Octadecene, reaction products with hexadecene, hydrogenated

Endpoint summary

Description of key information

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

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 May 2021 - 21 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar rat as a rodent is one of the standard strains for repeated dose toxicity studies

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Han:WIST rats
Source: Toxi-Coop Zrt., H-1122 Budapest, Magyar Jakobinusok tere 4B
Hygienic level at supplier: SPF
Hygienic level during
the study: Standard housing conditions
Justification of strain: The Wistar rat as a rodent is one of the standard strains for repeated dose toxicity studies
Number of groups: 4 dose groups
Number of animals: 40 male and 40 female rats, 10 animals/group/sex.
Sex: Male and female. The females were nulliparous and non-pregnant
Age of animals at start: Young adult rats, approximately 7-8 weeks old.
Body weight at the start: Did not exceed ± 20% of the mean weight at onset of treatment, males: 241 – 281 g; females: 167 – 194 g
Acclimatisation period: At least 6 days

HUSBANDRY

Animal health: Only healthy animals were used for the test. The health status was certified by the Veterinarian.
Cage type: T3H/T4 polycarbonate
Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20 – 24 °C (target: 22 ± 3 °C)
Relative humidity: 37 – 67 % (target: 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing / Enrichment: The animals were group housed. “SAFE 3/4-S-FASERN” certified wooden chips (batch number: 03027201125 / 03027210315, expiry date: 11 November 2023 / 15 March 2024) produced by J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) and “Sizzle pet” nest material (batch number: 491607 / 201016/02, expiry date: 05 August 2023 / 01 December 2023) produced by LBS (Serving Biotechnology) Ltd. (Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH, United Kingdom) were available to animals during the study. Fresh bedding was provided for the animals as frequently as appropriate/practical, but at least twice weekly. Copies of the Certificates of Analyses are archived with the raw data.

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

FOOD AND WATER SUPPLY

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

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

Name: SM Rat/Mouse, Breeding & Maintenance, 10 mm, autoclavable
Manufacturer: ssniff Spezialdiäten GmbH (D-59494 Soest, Germany)
Batch number: 211 72850 / 233 77046
Expiry date: 31 May 2021 / 30 September 2021

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

RANDOMISATION

Before start of dosing, the animals were assigned to their respective dose groups by randomisation based on body weights. All animals were within 20% of the overall mean at the start of the study. Animals were randomly allocated to the control and dose groups based on the most recent body weight. Males and females were randomised separately.

ANIMAL IDENTIFICATION

Each animal was individually identified by unique numbers written on the tail with an indelible pen. The numbers were given on the basis of NEXTREAT Laboratories' master file, for each animal allocated to the study.

The animal number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group.

The housing boxes were identified by cards holding information on the study code, the sex of animals, the dose group, the cage number and the individual animal number.
 

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. A constant dose volume of 1.5 mL/kg bw was administered to all animals in all groups.

Vehicle:

corn oil

Details on oral exposure:

The dose formulations were administered daily starting from Day 0, for 90 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. A constant dose volume of 1.5 mL/kg bw was administered to all animals in all groups.

The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight. Control animals were treated concurrently with the vehicle only.

The first day of dosing of each animal was regarded as Day 0.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation and analysis of formulations

The test item was formulated in the vehicle (corn oil) at the appropriate concentrations.

The test item was formulated in the vehicle (corn oil) at the appropriate concentrations. Formulations were prepared up to 7 days before use (formulation were kept closed, at room temperature until use). Stability of the test item in the vehicle was assessed during the analytical method validation [3]. In that study, the formulation samples in the 50-770 mg/mL concentration range (using corn oil as vehicle) were proven as being stable for at least 7 days when stored at room temperature 22±3°C.

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

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

Duration of treatment / exposure:

90 day

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

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:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The objective of this study was to obtain information on the possible toxic effects of the test item following repeated (daily) administration by oral gavage to rats at 4 dose levels for 90 days, according to OECD No. 408 guideline [1].

The 90-day study provides information on the possible health hazards likely to arise from repeated exposure over a prolonged period of time covering post-weaning maturation and growth into adulthood of the test animals. The aim of the study was to provide information on the major toxic effects, indicate target organs and the possibility of accumulation of test chemical, and to provide an estimate of a no-observed-adverse-effect level (NOAEL) of exposure which can be used in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.

Identification of the potential of the test item to cause neurotoxic, endocrine, immunological or reproductive organ effects, which may warrant further in-depth investigation was also the objective of this study.

The dose levels were selected by the Sponsor in consultation with the Study Director, based on the results from an OECD 422 reproductive toxicity study [2] with the aim of inducing toxic effects but no death or suffering at the highest dose, and to obtain a No Adverse Effect Dose Level (NOAEL) at the lowest dose.

Observations and examinations performed and frequency:

IN-LIFE PROCEDURES
Clinical observations

Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

General clinical observations were made once a day at approximately the same time with minor variations, toward the end of the working day.

Detailed clinical observations were made on all animals outside the home cage in a standard arena at least prior to the first treatment (to allow for within-subject comparisons) and weekly thereafter, in the morning hours.

Observations were performed on the skin, fur, eyes and mucous membranes, occurrence of secretions and excretions, autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behaviour pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagia/self-mutilation, backward motion, abnormal vocalization, aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (hunchback posture, etc.), gait, posture and response to handling and to environmental stimulation. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body weight

The body weight of all animals was measured weekly with a precision of 1 g.

Food consumption

The determination of food consumption was performed weekly (on body weight measurement days). The remaining, non-consumed food was weighed with a precision of 1 g. The measurement was performed on cage level. The daily mean food consumption per animals was calculated based on their body weight (g/animal/day, and g/ kg animal/day).

Ophthalmology evaluation

Ophthalmoscopic examination was conducted in all animals before treatment and in the Control (Group 1) and High dose (Group 4) animals towards the end of the treatment period (Week 12).

Mydriasis was produced after instillation of cyclopentolate eye drops into the conjunctival sac. The evaluation was performed by external examination and with an ophthalmoscope.

Neurological assessment (Functional Observation Battery)

Towards the end of the treatment period, during Week 12/13, each animal was subjected to the functional observation battery, including measurements of sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), landing foot splay, fore/hind grip strength and motor activity assessment.

Sensory reactivity

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 was tested. A modified Irwin test was performed.

Parameters such as, but not limited to 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/or vocalisation were evaluated.

Landing foot splay

To measure the landing foot splay, the fore and 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. This procedure was performed three times for each rat. The distance between the two resulting ink spots of the hind paws was measured.

Grip strength

Fore/hind grip strength measurements was conducted using a grip strength meter, an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and pulled back until they release the bar; the device measured the maximum grip strength. The procedure was repeated with the hind limbs with the appropriate grip support. This procedure was performed three times for each rat. The results were tabulated with individual and mean data.

Motor activity

Locomotor activity was recorded by placing the animals individually into an open-field for at least 1-hour observation time. Recording was made for a duration of at least 45 minutes, under dim-light and undisturbed conditions. The recorded video was processed and the calculated data was evaluated for distance travelled in 5 minute segments.
 
Examination of vaginal smears

Prior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

Sacrifice and pathology:

Terminal procedures

Euthanasia

Necropsy and macroscopic examination was performed on all animals, at the end of treatment period, on Day 90 (after the sample collection for clinical pathology evaluation). The animals were euthanized by exsanguination under pentobarbital anaesthesia.

Macroscopic examination

After exsanguinations, the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

Organ weight measurement

The following organs were trimmed of fat and weighed in all surviving animals:
With precision of at least 0.01g:
Brain
Epididymides
Heart
Kidneys
Liver
Prostate
Seminal vesicles with
coagulating glands
Spleen
Testes
Thymus
Uterus including cervix

With precision of at least 0.001g
Adrenal glands
Ovaries
Thyroid with parathyroid glands
Pituitary gland

Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported. If any significant difference in size is noted between paired organs, an individual weight of each organ was recorded.

Histopathology

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

Organs were preserved in 10% buffered formalin solution. Before preservation in
10% buffered formalin solution, the eyes with the optic nerve and the testes with epididymides were fixed in modified Davidson’s fixative.

The retained tissues and organs were embedded in paraffin wax, sections were cut at 3-5 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin and examined by light microscope. Full histopathology was performed in Groups 1 (Control) and 4 (High dose) In addition, any organs or tissues with macroscopic abnormalities were subjected to histological examination from all groups.

Other examinations:

CLINICAL PATHOLOGY

At the end of the treatment period, prior to scheduled necropsy on Day 90, clinical pathology investigations (haematology, coagulation, clinical biochemistry and urinalysis) were conducted in all animals.

After an overnight period of fasting of animals, 4 blood samples were collected by heart puncture under pentobarbital anaesthesia, one for haematology (in tubes K3-EDTA as anticoagulant, 1.6 mg/mL blood) and clinical chemistry and T4 analysis (in tubes with Li-Heparin as anticoagulant, 20 I.U./mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for TSH, T3 and cholesterin analysis.

Haematology and blood clotting times

The following parameters were evaluated in all animals euthanized at termination:

RBC –Erythrocytes
WBC – Total leukocytes
Hb – Haemoglobin concentration
HCT – Haematocrit
MCV – Mean Corpuscular (erythrocyte) Volume
MCH – Mean Corpuscular (erythrocyte) Haemoglobin
MCHC – Mean Corpuscular (erythrocyte) Haemoglobin Concentration
RDW – Red blood cell distribution width
Thromb – Thrombocytes (Platelets)
MPV – Mean platelet volume
Ret – Reticulocyte count
Neuts – Neutrophils
Lymphs – Lymphocyte
Monos – Monocyte
Basos – Basophil
Eosins – Eosinophil

APTT – Activated Partial Thromboplastin Time
PT – Prothrombin Time

Clinical chemistry

The following parameters were evaluated in all animals euthanized at termination:

• GLU – Blood sugar concentration
• T-Bili – Total Bilirubin concentration
• Urea – Urea concentration
• BUN – Blood urea nitrogen
• Chol – Total Cholesterol concentration
• HDL – High density lipoprotein cholesterol
• LDL – Low density lipoprotein
• Creat – Creatinine concentration
• P – Phosphorus concentration
• Na – Sodium concentration
• K – Potassium concentration
• Ca – Calcium concentration
• Cl – Chloride concentration
• TP – Total Protein concentration
• Alb – Albumin concentration
• A:G – Albumin:globulin ratio (calculated)
• AST – Aspartate Aminotransferase activity
• ALT – Alanine Aminotransferase activity
• GGT – Gamma-Glutamyl transferase activity
• ALKP – Alkaline Phosphatase activity
• TBA – Total bile acids

Thyroid hormone analysis

The following parameters were evaluated in all animals euthanized at termination:

• T4 – Thyroxine
• T3 – Tri-iodothyronine
• TSH – Thyroid-stimulating hormone

After centrifugation, the resulting plasma or serum (depending on the method used for measurement) was divided in at least two aliquots (volume target of at least 125 μL for the first aliquot and at least 75 µL for the second aliquot) and stored in a freezer (-20±5 °C) until analysis.

The first set was used for the hormone analysis; the second set was stored frozen for other possible hormone measurements, which might be triggered based on results observed for other endpoints.

Any sample not required for analysis will be discarded following acceptance of the draft study report.

Urinalysis

Urine collection was conducted over approximately 16 hours, during an overnight period of fasting of animals, which were placed in metabolic cages.

The evaluation of the urine samples was performed by observation (e.g. colour, appearance) or test strips as applicable. The following parameters were evaluated:

• LEU – Leukocyte
• NIT – Nitrite
• pH
• PRO – Protein
• GLU – Glucose
• UBG – Urobilinogen
• BIL – Bilirubin
• KET – Ketones
• BLD/ERY – Blood/Erythrocytes
• SG – Specific Gravity
• SED – Sediment Microscopic examination
• VOL – Volume
• Colour/appearance

Statistics:

Data was recorded on data collection sheets taken from the relevant SOPs, then tabulated using Microsoft Excel.

Descriptive statistics (mean, standard deviation, %versus control) were calculated for the continuous variables and frequency and percentage was calculated for categorical variables using Microsoft Excel.

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

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

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

Clinical signs:

no effects observed

Description (incidence and severity):

Administration of the test item for 90 consecutive days did not cause any clinical signs.

Mortality:

no mortality observed

Description (incidence):

No mortality was noted under the duration of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Daily administration of the test item did not cause any test item related effect in the body weight or body weight gain of the animals in comparison with the control. The body weight and body weight gain corresponded their sex, age and strain. Statistically significant changes noted in the group means compared to the control at a few isolated cases were considered incidental, without any biological and toxicological relevance

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 in any of the sexes when compared to control data. The statistically significant value in the male High dose group between Day 70-77 (p<0.05) is considered incidental, toxicologically not relevant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmological evaluation of the animals did not reveal any test item related effect. No changes were observed neither in the control nor in the high dose groups.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology and blood clotting times

Examination of haematology parameters did not reveal any test item related effect on the organs of the hematopoietic system, or cellular elements of the blood. Also, no effects were noted in major blood clotting parameters.
A slight, but statistically significant increase (+4.9, p<0.01) in red blood cell count (RBC), furthermore in haematocrit (HCT) and haemoglobin (HGB) concentration (+3.7, p<0.01,
+3.2, p<0.01, respectively) was observed in high dose males, however the majority of the individual values were within the control range in case of red blood cell count, and each values in case of the haematocrit and haemoglobin concentration, therefore, it is considered as incidental and not a test item related effect. A statistically significant decrease in platelet count (PLT) was noted in males in the low dose group, which, in the lack of dose response and further adverse consequences in blood clotting parameters was also considered incidental without toxicological relevance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry

Administration of the test item did not cause any test item related effect in the measured clinical biochemistry parameters.

In high dose males, a +65% elevation in aspartate aminotransferase (AST) group mean value was noted, however each of the individual values was within the historical control range (94-410 U/L), while the control range of this present study is 75-147 U/L, so the change is considered to be the outcome of the incidentally low study control values rather than a test item related effect.

Other variations attaining statistical significance occasionally, such as increased glucose concentration (GLU, p<0.01) group mean in mid dose males, decreased globulin concentration group mean (GLOB, p<0.05) in low dose females, decreased albumin (ALB, p<0.05) and total bilirubin concentration (TBIL, p<0.01) group means in mid dose females and decreased total protein concentration (TP, p<0.01) group mean in low and mid dose females remained minor, and/or no consistent dose or gender response was observed, with most of the individual values within the control range, therefore could not be ascribed to test item administration.

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis

Compared to the control values, there were no toxicologically significant differences in the assessed endocrine parameters (TSH, T4 and T3 hormone plasma concentrations). In comparison with the control, a statistically significant increase (+14.2%, p<0.01) was noted in
T3 concentration in mid dose male animals. Though, no dose response was noted, and the majority (7/10) of the individual values were within the study control range, therefore it is considered incidental and not test item related change, with no toxicological relevance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item related changes were observed in the examined urine parameters.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Sensory reactivity

A modified Irwin test performed in all animals, did not reveal any test item related effect.

Grip strength

No effect of the test item on the grip strength of the animals was detected. The statistically significant decrease in low dose males compared to the control (-15.28%, p<0.01) in hind legs is considered not test item related, but incidental, without toxicological relevance.

Landing foot splay

Landing foot splay assessment did not reveal any effect which could be ascribed to the administration of the test item. A -15.86%, statistically significant (p<0.05) decrease in 1st drops group mean was noted in high dose females in comparison with the control, however when all individual values are taken into consideration (1st, 2nd and 3rd drops), it seems that only two values of thirty are out of the control range, and the vast majority of the individual means of the three drops is also within the control range. Therefore this change is considered not test item related, but incidental.

Landing foot splay

Landing foot splay assessment did not reveal any effect which could be ascribed to the administration of the test item. A -15.86%, statistically significant (p<0.05) decrease in 1st drops group mean was noted in high dose females in comparison with the control, however when all individual values are taken into consideration (1st, 2nd and 3rd drops), it seems that only two values of thirty are out of the control range, and the vast majority of the individual means of the three drops is also within the control range. Therefore this change is considered not test item related, but incidental.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item related effects on organ weights.

In males, an elevation in thymus weight adjusted to the body and brain weight (+14.5, +13.1, p<0.05) was observed, without any effect in absolute organ weight. As no dose response was noted, it is considered a no test item related, incidental change, without toxicological relevance.
In female animals, statistically significantly higher than control group mean was observed in absolute weight of ovaries (+12.8%, +14.4, p<0.05) in the low and mid dose groups. Similar changes in the same dose groups were noted when ovaries’ weight was adjusted to the brain weight (+11.1%, +14.3, p<0.05), and also when adjusted to the body weight, however at the latter case, only the mid dose group was affected. In the lack of dose response and pathology findings, these changes are considered incidental, without toxicological relevance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related macroscopic findings were observed at the terminal necropsy.

Red multifocal discoloration of the glandular mucosa of the stomach in 1/10 low dose, and in 1/10 high dose male animals, multifocal pale discoloration of the spleen in 1/10 low dose male, single clear cyst in the pituitary in 1/10 low dose male, bilateral small testis, epididymis and adrenal glands in 1/10 mid dose male animals were restricted only to one animals therefore were considered incidental and not associated with the administration of the test item

Single, clear cysts in the liver in 1/10 high dose male and 1/10 high dose female were considered to be incidental or spontaneous changes.

Unilateral/bilateral pelvic dilation of the kidney in 3/10 control males, in 1/10 control and in 1/10 high dose females being a common sporadic macroscopic finding in laboratory rats without toxicological significance.

Bilateral dilation with clear fluid of the body and horns of the uterus observed in 4/10 control, 5/10 low dose, 4/10 mid dose and 4/10 high dose female. As this is known as a common sign of oestrus, it was not considered as test item-related effect.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Sensory reactivity

A modified Irwin test performed in all animals, did not reveal any test item related effect.

Grip strength

No effect of the test item on the grip strength of the animals was detected. The statistically significant decrease in low dose males compared to the control (-15.28%, p<0.01) in hind legs is considered not test item related, but incidental, without toxicological relevance.

Landing foot splay

Landing foot splay assessment did not reveal any effect which could be ascribed to the administration of the test item. A -15.86%, statistically significant (p<0.05) decrease in 1st drops group mean was noted in high dose females in comparison with the control, however when all individual values are taken into consideration (1st, 2nd and 3rd drops), it seems that only two values of thirty are out of the control range, and the vast majority of the individual means of the three drops is also within the control range. Therefore this change is considered not test item related, but incidental.

Motor 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 were some isolated, statistically significant changes such as a -54.5% (p<0.05) group mean decrease of animal movement from 50 to 55 minutes in the mid dose males, a +63.3% (p<0.05) group mean elevation of animal movement from 35 to 40 minutes in the low females, and a -54.0% (p<0.05) group mean decrease of animal movement from 45 to 50 minutes in the high dose female group. As no dose response was noted, and the pattern of the movements’ intensity under the whole duration of the test was very similar in all dose groups, these changes were considered toxicologically irrelevant, and not test item related.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related microscopic changes were observed in the investigated reproductive and other organs of the animals.

The pyelectasia in the kidney noted in 3/10 control males, 1/10 control and 1/10 high dose females without other histopathological lesions is a common slight individual disorder in laboratory rats, without toxicological significance.
The dilatation of the uterine horns in 4/10 control, 5/10 low dose, 4/10 mid dose and 4/10 high dose females is a slight neuro-hormonal phenomenon in connection with the sexual function – proestrus phase – of the inner genital organs.
 

Focal or multifocal reddish discolorations observed in the mucous membrane of the stomach at the gross pathological examination were identified as focal congestion (without hemorrhage, inflammation or erosion) in 1/10 low dose, and 1/10 high dose male. This finding could be in connection with possible mechanical irritation or with the physiological process of digestion.
The hepatic cyst (1/10 high dose male and female) are found more commonly in the liver of aged rats and cause no obvious change in the surrounding hepatocytes.
Cyst in the pituitary (1/10 low dose male) and lymphoid hyperplasia in the spleen (1/10 mid dose male) regarded as incidental or common spontaneous lesion.
In the macroscopically decreased in size testes, epididymides and adrenal glands no histopathological lesions were observed to correlate with necropsy findings. In the affected testes (1/10 high dose male) the histological picture of active spermatogenesis, and in the epididymides storage of mature spermatozoa were detectable

Histopathological findings: neoplastic:

no effects observed

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: No effects noted on the observed parameters at the highest dose tested

Critical effects observed:

no

DOSE FORMULATION ANALYSIS

Concentration and homogeneity of the dosing formulations were determined three times during the study.

 

Based on the results, all test item formulations were shown to be homogeneous and in the range of 92 to 101% of nominal concentrations. No test item was detected in the negative (vehicle) control sample. Based on these results, formulations were considered suitable for the study purposes.

Summary of analytical results

Nominal concentration (mg/mL)	Measured concentration ± 95% confidence interval (mg/mL)	Percentage of the nominal concentration (%)
1^st Analytical sampling (Sampling: 25 May 2021)
Control	not detectable	-
66.7	69.78±1.95	105%
200	192.38±1.77	96%
666.7	646.75±14.49	97%
2^nd Analytical sampling (Sampling: 06 July 2021)
Control	not detectable	-
66.7	63.13±1.00	95%
200	191.97±2.93	96%
666.7	640.30±8.12	96%
3^rd Analytical sampling (Sampling: 16 August 2021)
Control	not detectable	-
66.7	71.40±1.39	107%
200	196.84±19.38	98%
666.7	648.42±45.95	97%

Conclusions:

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

No test item related adverse effect on body weight and body weight gain, food consumption, clinical biochemistry, haematology and urinalyis parameters were noted. TSH, T4 and T3 hormone tests did not show any effect on the thyroid part of the endocrine system. Functional Observational Battery did not reveal any neurotoxic potential of the test item. No treatment related alterations were found during the ophthalmological examinations.

No test item related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation in any of the organs examined.

There were no test item effects on the measured organ weights.

Based on the results of this presented study, the No Observed Adverse Effect is established at 1000 mg/kg bw/day.

Executive summary:

A 90-days study was performed with SynNova^® Base Oil (test item) to assess the toxic potential of the test item when administered by oral gavage for 90 consecutive days.

 

40 male and 40 female Han:WIST rats were treated according to the following experimental design:

 

Group No.	Group Designation	Dose level (mg/kg bw/day)	Concentration (mg/mL)	Dose volume (mL/kg bw)	Number of animals
					Males	Females
1	Control	0	0	1.5	10	10
2	Low Dose	100	66.7		10	10
3	Mid Dose	300	200		10	10
4	High Dose	1000	666.7		10	10

 

The control group was treated with the vehicle only (corn oil).

 

The dose levels were selected by the Sponsor in consultation with the Study Director, based on the results from an OECD 422 reproductive toxicity study [2] with the aim of inducing toxic effects but no death or suffering at the highest dose, and to obtain a No Adverse Effect Dose Level (NOAEL) at the lowest dose.

 

Parameters measured during the study included signs of morbidity and mortality twice daily, daily general observation or weekly detailed observation of clinical signs, weekly body weight and food consumption. Ophthalmology examination was performed once before the start of dosing and toward the end of the study (week 11). During week 12, neurotoxicity was assessed by performing a Functional Observation Battery (testing of sensory reactivity, landing foot splay, grip strength and locomotor activity. Vaginal smears of female animals was examined, and oestrus cycle was determined on the day before necropsy. At the end of the treatment period, prior to scheduled necropsy on Day 90, clinical pathology investigations (haematology, coagulation, clinical biochemistry and urinalysis) was conducted in all surviving animals. At termination, necropsy with macroscopic examination was performed. Selected organs were weighed and preserved in appropriate fixatives from the adult animals. A detailed histological examination was performed on the selected organs.

 

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

 

No test item related adverse effect on body weight and body weight gain, food consumption, clinical biochemistry, haematology and urinanalysis parameters were noted. TSH, T4 and T3 hormone tests did not show any effect on the thyroid part of the endocrine system. Functional Observational Battery did not reveal any neurotoxic potential of the test item. No treatment related alterations were found during the ophthalmological examinations.

 

No test item related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation in any of the organs examined.

 

There were no test item effects on the measured organ weights.

 

Based on the results of this presented study, the No Observed Adverse Effect is established at 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

7

Species:

rat

Quality of whole database:

K1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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

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). 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.

 

SynNova® Base Oil - 90-Day Oral (Gavage) Toxicity Study in Rats (OECD 408)

A 90-days study was performed with SynNova® Base Oil (test item) to assess the toxic potential of the test item when administered by oral gavage for 90 consecutive days.

No test item related adverse effect on body weight and body weight gain, food consumption, clinical biochemistry, haematology and urinanalysis parameters were noted. TSH, T4 and T3 hormone tests did not show any effect on the thyroid part of the endocrine system. Functional Observational Battery did not reveal any neurotoxic potential of the test item. No treatment related alterations were found during the ophthalmological examinations.

No test item related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation in any of the organs examined.

There were no test item effects on the measured organ weights.

Based on the results of this presented study, the No Observed Adverse Effect is established at 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the available information for the substance classification is not required in accordance with Regulation 1272/2008 (CLP)