Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 October 2014 to 19 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD test guidelines in compliance with GLP and reported with a valid GLP certificate.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The measured range of the temperature in the animal room was out of the targeted range (actual range: 20.1 – 25.1 °C, target range: 22±3°C) These deviations have no effect on the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Water white liquid
Details on test material:
Name: NovaSpec base oilBatch/Lot No.: TS13732CAS number: 1472010-43-7Chemical name: Alkenes, C-10-16a-, mixed with (6E)-7,11-dimethyl-3-methylene-116,10-dodecatriene, dimers, tetramers and trimers, hydrogenatedPurity: 100%Manufacture date: 06 June 2014Expiry date: 06 June 2016Description: Water white liquidStorage condition: Controlled room temperature (15-25 oC below 70 RH%)Safety Precautions: Routine safety precautions for unknown materials (lab coat, gloves, safety glasses and face mask) were applied to assure personnel health and safety.No correction for purity of the test item was applied.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: Crl:WI rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony. Males and females originated from different units to avoid subsequent brother/sister matings.
Hygienic level: Standard laboratory conditions during the study
Number of animals: 48 male, 48 female rats, 12 animals/sex/group, 4 groups (main animals); 10 male and 10 female rats, 5/sex/group (recovery group) and 12 males and 12 females rats in the positive control group for the micronucleus test).
Age of animals: Young adult rats, 10-11 weeks old at starting and 12-13 weeks at mating.
Body weight range: Males: 327 g – 478 g, Females: 207 g - 273 g;
Acclimation period: At least 5 days
Husbandry
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian. Females were nulliparous and non-pregnant.
Room number: 508
Cage type: Type II and/or III polypropylene/polycarbonate
Bedding: Lignocel® and GRADE 5 type wooden chips were available to the animals.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.1 – 25.1 °C (target range 22±3°C)
Relative humidity: 31 – 64 % (target range 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 5 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 allowed digging and other normal rodent activities (i.e. nesting).
Food and water supply
Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36, Hungary). The quality control results are retained in the archives at CiToxLAB Hungary Ltd.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 parental animal (P Generation) was identified by a number unique within the study written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd. The animal number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section.The boxes (cages) were marked by identity cards, with information at least about study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery. Boxes were arranged in such a way that possible effects due to cage placement were minimized.Identification of the new-borns (Offspring, F1 Generation) was performed by permanently marking of the tips of the digits on the day of birth. (DETAILED IN TABLE FOR SEE ANY OTHER INFORMATION)
Randomization
All adult animals were sorted according to body weight by computer and divided in to weight ranges. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that the mean group weights were similar. The grouping was controlled by SPSS/PC software, according to the actual body weight verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
The vehicle was selected by the Study Director in consultation with the Sponsor based on the trial formulations performed in the Central Dispensary of CiToxLAB Hungary Ltd..
Name: Poly(ethylene glycol) 400
Lot No.: BCBL5307V/BCBM8497V//BCBK 9981V
Manufacturer: Sigma-Aldrich Co.
Expiry Date: 31 January 2015/31 January 2016/
Storage: Room temperatureName: 0.2% Polysorbate 80
Lot No.: BCBL9041V/BCBN3690V
Manufacturer: Sigma-Aldrich Co.
Expiry Date: 30 November 2014/31 July 2016
Storage: Room temperature under inert gas
Preparation of the vehicle
For the preparation of 100 g (approximately 100 mL) vehicle, 0.2 g Polysorbate 80 was weighed by analytical balance into 99.8 g PEG 400, and was stirred by a magnetic stirrer.The test item was formulated in the vehicle as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared for 7 days and kept in refrigerator pending dosage in the first 14 days of the study, and daily afterwards.
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. Brother/sister matings were avoided. Females remained with the same male until copulation occurred, for up to 12 days (except two females from the high dose group cohabited with a other males from the same dose group after a 14 days unsuccessful mating period). Vaginal smears were 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 caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed at the Test Site using a gas chromatographic method (internal ID at the test site: FPBSTUDY-113-MEAS) to determine the test item content.Top, middle and bottom duplicate samples were taken into 50 mL polypropylene centrifuge tubes from each concentrations at four occasions (Week 1, Week 3 two occasions, Week 3 and Week 6), one set to analyze (which was collected in duplicates) and one set as a back-up, if required for any confirmatory analyses from all dose groups. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements. Further samples were taken in the same manner several times (from Day 20 until Day 27, from Day 29 until 36, and from Day 38 until Day 54 daily only from the high dose formulations for confirmatory purposes.Top, middle and bottom duplicate samples were taken into 50 mL polypropylene centrifuge tubes from test item formulations, one set to analyze and one set as a backup.Analysis of test item formulations was performed in the Fumoprep Kft. At the first instance, the first set of samples taken on Day 0, Day 17/18 and Day 37 were analyzed.For confirmatory purposes, back up samples were also measured later.The positive control material (cyclophosphamide) was dissolved in physiological saline (10 mg/mL) for the treatment. The solution was prepared just before the treatment. The test item solutions were given to assure the same dosing volumes in rat (2 mL/kg bw).No dose formulation analysis was performed from the positive control solutions.

Analytical Method
60 g/L stock solution Approx. 600 mg Nova Spec base oil test item was weighed by analytical balance into 10 mL volumetric flask and filled up with heptane. The solution was mixed well.
Preparation of the vehicle (0.2% Polysorbate in PEG)
Approx. 0.2g Polysorbate 80 was weighed by analytical balance into approx. 99.8 g PEG-400 and was mixed with magnetic stirrer.QC sample preparation500 mg/mL formula was prepared as follows: 250 mg of NovaSpec Base Oil was weighed with analytical precision into 50 mL centrifuge tube and 215 mg PEG-400 (0.2% Polysorbate) was added. The QC samples were prepared the samples.Preparation of the samples0.5-1.0 mL formula was received (weighed with analytical precision) in 50 mL centrifuge tubes. 10 mL n-Heptane was added at least 0.01 mg precision and 10 mL HPLC grade water was pipetted into the tube. The tubes were shaken manually and sonicate for 15 min. approx. 2 mL were centrifuged with 15000 rpm for 2 min. After the centrifuging the upper phase was diluted with n-Heptane into the dynamic range of the calibration (5x dilution for the 150 mg/mL and 10x dilution for the 500 mg/mL levels).
Chromatographic System
Column: HP-1; 5 m x 0.53 mm; 2.65 μm (CGC-01/03)
Mobile Phase: H2
Flow Rate: 15 mL/min
Split Ratio: 1:5
Detection: FIDTemperature: 160-300
Init Temp: 160°C (0 min) 10°C/min to 300°C (5 min)
Acquisition time: 20.00 min
Injection volume: 0.5 μL
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period).Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (4 days post-partum dosing). The day of birth is defined as Day 0 post-partum.
Frequency of treatment:
Animals were administered the dosing solutions daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe.
Details on study schedule:
Dosing schemes in table form (See Any other information)
Doses / concentrationsopen allclose all
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:
48 male, 48 female rats, 12 animals/sex/group, 4 groups (main animals); 10 male and 10 female rats, 5/sex/group (recovery group).
Control animals:
yes, concurrent vehicle
Details on study design:
Justification of species/strain: The rat is regarded as a suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for dose range finding study (study code: 14/328-220PE).Rationale for dose selection and route of administration
The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, including the results of a repeated dose range finding study in the rat (CiToxLAB study code 14/328-220PE). The aim was to induce toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The selected highest dose of 1000 mg/kg bw/day is a limit dose for this study type.The oral route was selected as it is a possible route of exposure to the test item in humans.

Dosing procedure
Test item or vehicle control-treated groups Groups 1-4 Main animals were administered the dosing solutions daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 2 mL/kg bw was administered to all animals. The actual volume to be administered was calculated and adjusted based on most recent individual body weight.Dosing of both sexes beginned after at least 5 days acclimatisation, animals were dosed for 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.
Positive control:
Postive Control not required for this study type.

Examinations

Parental animals: Observations and examinations:
Clinical observations and functional observation battery (FOB)
Main and Recovery animals: Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and end of the working day.General clinical observations were conducted once a day, during the treatment period in the afternoon at approximately the same time with minor variations as practical during the working day.Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration as applicable.Detailed examinations were conducted once before the first exposure (to allow for within-subject comparisons), then weekly at the morning time. These observations were made outside the home cage in a standard arena, at similar times as practical.Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, 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 assessed. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.On gestation day GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).Main animals, randomly selected 5 males and 5 females: Assessment of any potential test item related neurotoxicity was performed during the last exposure week on five selected main males and females.Selected animals were subjected to the functional observation battery, including quantitative assessment of grip strength (manual and instrumental), and measurement of landing foot splay and fore/hind limb grip strength. Qualitative and quantitative assessment of motor activity were measured.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 at 3 times for each animal on each test day. The procedure was repeated with the hind limbs and the appropriate grip support. The results were tabulated with individual and mean data.Manual assessment of sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity were conducted and the general physical condition and behaviour of animals was 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, 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 using a scoring system, where score “0” is given when the behaviour or reaction of the animal is considered normal, and -1 or -2, or +1 and +2 is given if the response is less, or heightened than expected in an untreated animal.Quantitative assessment of motor activity was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany).Locomotor activity were monitored by placing each animal individually into an open field for 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 are made to produce the appropriate parameters. Obtained data was evaluated for distance travelled in 5 minute segments in the control and high dose groups.Recovery animals: Neurotoxicity evaluation was similarly conducted in the Recovery animals towards the end of the recovery period.Body weight measurementAll adult animals were weighed with accuracy of 1 g for randomization purposes, then on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on post partum Days PPD0 (within 24 hours after parturition), PPD4 and before termination. Body weights of the female animals was additionally taken on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data was not evaluated statistically.

Food consumption measurement
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 and weekly afterwards.

CLINICAL PATHOLOGY
Randomly selected five males and females from the main animals, and all recovery animals.All animals selected for blood sampling were fasted (overnight period of food deprivation). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy. Three samples were taken from each animal: one for haematology (1.2 mL blood, in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (1.4 mL blood in tubes with sodium citrate as anticoagulant) and one to obtain serum (approximately 1 mL blood as practical, in tubes with no anticoagulant) for clinical chemistry.
Oestrous cyclicity (parental animals):
Not examined - Screening study
Sperm parameters (parental animals):
Not examined - Screening study
Litter observations:
Main Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. Any evidence of abnormal deliveries was 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.Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND0 or PND1) and on PND4, with accuracy of 0.01g. All litters were checked daily for the number of viable and dead pups.All pups were culled on PND4.
Postmortem examinations (parental animals):
Terminal procedures and macroscopic evaluation
Gross necropsy was performed on each animal irrespective of the date of death.Terminally (one day after the last treatment), all animals were sacrificed under anaesthesia by exsanguination.The external appearance was then examined and the cranium, thoracic and abdominal cavities were opened. The appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.The number of implantation sites and of corpora lutea were recorded for the main females.Organ weight measurements
At the time of termination, body weight and weight of the following organs of all adult animals was 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) was calculated and reported.Tissue preservation and microscopic evaluationThe weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerves were retained in modified Davidson’s fixative, testes with epididymides in Bouin’s solution, and all other organs in 10% buffered formalin solution.In addition, on completion of the macroscopic examination the following tissues and organs were retained from all animals.The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.For the adult animals, detailed histological examination were performed as follows:• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),• all macroscopic findings (abnormalities), except of minor order from all animals• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the control and high dose group and of all males that failed to sire and all females that failed to deliver healthy pups.Special attention was paid to 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.
Postmortem examinations (offspring):
Dead pups and pups euthanized on PND4 were carefully examined externally for gross abnormalities.
Statistics:
The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.
Reproductive indices:
Data was recorded to provide information on:Male mating index, Male fertility index, Female mating index, Female fertility index and Gestation index
Offspring viability indices:
Data was recorded to provide information on:Survival Index of pups on postnatal Days 0 and 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

Mortality
There was no mortality during the study.
Clinical observation
No test item related adverse effects or systemic clinical signs were noted following daily administration of the test item by oral gavage.Red discharge from the eyes was observed in one male in the Low dose group (100 mg/kg bw/day) on Day 20. The vaginal prolapse was noted in one Low dose female from day 40 (progressed to the prolapse of uterus), one Mid dose female from Day 29 and a prolapse of uterus was seen in one High dose female from Day 38. This was accompanied by piloerection at Mid and High dose.All the signs detailed above are considered incidental with no toxicological relevance.

Neurological assessment
There were no treatment related effects.There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.There was no effect of treatment noted during the assessment of foot splay, grip strength or motor activity. The total distance travelled was comparable to the control for both sexes in the high dose group. The statistically significantly higher than control total travelled distance group mean value observed in recovery females is considered incidental and not toxicologically meaningful.

Body weight and body weight gain
There were no test item related adverse effects noted on the body weight and body weight gain values following daily administration of the test item at dose levels up to and including 1000 mg/kg bw/day.The statistically significantly lower than control body weight gain group mean value was noted in recovery females from Day 35 until Day 42, but considered incidental with no toxicological relevance.

Food consumption
There were no test item related differences in the food consumption in test item treated groups when compared to the Control. Small but statistically significantly changes such us higher than control group mean value in High dose males (1000 mg/kg bw/day) from Day 21 until Day 27 and higher than Control group mean value in females from Day 14 until GD0 are considered incidental with no toxicological relevance.

Clinical pathologyHaematology
There were no test item related differences. Statistically significant differences in large unstained cells group mean value in the Low dose females (100 mg/kg bw/day) was considered incidental. Statistically significantly higher (p<0.05, +60%) than Control monocyte percent (MONO%) group mean value was measured in the High dose group in females (1000 mg/kg bw/day). However, it is ascribed to lower control values rather than to the effect of treatment (historical control mean of CiToxLAB Hungary Ltd. Is 3.129 %). The actual control mean is 2.020 %, and the High dose mean is 3.240 %, which are similar to the historical mean.

Clinical chemistry
There were no test item related differences.In males, statistically significantly lower than control albumin (p≤0.05, -6%; p≤0.01, -8%) and total protein (p≤0.05, -3%; p≤0.01, -5%) group mean values were noted in the Mid and High dose group. The group mean values in the Mid and High dose groups were comparable with the historical control mean of CiToxLAB Hungary Ltd. while the study control values were higher. Therefore the changes were ascribed to a relatively higher study control rather than a test item related effect. (study control is 31.56 g/L for albumin, the historical control mean is 29.80 g/L, while the mid and low dose mean values are 29.78 g/L and 30.70 g/L respectively. For the total protein the study control is 59.44 g/L, while the historical mean is 56.50 g/L, and the group mean in the mid and high dose groups are 57.54 g/L and 57.96 g/L respectively).

Urinalysis
There were no test item related differences.

Reproductive ability assessment and indices
There was no effect of treatment on reproductive parameters.There were no differences between the control and test item treated groups with regard to reproductive ability or in the mating or gestation indices. The mating, fertility and gestation indices were comparable with the control in the test item-treated groups.Two males from the High dose group did not mate but the females were permanently in dioestrus phase of their sexual cycle (unresponsive to mating attempts). No histopathology alterations were found in the male reproductive organs, therefore the causative role of the test item is excluded. There was a relatively lower number of pregnant females with liveborns on PND0 and PND4 in the Mid dose group, however the values in the Low and High dose groups were comparable with the control, therefore it is considered incidental and with no toxicological relevance.Test item administration was considered to have no impact on the duration of the mating period. Group mean pre-coital intervals were 2.8 2.4, 2.3 and 2.8 days in the Control, Low, Mid and High dose groups, respectively. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within up to 5 days of pairing (cohabitation) in the majority of the animals.

Evaluation of the gestation, parturition and post-partal period
There was no effect of treatment noted during gestation, parturition or the post-partal period.The mean duration of pregnancy was comparable in the Control and test item treated groups. All parturitions were normal except one animal in the Mid dose group, where a prolonged parturition was noted without any live born pups. The number of corpora lutea and number of implantation sites was comparable to the control mean at all dose groups. There were no significant differences or effects that could be ascribed to treatment on the pre-implantation, post-natal or total mortality values (litter mean and %) at up to and including 1000 mg/kg bw/day.

PATHOLOGY EVALUATION AND ORGAN WEIGHTS

Macroscopic Findings
No test item related macroscopic findings were observed up to the dose level of 1000 mg/kg bw/day.Pale discoloration/pelvic dilation of the kidney, enlargement of the thyroids/parathyroids, enlarged right testis, red discoloration of the lungs, dilatation/enlargement of the uterus or spleen, red discoloration of the glandular stomach, were observed without meaningful incidence and were considered to be incidental or a common background.

Microscopic Findings
No test item related microscopic changes were noted at a dose level of 1000 mg/kg bw/day.Minimal focal myocardial degeneration in 2/5 Control males, minimal focal interstitial cell infiltrate in 2/5 Control and 1/5 High dose males were seen in the heart. Minimal chronic progressive nephropathy in 1/7 High dose male, minimal unilateral interstitial inflammation in 1/5 Control male, minimal unilateral pyelitis in 1/5 Control female, mild unilateral pelvic dilatation in 2/7 High dose males, minimal focal unilateral tubular basophilia in 1/8 High dose male were observed in the kidney. Diffuse moderate edema of the lungs in 1/6 Control female and mild periportal fibrosis of the liver in 1/5 Control female were also noted. In the spleen, minimal to moderate extramedullaryhematopoiesis was recorded in 3/5 Control and 4/5 High dose males and 2/5 Control, 3/3 Mid dose and 5/5 High dose females. Minimal focal erosion of the glandular stomach in 1/5 High dose female, moderate tubular dilatation of the right testis in 2/2 Mid Dose males, moderate uterine dilatation in 1/3 Mid dose female, and pyometra in 1/2 Low dose female were detected.These changes were incidental or considered to be a common background observation.

Organ weight
There were no treatment related adverse effects on organ weights.Statistically significantly higher than control absolute (p≤0.01, +21%) and relative (p≤0.01, +19% related to body weight and p≤0.01, +20% related to brain weight) adrenal weight group mean values were noted in male animals in the High dose group (1000 mg/kg bw/day). The causative role of the test item cannot be excluded, however, the majority of individual results were within the historical control range of CiToxLAB Hungary Ltd., and no association with clinical pathology or pathology effects was noted. These are therefore considered as a not adverse.Other occasional statistically significant differences were not dose-related and therefore considered incidental and of no toxicological significance.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: general toxicity, reproduction parameters, and for the development of offspring F1 generation

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

Mortality and clinical observations
Administration of the test item to parental generation at doses of up to 1000 mg/kg bw/day did not cause mortality or any adverse effects considered related to treatment in the F1 generation. No abnormal behaviour of the pups was noted. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. The occurrence of signs noted (haemorrhage, cyanotic, cold to touch) was comparable with the study and/or historical control level, therefore considered incidental or background, without toxicological relevance.The number of viable pups on PND 0 and 4 was comparable to the control values, no statistically significantly difference was noted in comparison with the control.The incidence of mortality during 4 postnatal days was negligible and was 3 of 156 (Control) 2 of 135 (Low dose), 1 of 118 (Mid dose) and 5 of 137 (High dose).The survival indices on PN Days 0 and 4 were similar in all groups.The sex ratios did not differ significantly between the control and treatment groups.

Body weight and body weight gain
There was no effect of treatment on the offspring body weight or body weight gain.When evaluated on per litter basis, the mean body weight and body weight gain in the F1 generation evaluated on PND 0 and 4 were similar in all treated groups and the values were comparable to controls. Slight variability noted (lower than control body weight and body weight gain in the Low dose group on Day 0 and Day 4 and from Day 0 to Day 4, and higher than control body weight on Day 0 in the mid dose group were considered incidental without toxicological relevance.Macroscopic FindingsNo test item related gross changes were observed in the pups.Two Control pups, 4 Low Dose and 3 High Dose pups were found dead.Hemorrhage (neck, head, cranium, nasal area, sacrum, both hindlimbs, snout/both, right cheeks) in 4/5 Control and one Low Dose, 5 Mid Dose pups and 7 High Dose pups; 2 Control, one Low Dose, one Mid Dose and 4 High Dose pups were cannibalized; 2 Control, one Low Dose, one Mid Dose pups and 3 High Dose pups were cold; 2 High Dose pups were cyanotic and 5 Mid Dose pups were autolysed. The severity or frequency of these signs did not exceed control level, and are considered as incidental or background.

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: No effects noted.
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no
Relation to other toxic effects:
not specified
Dose response relationship:
no
Relevant for humans:
no

Any other information on results incl. tables

Summary of male reproductive ability

 

Control

100 mg/kg

300 mg/kg

1000 mg/kg

Number of treated males

12

12

12

12

Number if males paired

12

12

12

12

Number of mated males

12

12

12

10

Male mating index

100

100

100

83

Male fertility index

100

92

100

100

 

Summary of female reproductive ability

 

Control

100 mg/kg

300 mg/kg

1000 mg/kg

Number of females paired

12

12

12

12

Number of sperm positive females

12

12

12

12

Number of pregnant females

12

11

12

12

Pregnant females with live borns on PND0

12

11

9

11

Pregnant females with live borns of PND4

11

11

9

11

Female mating index

100

100

100

100

Female fertility index

100

92

100

100

Gestation index

100

100

75

92

 

Post-natal mortality (No. = live birth minus alive on PND 4)

 

Number of dams

Post-natal loss

Control dose group

Low dose group

Mid dose group

High dose group

0

9

9

8

6

1

3

2

1

4

2

0

0

0

1

≥3

0

0

0

0

Group mean (%)

0.25

0.18

0.11

0.45

Survival index PND0 (%)

100

100

100

98.99

Survival index on PND4 (%)

90.35

99.23

99.31

93.75

 

Summary of Delivery and Viable Pups Per Sex on Post-natal Days 0 and 4 (Total Numbers)

 

Dose (mg/kg bw/day)

Control

100

200

1000

No. of Dams

 

12

11

10

11

No. of Pups Born

Sum:

157

138 NS

123 NS

139 NS

No. of Live Born

(Data compared to no. of pups born)

Sum:

%:

156

99.4

135 NS

97.8

118* CH2

96.9

138NS

99.3

PN0

No. of viable pups

(Data compared to no. of live born)

Sum:

%:

156

100.0

135 NA

100.0

 

 

Dead pups (born alive)

 (Data compared to no. of live born)

Sum:

%:

0

0.0

0 NA

0.0

0 NA

0.0

1 NS

0.7

Male

(Data compared to no. of viable pups)

Sum:

%:

98

62.8

79 NS

58.5

69 NS

58.5

71 NS

51.8

Female

 (Date compared to no. of viable pups)

Sum:

%:

58

37.2

56 NS

41.5

49 NS

41.5

66 NS

48.2

PN4

No. of viable pups

(Data compared to no. of viable pups)

Sum:

%:

153

98.1

133 NS

98.5

117 NS

99.2

132 NS

96.4

Dead pups (born alive)

(Data compared to no. of live born)

Sum:

%:

3

1.9

2 NS

1.5

1 NS

0.8

6 NS

4.3

Male

 (Data compared to no. of viable pups)

Sum:

%:

97

63.4

77 NS

57.9

69 NS

59.0

69 NS

52.3

Female

(Data compared to no. of viable pups)

Sum:

%:

56

36.6

56 NS

42.1

48 NS

41.0

63 NS

47.7

Remarks:                                           NS = Not significant

* = p<0.05 Chi Square                  NA = Not applicable

** = p<0.01 Chi Square                - = No data

 

Summary of Body Weight and Body Weight Gain Data Means (Viable Pups) and Clinical Observations Data on Post-natal Days 0 and 4 (g)

TIME

 

DOSE (mg/kg bw/day)

Post-natal day

Control

100

300

1000

Body weight: Male & Female (g)

0

MEAN

SD

N

±%

6.527

0.52

156

6.250**

0.56

135

-4.2

6.670*

0.61

118

2.2

6.497 DN

0.58

137

-0.5

4

MEAN

SD

N

±%

10.979

1.42

153

9.953**

1.01

133

-9.3

11.141

0.91

117

1.5

10.932 U

1.32

132

-0.4

 

 

Body weight gain: Male & Female (g)

0 – 4

MEAN

SD

N

±%

4.444

1.13

153

3.708**

0.86

133

-16.6

4.466

0.84

117

0.5

4.411 U

1.04

132

-0.8

 

 

Pup Mean Weight/Litter (g)

0

MEAN

SD

N

±%

6.553

0.39

12

6.392

0.64

11

-2.5

6.684

0.45

9

2.0

6.570 NS

0.45

11

0.3

4

MEAN

SD

N

±%

11.254

1.40

11

10.252

1.07

11

-8.9

11.146

0.60

9

-1.0

11.137 NS

1.26

11

-1.0

 

 

Pup Mean Weight Gain/Litter (g)

0 – 4

MEAN

SD

N

±%

4.665

1.06

11

3.875

0.65

11

-16.9

4.463

0.45

9

-4.3

4.527 NS

0.88

11

-3.0

Occurrent remarks:                                                       U = Mann-Whitney U-test Versus Control

±% = Percent Deviation Versus Control                 DN = Duncan’s multiple range test

NS = Not significant

* = p<0.05

** = p<0.01

Applicant's summary and conclusion

Conclusions:
Administration of NovaSpec Base Oil in PEG 400 + Polysorbate 80 via oral gavage to Wistar rats at the dose levels of 100, 300 and 1000 mg/kg bw/day was not associated with any effect on reproduction in the parent males and females, or on development of offspring. Also, no adverse effects were noted in general toxicology parameters. At the dose level of 1000 mg/kg bw/day, elevation of adrenal absolute and relative weights was noted, however considered as not adverse in the lack of any corresponding clinical pathology or histopathology effect.In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) of NovaSpec Base Oil for general toxicity, reproduction parameters, and for the development of offspring F1 generation, was considered to be 1000 mg/kg bw/day.
Executive summary:

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat according to OECD 422 and OECD guidance document 43 was performed in order to obtain information on the possible toxic effect of the test item following repeated (daily) administration by oral gavage to Wistar rats at 3 different dose levels. The study also provided initial information on the 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 4 post-partum.

 

The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, with the aim of inducing toxic effects but ideally no death or suffering at the highest dose (a limit dose for this study type) and a NOAEL at the lowest dose.

 

Twelve Wistar rats per sex/group were assigned to 4 Main study groups.

Additional 5 Wistar rats per sex/group were assigned to a Control and High dose Recovery groups.

Recovery animals scheduled to follow-up observations were not mated but kept without treatment for at least 14 days after the first scheduled euthanasia of dams to detect possible delayed occurrence, or persistence of, or recovery from toxic effects.

The control group was treated with the vehicle only.

 

Analysis of all dose formulations was performed on samples collected on Week 1, Week 3 two occasions, Week 3 and Week 6. The actual concentrations varied between 70% and 105% of the nominal in the Low dose group, 72% and 103% in the Mid dose group, and 79 and 105% in the High dose group. No test item was detected in the control samples. However there was a relatively high individual variations in homogeneity between formulations, the mean concentrations were 45.9 mg/mL (92% of the nominal, n=18, SD = 4.10) for the Low dose group, 139.6 mg/mL (93% of the nominal n=18, SD=12.36 for the Mid dose group, and 458.69 mg/mL (92% of the nominal, n=54, SD=26.95) in the High dose group, therefore considered to be suitable for the study purposes, providing the expected exposure of the animals.

 

Parameters monitored during the study included signs of morbidity and mortality twice daily, observation of clinical signs, body weight and body weight gain, and food consumption. Five selected males and females from the main group, and all recovery animals were subjected to a clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment in five selected males and females from the main group, and in all recovery animals. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult Main animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals.

 

For the Main adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

 

There was no mortality during the study.

No test item related adverse effects or systemic clinical signs were noted following daily administration of the test item.

Neurological examinations did not reveal any test item related adverse effect.

No test item related adverse effects were noted on body weights or body weight gains.

No effects were observed on the food consumption.

There were no toxicologically relevant effects noted at haematology measurements.

Clinical chemistry measurements did not reveal any test item related adverse effect.

No adverse effects were noted at urinalysis.

There was no effect of treatment noted during evaluation of the reproductive parameters. There were no adverse effects on the F1 offspring viability, clinical signs or development.

No test item related effects were observed at necropsy.

Histopathology examinations on selected tissues did not reveal any test item related effects in the High dose group.

There were no toxicologically significant effects observed in organ weights, however statistically significantly higher than control (+21%) absolute and relative adrenal weight was noted in the High dose group. However, no corresponding effects in clinical pathology or histopathology were noted.

 

Conclusion

Administration of NovaSpec Base Oil in PEG 400 + Polysorbate 80 via oral gavage to Wistar rats at the dose levels of 100, 300 and 1000 mg/kg bw/day was not associated with any effect on reproduction in the parent males and females, or on development of offspring. Also, no adverse effects were noted in general toxicology parameters. At the dose level of 1000 mg/kg bw/day, elevation of adrenal absolute and relative weights was noted, however considered as not adverse in the lack of any corresponding clinical pathology or histopathology effect.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) of NovaSpec Base Oil for general toxicity, reproduction parameters, and for the development of offspring F1 generation, was considered to be 1000 mg/kg bw/day.

The No Observed Effect Level was determined to be at 1000 mg/kg bw/day in females, and at 300 mg/kg bw/day in males, due to the changes in adrenal absolute and relative weights.