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Effects on fertility

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Reference
Endpoint:
toxicity to reproduction
Remarks:
other: combined repeated dose toxicity study with reproductive /developmental toxicity screening test
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
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)
Route of administration:
oral: gavage
Vehicle:
water
Details on mating procedure:
Mating began 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, within 5 days from the onset of pairing. A vaginal smear was prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (and as Day 0 of pregnancy). Sperm positive females were caged individually.

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.
Details on study schedule:
Dosing Scheme Male:
A- accclimation at least 5 days,
PM- pre-mating period 14 days,
M- mating/post-mating period at least 14 days - last week of treament : FOB (5 animals/group), prior to or at necropsy (day 28): hematology, coagulation, clinical chemistry and urinalysis, at necrosy organ weight determination (5 animals/group), all males: necropsy with organ weight determination.

Dosing Scheme Female:
A- accclimation at least 5 days,
PM- pre-mating period 14 days
M- mating up to 5 days
G- gestation 22-23 days
Delivery
PP/PN- lactation period at least 4 days - FOB and pups necropsy
PP/PN- PND5 - prior to or at necropsy: hematology, coagulation, clinical chemistry and urinalysis, at necrosy organ weight determination (5 animals/group) all females: necropsy with organ weight determination.
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 male and 12 female
Control animals:
yes, concurrent vehicle
Details on study design:
No correction for purity of the test item was applied. The test item was formulated in the vehicle at the appropriate concentrations of 30, 80 and 200 mg/mL according to the dose level at constant volume of 5 mL/kg bw. Formulations were prepared and used according to stability assessment results within 7 days while stored refrigerated.
Parental animals: Observations and examinations:
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.
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).
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).
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

Litter observations:
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.
Pups were culled on PND4.
Postmortem examinations (parental animals):
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.
Postmortem examinations (offspring):
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.
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.
Reproductive indices:
Parental Males
- Number of pairings
- Number of fertile pairings
- Number of infertile males
- * Male mating index
- * Male fertility index


Parental Females
- Number of pairings
- Number of pregnant females
- Number of sperm positive, but non-pregnant females
- Number of non mated females
- * Female mating index
- * Female fertility index
- * Gestation index
- Duration of pregnancy (days)
- Number of Corpora lutea / dams
- Number of implantations / dams
- Number of dams with live pups Day 0 and 4
- * Pre-implantation mortality
- * Intrauterine mortality
- * Total mortality (intra and extra uterine mortality)
Offspring viability indices:
- Mean pup body weight (per pup within the group and per litter) on PND 0 and 4
- Mean pup body weight gain (per litter) between postnatal Days 0-4
- Number of live births per litter, and number of viable pups per litter on postnatal Days 0 and 4
- * Survival Index of pups on postnatal Days 0 and 4
- * *Sex ratio % (on postnatal Days 0 and 4)
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Compared to the control means, the following tendencies were observed during clinical chemistry examination:
• Cholesterol concentration was increased in females at 400 and 1000 mg/kg bw/day, (p<0.05 and p<0.01, respectively). See overview Table 9 below.
•Total protein concentration was slightly higher in all test item treated groups in both males and females corresponding to slight increase in albumin concentration.
•Creatinine concentration was higher than control mean in males at 1000 mg/kg bw/day (p<0.05).
•Bile acid concentrations were higher in females in low and high groups (p<0.05).

The above listed changes in the serum chemistry parameters were of small magnitude and were within the normal historical ranges, and were not associated with any changes in the other indicative parameters, in the weight of kidneys (males) and liver (females) or histopathology. The lack of any clear dose response or consistent differences confirms that they were not considered to indicate any adverse effect of the test item.

Other differences observed between the Control and treated groups, occasionally attaining statistical significance, were regarded as incidental or individual findings, which were not related to treatment. The values were generally comparable with the expected physiological range or were with no toxicological significance.

Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Sex:
male/female
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
In one stillborn pup at 1000 mg/kg bw/day (4505/13), absence of tail was observed. Although it was observed only in one pup out of 192, the causative role the test item cannot be excluded.
Reproductive effects observed:
not specified
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.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP study reliability 1

Justification for classification or non-classification

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. Hence, no classification is required according to CLP (1272/2008/EC)