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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2014

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:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Diet and water: 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.
The food and water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Husbandry:
Cage type: Type II and/or III polycarbonate (dimensions in mm 230 x 380 x h 180 and 380 x 380 x h 180, respectively)
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Details of bedding quality are reported in Appendix 6.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.9 - 24°C
Relative humidity: 36 - 66 %
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 3 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.
The temperature and relative humidity were recorded twice daily during the study and were within acceptable ranges.
- IN-LIFE DATES: animal arrival: 10 July 2014, start of treatment 16 July 2014, end of treatment: 30 august 2014, end of experiment: 31 august 2014 (necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test item in the vehicle was assessed in the conditions employed on the study in the concentration range of 10 mg/mL-200 mg/mL by Fourier-transformation infrared spectroscopy (non GLP investigation). According to the results the test item is stable over 7 days in refrigerator (2-8°C) and 6 hours storage at room temperature.
Analysis of test item concentration in formulations was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. using a validated UV spectrophotometric method. Duplicate samples were taken from test item formulations three times during the study (during the first and last weeks of treatment as well as at the beginning of the mating period). One set was analyzed and one set was retained as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements.
Duration of treatment / exposure:
Dosing of both sexes began after a minimum of five days acclimation and 2 weeks before mating. The treatment continued up to and including the day before necropsy. Mating began after the animals attained full sexual maturity. Dosing continued in both sexes during the mating period.

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post- mating period), then they were euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum.
Frequency of treatment:
The dosing solutions (test item or vehicle) were administered once daily by oral gavage, using a tipped gavage needle attached to a syringe. A constant volume of 5 mL/kg bw was administered to all animals. The actual dose volume was calculated and adjusted based on the most recent individual body weight. Control animals were treated concurrently with the vehicle only.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
other: dose level
Remarks:
Doses / Concentrations:
400 mg/kg bw/day
Basis:
other: dose level
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
other: dose level
No. of animals per sex per dose:
12 males 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
Male and female Wistar rats were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period, up to and including postpartum/lactation Day PPD4.
The control animals were treated with the vehicle only (distilled water).
Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, at least weekly body weight and food consumption, and clinical pathology evaluation, including haematology with coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity was performed during the last week of the treatment. In addition, the reproductive performance, pregnancy, parturition and postpartum /lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until post natal Day 4 (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 adult animals, histopathological examination was performed on the selected list of retained organs in the Control and High dose. Reproductive endpoints were measured on all animals, clinical pathology and non-reproductive organ histopathology was performed on 5 adults per sex.

Analysis of test item formulations for concentration was performed three times during the treatment period (during the first and the last week of the treatment period and at the beginning of the mating period), using validated UV spectrophotometric method. All formulations were found to be in the range of 97 to 105% of nominal concentrations and therefore considered acceptable. No test item was detected in the control samples.

Examinations

Observations and examinations performed and frequency:
- Clinical observations and functional observation battery (FOB): Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and end of the working day. General clinical observations were made once a day.
On gestation day 13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including onset, degree and duration of signs as applicable.
More detailed examinations were made once before prior to treatment (to allow for within-subject comparisons), then weekly in the morning. These observations were performed outside the home cage in a standard arena, at similar times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or 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); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were observed during the study.

- In selected animals, 5 males and 5 females/group:
Assessment of any potential test item related neurotoxicity was performed during the last week of treatment. Selected animals were subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay and fore/hind grip strength. Motor activity was 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 of the hind paws was measured.
Fore/hind grip strength measurements were conducted 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 test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support; results are tabulated with individual and mean data.
Sensory reactivity to different types of stimuli (auditory, visual and proprioceptive) was investigated and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, 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.
Motor activity assessment was conducted using the Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1 hour during which a DVD recording of movement was made. Recording was made for a duration of 60 min, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software. In the first instance the data from the high dose and control groups were evaluated for distance travelled in 5 minute segments.

- Body weight measurements
All adult animals were weighed with an accuracy of 1 g for randomization purposes, then on Day 0, twice a week thereafter and at termination.
Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition) and PPD4 (before termination).
- Food consumption measurement
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then weekly (on the days of body weight measurements).
- CLINICAL PATHOLOGY
Clinical pathology blood and urine samples evaluation was conducted in all selected animals (subgroup B, 5 males and 5 females per group) prior to necropsy.
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 (approximately 1.2 mL blood, in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (approximately 1.4 mL blood in tubes with sodium citrate as anticoagulant) and one to obtain serum (approximately 1 mL blood, in tubes with no anticoagulant) for clinical chemistry.
Haematology parameters observed: RBC Red Blood Cell (erythrocyte) count, (1012/L) M/µL, WBC White Blood Cell (leukocyte) count, (109/L) K/µL , Hgb Haemoglobin concentration, (g/dL), Hct Haematocrit (relative volume of erythrocytes) (%), MCV Mean Corpuscular (erythrocyte) Volume (fL), MCH Mean Corpuscular (erythrocyte) Haemoglobin, (pg), MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, (g/dL), RDW Red Cell (erythrocyte) volume (%), Plt Platelet (thrombocyte) count, (109/L) K/µL, MPV Mean Platelet Thrombocyte volume (fL), RETIC % Reticulocyte count (%), NE % Neutrophil (%),LY % Lymphocyte (%),MO % Monocyte (%),BA % Basophil (%), EO % Eosinophil (%),LUC % Large Unstained Cells (%) + coagulation: APTT Activated Partial Thromboplastin Time (sec), PT Prothrombin Time (sec)
Clinical chemistry: Glucose Blood sugar concentration (mmol/L), T-BIL Total Bilirubin concentration (μmol/L), Urea Urea concentration (mmol/L), Chol. Cholesterol concentration (mmol/L), Creat. Creatinine concentration (μmol/L), Phos. Phosphorus concentration (mmol/L), Na+ Sodium concentration (mmol/L), K+ Potassium concentration (mmol/L), Ca++Calcium concentration (mmol/L), Cl- Chloride concentration (mmol/L), Tot. Prot. Total Protein concentration (g/L), Alb. Albumin concentration (g/L), A/G Alb/glob ration, AST/GOT Aspartate Aminotransferase activity (U/L), ALT/GPT Alanine Aminotransferase activity (U/L), ALKP Alkaline. Phosphatase – activity (U/L), Bile acids (µmol/L)
Urinalysis: LEU / Leukocyte, NIT / Nitrite, pH, PRO / Protein, GLU / Glucose, UBG / Urobilinogen, BIL / Bilirubin, KET / Ketones, BLD / ERY Blood/Erythrocytes, SG / Specific Gravity, SED / Sediment, VOL / Volume, Colour/appearance


Sacrifice and pathology:
Gross necropsy was performed on each animal, terminally one day after the last treatment. Animals were euthanised under pentobarbital anaesthesia by exsanguinations.

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

The number of implantation sites and of corpora lutea was recorded in the females.
At the time of termination, body weight and weight of the following organs were determined:
All adult animals
- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain,
- With a precision of 0.001 g: ovaries

Subgroup 5 males and 5 females/group
- With a precision of 0.01 g: heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenal glands, pituitary
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight were reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised.

Gross necropsy was performed on each animal, terminally one day after the last treatment. Animals were euthanised under pentobarbital anaesthesia by exsanguinations.
After exsanguination the external appearance was examined, the cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.
The number of implantation sites and of corpora lutea was recorded in the females.
Macroscopic examination: Lungs with bronchi , Skeletal muscle (quadriceps), Adrenal gland, Lymph node, Small intestine, Animal identification, Ovary,Spinal cord (three levels), Aorta, Oviduct, Spleen, Brain, Pancreas, Sternum with marrow, Epididymis, Pituitary, Stomach, Eye with the optic nerve, Prostate, Testis, Oesophagus, Salivary gland (including mandibular, sublingual and parotid glands), Thymus, Femur with marrow, Thyroid with parathyroid gland, Heart, Tongue, Kidney, Sciatic nerve, Trachea, Large intestine, Seminal vesicle with coagulating gland, Urinary bladder, Extraorbital lachrymal gland, Uterus, Harderian gland, Skin, subcutis and mammary gland area (inguinal), Vagina, Liver.
For the adult animals, detailed histological examination was performed as follows:
• on retained organs in the Control and High dose groups (5 animals/sex/group),
• liver of female 4504, High dose group, due to macroscopic finding
• on the retained reproductive organs of all animals of the control and high dose group.
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.
Examination of tissues from Low and Mid groups was not performed as no treatment-related changes were found in High dose group.
Pups euthanized at PND 4 were carefully examined externally for gross abnormalities. Dead pups were subjected to necropsy with macroscopic examination in order to identify the probable cause of death.
Other examinations:
- Observation of the delivery process, offspring and nursing instinct
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. No evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy.
Dams were observed to record whether they formed a nest from the bedding material and covered their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach.
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 significantly smaller than normal pups) and the presence of gross abnormalities. Observations are reported individually for each adult animal.
In addition to the observations on parent animals, the pups (offspring) were monitored for any behavioural changes. Live pups were counted, sexed and weighed individually within 24 hours of parturition (ex. Day 0 or 1 post-natal, PND0 or 1) and on PND4, with accuracy of 0.01g. All the litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination in order to identify the possible cause of death.

Statistics:
The statistical evaluation of appropriate data 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. Getting 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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
There was no mortality during the study.
There were no clinical signs related to treatment.
There were no toxicologically significant changes in the animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or treated groups. The results of the landing foot splay test, fore and hind limb grip strength evaluation and motor activity measurements were comparable with the control mean in all treated groups in males and females.
There was no adverse effect on body weight or body weight gain.
There was no effect of treatment on food consumption.
There was no effect of treatment on haematology parameters, blood coagulation or urinalysis parameters. For clinical chemistry parameters, slightly higher serum protein values were measured in both sexes, slightly increased creatinine in males at 1000 mg/kg bw/day and slightly increased cholesterol concentration in females at 400 and 1000 mg/kg bw/day. The changes were of low magnitude, values were within the normal historical range, were not consistent between sexes and not associated with any morphological changes, therefore were not considered to be adverse.
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.
There were no treatment related macroscopic findings.
There was no adverse effect of treatment on organ weights. Slightly higher liver weights (by 14% for body weight relative value) in males at 1000 mg/kg bw/day, without any structural or relevant clinical pathology changes were not considered to be adverse.
There were no treatment related microscopic parameters at histopathology of any organ or tissue.

Effect levels

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Administration of the test item Carbohydrates and Sugars, hexitols, anhydro - LAB 4623 at dose levels of 150, 400 and 1000 mg/kg body weight/day to Wistar rats for 28 consecutive days (males) and throughout gestation period, up to and including postpartum/lactation Day PPD4 (females) was associated with no evident general toxicity of parent generation and no reproduction and developmental toxicity was observed.

The NOAEL for parental, developmental toxicity as well as reproductive performance and fertility was 1000 mg/kg bw/day in male and female Wistar rats.
Executive summary:

The subject of this study was the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat according to OECD 422 and OECD guidance document 43. The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing. The purpose of this study is to obtain information on the possible toxic effects of the test item following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels. The study also included a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy and parturition, and also on the development of the F1 offspring from conception to Day 4 post-partum.

The dose levels were 0, 150, 400 and 1000 mg/kg bw/d, and vehice use was water. There was no mortality during the study. There were no clinical signs related to treatment. There were no toxicologically significant changes in the animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or treated groups. The results of the landing foot splay test, fore and hind limbgrip strengthevaluation and motor activity measurements were comparable with the control mean in all treated groups in males and females. There was no adverse effect on body weight or body weight gain. There was no effect of treatment on food consumption. There was no effect of treatment on haematology parameters, blood coagulation or urinalysis parameters.

For clinical chemistry parameters, slightly higher serum protein values were measured in both sexes, slightly increased creatinine in males at 1000 mg/kg bw/day and slightly increased cholesterol concentration in females at 400 and 1000 mg/kg bw/day. The changes were of low magnitude, values were within the normal historical range, were not consistent between sexes and not associated with any morphological changes, therefore were not considered to be adverse. 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. There were no treatment related macroscopic findings. There was no adverse effect of treatment on organ weights. Slightly higher liver weights (by 14% for body weight relative value) in males at 1000 mg/kg bw/day, without any structural or relevant clinical pathology changes were not considered to be adverse.

There were no treatment related microscopic parameters at histopathology of any organ or tissue.