 1383 393 5 -	559.9 249.0 724 122 5 -28.0	585.6 227.5 889 287 5 -24.7	556.6 316.2 1109 302 5 -28.4
LMA Dist. 25-30 min (cm)	Mean SD Max Min N %Diff	761.1  I^1 264.0 1114 512 5 -	574.6 241.1 845 197 5 -24.5	648.3 360.4 1218 223 5 -14.8	646.6 347.4 1190 347 5 -15.0
LMA Dist. 30-35 min (cm)	Mean SD Max Min N %Diff	667.2  I^1 369.4 1063 247 5 -	629.8 293.3 849 134 5 -5.6	567.8 410.1 1236 206 5 -14.9	655.0 125.3 737 434 5 -1.8
LMA Dist. 35-40 min (cm)	Mean SD Max Min N %Diff	661.6  I^1 228.7 910 410 5 -	646.0 191.4 913 472 5 -2.4	674.3 411.8 1355 346 5 1.9	588.3 189.3 905 412 5 -11.1
LMA Dist. 40-45 min (cm)	Mean SD Max Min N %Diff	629.2  I^1 259.5 962 313 5 -	516.0 227.4 759 184 5 -18.0	510.2 256.9 761 223 5 -18.9	478.0 184.2 655 211 5 -24.0
LMA Dist. 45-50 min (cm)	Mean SD Max Min N %Diff	650.5  I^1 176.7 876 481 1 -	632.8 129.7 833 469 5 -2.7	402.3 220.2 671 137 5 -38.2	445.6 134.1 635 291 5 -31.5
LMA Dist. 50-55 min (cm)	Mean SD Max Min N %Diff	392.5  I^1 141.1 491 156 5 -	443.2 196.6 639 156 5 12.9	354.6 160.2 600 193 5 -9.7	437.0 193.7 699 192 5 11.4
LMA Dist. 55-60 min (cm)	Mean SD Max Min N %Diff	582.0  I^1 136.3 802 429 5 -	469.3 172.2 655 262 5 -19.4	523.9 234.0 877 287 5 -10.0	549.9 171.5 847 416 5 -5.5
LMA Dist. 0-60 min (cm)	Mean SD Max Min N %Diff	9325.0I^1 2624.0 12525 6656 5 -	8074.1 2236.3 9913 4212 5 -13.4	8069.8 2689.69 12814 6136 5 -13.5	7996.3 1693.5 10870 6616 5 -14.2

1 [I – Automatic Transformation: Identity (No Transformation)]

 

Summary of Locomotor Activity Main Data (SMART)

Day: 49 Relative to Start Date

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
LMA Dist. 0-5 min (cm)	Mean SD Max Min N %Diff	1097.8R^1 188.1 1405 892 5 -	1351.6 493.6 2231 1083 5 23.1	1297.2 225.8 1582 1021 5 18.2	1609.1 905.6 3205 950 5 46.6
LMA Dist. 5-10 min (cm)	Mean SD Max Min N %Diff	629.9  L^2 68.8 724 562 5 -	706.6 28.7 746 679 5 12.2	673.6 177.5 941 444 5 7.0	785.8 252.5 1172 548 5 24.8
LMA Dist. 10-15 min (cm)	Mean SD Max Min N %Diff	599.5  I^3 215.5 973 434 5 -	433.4 167.2 631 169 5 -27.7	518.6 259.0 948 283 5 -13.5	521.7 221.4 826 237 5 -13.0
LMA Dist. 15-20 min (cm)	Mean SD Max Min N %Diff	465.5  I^3 236.1 714 140 5 -	465.3 220.8 803 268 5 -0.1	333.9 152.5 580 174 5 -28.3	316.5 91.3 427 178 5 -32.0
LMA Dist. 20-25 min (cm)	Mean SD Max Min N %Diff	492.0  I^3 187.4 769 262 5 -	275.7 254.8 649 99 5 -23.6	423.8 205.2 669 117 5 -13.9	433.2 136.3 609 302 5 -11.9
LMA Dist. 25-30 min (cm)	Mean SD Max Min N %Diff	534.6  I^3 227.1 715 160 5 -	336.8 247.1 705 86 5 -37.0	298.4 286.0 788 66 5 -44.2	411.3 237.6 760 143 5 -23.1
LMA Dist. 30-35 min (cm)	Mean SD Max Min N %Diff	389.4  I^3 158.9 614 172 5 -	415.2 192.4 608 139 5 6.6	190.4 190.9 464 53 5 -51.1	476.0 199.3 695 181 5 22.3
LMA Dist. 35-40 min (cm)	Mean SD Max Min N %Diff	504.6  I^3 188.6 668 200 5 -	573.4 140.2 797 451 5 13.6	301.3 353.6 884 67 5 -40.3	401.7 206.2 662 111 5 -20.4
LMA Dist. 40-45 min (cm)	Mean SD Max Min N %Diff	553.4  I,aa^5 76.7 676 474 5 -	511.7 117.8 694 368 5 -7.5	186.3  dd^4 119.0 328 67 5 -66.3	423.8 101.8 563 286 5 -23.4
LMA Dist. 45-50 min (cm)	Mean SD Max Min N %Diff	362.0  R^1 180.7 537 104 5 -	368.1 123.0 521 229 5 1.7	389.2 415.5 1028 93 5 7.5	380.8 136.2 581 239 5 5.2
LMA Dist. 50-55 min (cm)	Mean SD Max Min N %Diff	411.8  I^3 138.0 589 241 5 -	177.1 109.0 285 48 5 -57.0	506.5 328.9 951 65 5 23.0	321.1 142.6 460 83 5 -22.0
LMA Dist. 55-60 min (cm)	Mean SD Max Min N %Diff	398.5  I^3 151.0 637 231 5 -	242.3 227.3 529 70 5 -39.2	307.8 247.6 630 55 5 -22.8	258.6 116.9 386 95 5 -35.1
LMA Dist. 0-60 min (cm)	Mean SD Max Min N %Diff	6442.7I^3 1233.8 7799 4682 5 -	5960.0 898.3 6752 4412 5 -7.5	5248.7 1222.6 6704 3684 6 -15.7	6342.5 1529.4 8675 4870 5 -1.6

1 [R – Automatic Transformation: Rank]

2 [L – Automatic Transformation: Log]

3 [I – Automatic Transformation: Identity (No Transformation)]

4 [dd – Test: Dunnett 2 Sided p<0.01]

5 [I,aa – Automatic Transformation: Identity (No Transformation), (All Groups)

 

Summary of Locomotor Activity Recovery Data (SMART)

Day: 41 Relative to Start Date

Sex: Male		0 mg/kg bw/day	1000 mg/kg bw/day
LMA Dist. 0-5 min (cm)	Mean SD Max Min N %Diff	1061.6I^1 337.9 1485 688 5 -	1045.8 238.0 1322 791 5 -1.5
LMA Dist. 5-10 min (cm)	Mean SD Max Min N %Diff	939.9  I^1 358.6 1326 391 5 -	780.7 218.2 1155 626 5 -16.8
LMA Dist. 10-15 min (cm)	Mean SD Max Min N %Diff	754.0  I^1 234.2 1023 425 5 -	620.4 116.9 823 538 5 -17.7
LMA Dist. 15-20 min (cm)	Mean SD Max Min N %Diff	518.3  R^2 215.6 748 211 5 -	509.0 53.2 572 441 5 -1.8
LMA Dist. 20-25 min (cm)	Mean SD Max Min N %Diff	397.4  R^2 59.3 438 294 5 -	383.9 316.5 769 254 5 -3.4
LMA Dist. 25-30 min (cm)	Mean SD Max Min N %Diff	407.7  I^1 104.9 519 247 5 -	391.2 197.9 602 166 5 -4.0
LMA Dist. 30-35 min (cm)	Mean SD Max Min N %Diff	380.2  I^1 152.3 586 196 5 -	343.0 208.8 605 111 5 -9.8
LMA Dist. 35-40 min (cm)	Mean SD Max Min N %Diff	396.3  R^2 136.7 514 171 5 -	278.6 147.8 392 101 5 -29.7
LMA Dist. 40-45 min (cm)	Mean SD Max Min N %Diff	300.6  I^1 148.8 453 101 5 -	318.9 229.5 623 98 5 6.1
LMA Dist. 45-50 min (cm)	Mean SD Max Min N %Diff	410.2  I^1 116.2 548 257 1 -	341.6 177.6 611 143 5 -16.7
LMA Dist. 50-55 min (cm)	Mean SD Max Min N %Diff	339.8  I^1 122.5 508 213 5 -	340.2 226.9 689 61 5 0.1
LMA Dist. 55-60 min (cm)	Mean SD Max Min N %Diff	246.6  L^3 143.2 468 127 5 -	252.0 189.3 470 67 5 -4.8
LMA Dist. 0-60 min (cm)	Mean SD Max Min N %Diff	6172.3I^1 1408.0 7936 4448 5 -	5608.0 1677.4 8007 3677 5 -9.1

1 [I – Automatic Transformation: Identity (No Transformation)]

2 [R – Automatic Transformation: Rank]

3 [L – Automatic Transformation: Log]

 

Summary of Locomotor Activity Recovery Data (SMART)

Day: 41 Relative to Start Date

Sex: Female		0 mg/kg bw/day	1000 mg/kg bw/day
LMA Dist. 0-5 min (cm)	Mean SD Max Min N %Diff	1944.9I^1 357.7 2397 1402 5 -	1861.2 200.6 2179 1671 5 -4.3
LMA Dist. 5-10 min (cm)	Mean SD Max Min N %Diff	1235.0R^2 315.7 1556 844 5 -	1417.8 135.2 1586 1224 5 14.8
LMA Dist. 10-15 min (cm)	Mean SD Max Min N %Diff	1109.0L^3 352.6 1494 775 5 -	946.7 169.2 1148 742 5 -14.6
LMA Dist. 15-20 min (cm)	Mean SD Max Min N %Diff	1132.5R^2 284.8 1639 968 5 -	838.0 173.3 1003 582 5 1.5
LMA Dist. 20-25 min (cm)	Mean SD Max Min N %Diff	825.6  I^1 202.8 1173 696 5 -	838.0 173.3 1003 582 5 1.5
LMA Dist. 25-30 min (cm)	Mean SD Max Min N %Diff	805.6  I^1 199.7 1110 590 5 -	753.6 207.1 1028 545 5 -6.5
LMA Dist. 30-35 min (cm)	Mean SD Max Min N %Diff	694.0  I^1 229.0 1035 510 5 -	718.2 181.9 930 463 5 3.5
LMA Dist. 35-40 min (cm)	Mean SD Max Min N %Diff	967.5  I,a^4 238.9 1229 687 5 -	603.7  d^5 198.1 885 379 5 -37.6
LMA Dist. 40-45 min (cm)	Mean SD Max Min N %Diff	769.3  I^1 384.3 1147 282 5 -	603.4 373.4 1051 197 5 -21.6
LMA Dist. 45-50 min (cm)	Mean SD Max Min N %Diff	806.2  I^1 429.5 1518 429 5 -	518.6 349.1 908 136 5 -35.7
LMA Dist. 50-55 min (cm)	Mean SD Max Min N %Diff	504.7  I^1 392.5 1134 165 5 -	250.5 168.1 455 101 5 -50.4
LMA Dist. 55-60 min (cm)	Mean SD Max Min N %Diff	767.3  I^1 341.0 1244 299 5 -	716.3 187.8 971 543 5 -6.6
LMA Dist. 0-60 min (cm)	Mean SD Max Min N %Diff	11568.0 I^1 3198.2 16400 8644 5 -	10137.8 946.3 11629 9273 5 -12.4

1 [I – Automatic Transformation: Identity (No Transformation)]

2 [R – Automatic Transformation: Rank]

3 [L – Automatic Transformation: Log]

4 [I,a – Automatic Transformation: Identity (No Transformation). (All Groups) Test: Analysis of Variance p<0.05]

5 [d – Test: Dunnett 2 Sided p<0.05]

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 related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs.
The NOAEL for systemic toxicity of the parental generation 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 related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs.

 

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

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

OECD Guidelines for the Testing of Chemicals No. 474, Mammalian Erythrocyte Micronucleus Test, Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

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.

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:
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.

Route of administration:

oral: gavage

Vehicle:

Based on the dose range finding (DRF) study [3], 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 exposure:

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.

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.
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.
The positive control (Cyclophosphamide) group for the main animals (group 5) for the Mammalian Erythrocyte Micronucleus Test were mated and females allowed to deliver similarly to the Main animals, then treated with 60 mg/kg bw
Cyclophosphamide, administered by intraperitoneal injection approximately 24-hour prior to scheduled necropsy (for males, on Day 28 for necropsy on Day 29; females, on PND13 for necropsy on PND14).

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.

Post exposure period:

No post exposure observation period specified

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).
For the satellite group (Group 5), 5 animals/sex were included to serve as the positive control group for the Mammalian Erythrocyte Micronucleus Test (MNT).

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control animals were treated once with 60 mg/kg bw Cyclophosphamide (CP), administered by intraperitoneal injection approximately 24 hours prior to scheduled necropsy.

Name: Cyclophosphamide monohydrate
Supplier: Sigma-Aldrich Co.
Batch No.: MKCF1756
Expiry date: 31 October 2020
Storage condition: 5 ± 3°C

Tissues and cell types examined:

The micronucleus test (MNT) identifies substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualisation of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage.

Details of tissue and slide preparation:

At least three sets of bone marrow smears for MNT were prepared from the main animals, including the vehicle control and the positive control groups.
The bone marrow was collected from the right femur of the rats immediately after euthanasia (the left femur of Main and Recovery group animals were used for routine histopathology, the left femur of positive control animals was discarded) and flushed with foetal bovine serum (5 mL) using a syringe and needle. Cells were concentrated by a gentle centrifugation.
Smears of the cell pellet was made on standard microscope slides. Slides were air-dried at room temperature for approximately 24 hours. Dried slides were fixed in methanol for a minimum of 5 minutes and allowed to air-dry.
1) Two sets of slides will be stained with 10 % Giemsa solution for approximately 18-20 minutes. Slides will be thoroughly rinsed with distilled water, and then air-dried at room temperature for at least 12 hours. After staining, cover slips were mounted on them.
a) One set of Giemsa-stained slides were given unique code numbers for blinded evaluation (the code labels covered all unique identification markings on the slides to ensure that they were scored without bias).
At the first instance only the Control (Group 1) and High dose (Group 4) slides from the main animals and the positive control (Group 5) were evaluated.
At least 4000 polychromatic (immature) erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated (MN) cells, to be expressed as percent of micronucleated cells based on the first 4000 PCEs counted in the optic field. The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 1000 cells (immature erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs), in which the number of micronuclei are recorded in both types of erythrocytes.

Evaluation criteria:

Criteria for Identification of Micronucleated Erythrocytes:
- A bluish mauve strongly coloured uniform round or oval particle in the cell.
- The particle should be large enough for the colour to be recognisable, and it should be located inside the cells. Areas with micronucleus-like particles outside the cells should not be used for analysis.
- During focusing, the particle should stay uniform in colour /light refraction and shape within a large interval and focus in the same plane as the erythrocyte.
- The unit of damage is deemed to be the cell, and therefore cells with two or more micronuclei will be counted as single micronucleated cells.

Statistics:

If significance is plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group is made. If the group size is <5 then Fisher’s Exact Test is used, if the group sizes are bigger then the Chi-squared test is used; identifying differences of <0.05, <0.01 or <0.001 as appropriate.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No test item related effect was detected on the bone marrow erythrocytes when evaluated in the micronucleus test. The frequency of micronucleated polychromatic erythrocytes was comparable with control level in both males and females. The positive control group produced the expected, statistically significant and biologically relevant increase in the frequency of micronucleated polychromatic erythrocytes when compared to control in female, but not in males. However, this fact was considered not to adversely affect the results of the study.

Results of Micronucleus Test

Parameters	Dose group/ Concentration (mg/kg bw/day)				Positive control
	Control (0)	Low (100)	Mid (300)	High (1000)
Males
Number of evaluated animals	12	12	12	12	5
Number of PCE/1000 cells	329.6	NE	NE	340.1	219.2
Number of MN-NCE/1000 cells	0.3	NE	NE	1.0	0.6
Number of MN-PCE/4000 cells	10.2	NE	NE	8.0	13.4
Females
Number of evaluated animals	12	12	12	12	5
Number of PCE/1000 cells	402.6	NE	NE	401.3	295.6
Number of MN-NCE/1000 cells	0.2	NE	NE	0.2	0.4
Number of MN-PCE/4000 cells	6.6	NE	NE	6.4	26.4**

Notes: Group mean values were rounded to one decimal place. Scoring of PCE and MN-NCE was performed using 1000 cells in total (PCE+NCE), Scoring of MN-PCE was performed in 4000 PCEs

Positive control: Cyclophosphamide (60 mg/kg bw); NE: Not evaluated; NCE: Normochromatic erythrocytes; PCE: polychromatic erythrocytes

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

Conclusions:

No genotoxic effect of the test item was observed in the bone marrow erythrocytes micronucleus test.

Executive summary:

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.

In addition, the test item was evaluated for genotoxic effects by examination of micronuclei formation in bone marrow erythrocytes of treated and control animals in accordance with OECD Guidelines for the Testing of Chemicals No. 474, Mammalian Erythrocyte Micronucleus Test, Adopted 29 July 2016. A satellite positive control group was included in the study for micronucleus test.

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. Based on the results, 1000 mg/kg bw/day was selected as the High dose for this study.

 

The mammalian in vivo micronucleus test (MNT) is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled from bone marrow of animals, usually rodents.

 

The purpose of this test as part of the study is to determine whether the test item causes genotoxic effects resulting in the formation of micronuclei in erythrocytes as sampled from the bone marrow of treated animals.

 

The micronucleus test (MNT) identifies substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualisation of micronuclei is facilitated in these cells because they lack a main nucleus.

An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage.

At least three sets of bone marrow smears for MNT were prepared from the main animals, including the vehicle control and the positive control groups.

 

No test item related effect was detected on the bone marrow erythrocytes when evaluated in the micronucleus test. The frequency of micronucleated polychromatic erythrocytes was comparable with control level in both males and females. The positive control group produced the expected, statistically significant and biologically relevant increase in the frequency of micronucleated polychromatic erythrocytes when compared to control in female, but not in males. However, this fact was considered not to adversely affect the results of the study.

 

No genotoxic effect of the test item was observed in the bone marrow erythrocytes micronucleus test.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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.

Test material

Test material information

Constituent 1

Reference substance name:

Oligomerisation products of alpha-alkenes C16-18 (even numbered), hydrogenated, hydroisomerised

EC Number:

832-827-5

Cas Number:

2241366-04-9

Molecular formula:

Variable - UVCB

IUPAC Name:

Oligomerisation products of alpha-alkenes C16-18 (even numbered), hydrogenated, hydroisomerised

Test material form:

liquid

Details on test material:

Name: SynNova Base Oil
CAS number: 2241366-04-9
Batch/Lot number: TS20371/TS21270
Description: Liquid - water white colorless oil.
Purity: 100%
Expiry date: 25 February 2021/31 July 2021
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to ensure personnel health and safety.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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.

Effect levels (P0)

1

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

2

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

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

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.

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.