Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Key study: According to OECD 422. GLP study. The no observed adverse effect level (NOAEL) of the test item for systemic toxicity was determined to be 300 mg/kg b.w./day (highest dose tested). As for pups, based on number of stillbirths and mortality in group 2 and 3, it may be concluded that the test item affected pups survival. Therefore, on the grounds of the used doses, the no observed adverse effect level (NOAEL) for reproduction/developmental toxicity for pups can be determined on 33.3 mg/kg b.w./day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24.03.2016 to 22.01.2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Analytical purity: 93%
- Expiry date: 06.03.2018
Species:
rat
Strain:
Wistar
Remarks:
Cmdb:WI; outbred
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: from the husbandry of laboratory animals of the Experimental Medicine Centre at the
Medical University in Białystok,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 13-14 weeks old.
- Weight at study initiation: average body weights:
- group 0: males 403.50 g; females 243.75 g
- group 1: males 403.50 g females 243.92 g
- group 2: males 403.40 g; females 243.75 g
- group 3: males 403.50 g; females 243.92 g
- group 0 SAT: males 404.00 g; females 244.90 g
- group 3 SAT: males 404.10 g; females 245.10 g.
- Fasting period before study:
- Housing: Air-conditioned rooms. The animals were kept in plastic cages covered with wire bar
lids. The dimensions of the cages were 58 x 37 x 21 cm (length x width x height). During the main e
xperiment, there were 2 or 3 animals/cage. Each sex was kept separately.
- Diet (e.g. ad libitum): ad libitum access,
- Water (e.g. ad libitum): ad libitum access.
- Acclimation period: All animals were quarantined and observed for at least 5 days.

DETAILS OF FOOD AND WATER QUALITY: Food: Altromin 1324 P TPF (Phytoestrogen Deficient,
Total Pathogen Free) standard granulated fodder produced by Altromin Spezialfutter GmbH & Co.
KG, Lage, Germany, batch number: 1/17 -200418/0453. A certificate of analysis is included in the
report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25ºC
- Humidity (%): 43 - 81%
- Air changes (per hr): 13-18 times/h
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: Experimental starting date 13.06.2017 To: Experimental completion date 22.08.2017
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
During the main study solutions of the test item in a vehicle (corn oil) were prepared once a week.
Prepared solutions were stored in a fridge (temperature 4-8#C). During administration, solutions were
kept at room temperature and they were thoroughly mixed.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): most suitable vehicle, as the test item hydrolyses in water.
- Concentration in vehicle: 6.66 mg VL3/1 mL solution, 20 mg VL3/1 mL solution and 60 mg VL3/1mL solution
- Amount of vehicle (if gavage): the total volume administered was 0.5mL/100 g b.w. (including testitem)
- Lot/batch no. (if required): L5294 (Supplier BASSO FEDELE & FIGLI srl, ITALY)
- Purity: 100 % corn oil
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation was observed
- Proof of pregnancy: [vaginal plug referred to as day 0 of pregnancy
- Further mattings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Pregnant females were housed individually. After delivery, females were housed with their offspring. A label was attached to each cage. It provided information of the study code, the cage number, sex and numbers of the animals, the dose of the test item, the starting and ending dates of the study, day
0 of pregnancy (when appropriate) and the date of delivery (when appropriate)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
It was conducted by BLIRT S.A. Laboratory, Trzy Lipy 3/1.38, 80-172 Gdańsk, Poland, which holds the Good Laboratory Practice Certificate GLP. Solutions of the test item in a vehicle (corn oil) were prepared once a week. Two samples (2 x 2 ml) were collected from each batch of prepared solutions. One of them was frozen and sent in a pack of dry ice to BLIRT S.A. (27 samples in 9 batches) for analysis and the other one (spare sample) was archived in the freezer (-15 - -20°C).

TECHNIQUE AND TEST METHOD: Gas chromatography coupled with flame ionization detector (FID).

TEST PARAMETERS: Chromatographic column: ZB-5MS, 30 m x 0.25 mm x 0.25 μm, catalog no.:7HG-G010-11, serial no.: 221879, Phenomenex.
Temperature gradient: 175ºC (Injection port), 280ºC (detector), 25ºC/min column rate., Column flow:0.69 mL/min, Carrier gas: helium, Injection volume: 1 μL
Split ratio: 33.3 mg/kg b. w. (6.66 mg/mL of VL3 in corn oil) and 100 mg/kg b. w. (20 mg/mL of VL3 in corn oil): 1:50, 300 mg/kg b. w. (60 mg/mL of VL3 in corn oil): 1:100.

ACCEPTANCE CRITERIA: The relative standard deviation, RSD [%], of the VL3 content for 3 results for 33.3 mg/kg b. w. (6.66 mg/mL of VL3 in corn oil) dose is 6.67 %, so it is ≤ 10.0 %. The relative standard deviation, RSD [%], of the VL3 content for 3 results for 100 mg/kg b. w. (20 mg/mL of VL3 in corn oil) dose is 9.91 %, so it is ≤ 10.0 %. The relative standard deviation, RSD [%], of the VL3 content for 3 results for 300 mg/kg b. w. (60 mg/mL of VL3 in corn oil) dose is 0.48 %, so it is ≤ 10.0 %. The acceptance criteria are fulfilled.

QUANTITATIVE AND QUALITATIVE DETERMINATION: All average VL3 content in study samples were at the expected level.
Duration of treatment / exposure:
The test item/vehicle control was administered to males for 28 days. Females were dosed throughout the study. This included 2 weeks prior to mating, the variable time to conception, the duration of pregnancy, and at least 13 days after delivery (51 – 59 days). Males from groups 0 SAT and 3 SAT were treated for 28 days, females from groups 0 SAT and 3 SAT were treated for 53 days. Post treatment, the satellite groups were observed for 14 days.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
33.3 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Group 0, 1, 2, 3: 10 males, 12 females
Satellite groups: 10 males, 10 females
Control animals:
yes, concurrent vehicle
other: other: two satellite groups one receiving vehicle control and a second one receiving the highest dose level 300 mg/kg bw/d
Details on study design:
Two Dose range-finding studies were conducted.
First Dose range-finding study: administered 250, 500 and 1000 mg/kg bw/d (n= 5animals/sex/group) during 7 days. In haematological examinations, statistically significant decrease in the number of leukocytes in males from group 1 and females from group 1 and 3 were stated. Biochemical examinations revealed following statistically significant changes between the treated and the control groups: the decreased total protein concentration in males from group 1, the decreased albumin concentration in males from group 1, the decreased globulin concentration in males from group 1, the decreased calcium concentration in males from group 1, the decreased creatinine concentration in females from group 3.

Based on these results, a second Dose range-finding study was conducted: administered 31.3, 65.5,125 mg/kg b.w./day for 7 days.It was not observed treatment-related changes.
Based on both dose range finding studies, the doses of 33.3, 100 and 300 mg/kg b.w./day were selected for the main study.

- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: control and highest dose group in order to evaluate the reversibility, stability, or delay in the onset of possible harmful effects.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
Not conducted.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day or once a day (on days off)
- Cage side observations checked: General condition of the animals, i.e. the observation of all animals for morbidity and mortality was conducted.

DETAILED CLINICAL OBSERVATIONS: Yes, evaluation of skin, fur, eye and mucosa changes, the respiratory, circulatory, and autonomous and central nervous systems, somatic activity, and behaviour .
- Time schedule: before the experiment and then once a week.

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week during the entire experiment. Body weights of females were measured twice a week before gestation, on days 0, 7, 14, and 20 of gestation, after delivery (day 0), and day 4, 7, 10 and 13 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, food intake by parental males was measured once a week during the pre-mating period. Food intake
by parental females was measured once a week during the pre-mating period, on days 0, 7, 14, and 20 of gestation, on day 0, 4, 7 and 13 post-partum. Food intake by females and males from satellite groups was measured once a week during the entire experiment.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (g/100 g b.w./day)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: the day before the introduction of the animals to the experiment and one day before euthanasia.
- Dose groups that were examined: all adult animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of the experiment, blood was collected for clinical-chemical examinations.
- Anaesthetic used for blood collection: Yes, anesthetized with a mixture of xylazine and ketamine at the doses of 10 mg xylazine/kg b.w. and 100 mg ketamine/kg b.w.
- Animals fasted: Yes, prior to the clinical-chemical examinations, the animals were fasted for about 18 – 19 hours. Blood samples were taken from the heart as part of the procedure for euthanasia of the animals.
- How many animals: 5 adult males and 5 adult females from each group (0, 1, 2, 3, 0 SAT, and 3 SAT). Additionally 5 other males (from all groups) and 7 other females (from group 0, 1, 2, 3) or 5 females (from 0 SAT and 3 SAT group) were subjected to hematological (without bone marrow) biochemical and enzymatic examinations.. The level of thyroid hormone, i.e. total T4 (TT4) in serum was determined in all adult males after termination.
- Parameters examined: numbers of leukocytes, erythrocytes, and thrombocytes, the concentration of hemoglobin, hematocrit, MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin weight), and MCHC (mean corpuscular hemoglobin concentration), number of reticulocytes, circulatory blood and bone marrow microscopic examinations, Prothrombin time (PT) AND Partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of the experiment, blood was collected for clinical-chemical examinations.
- Animals fasted: Yes, prior to the clinical-chemical examinations, the animals were fasted for about
18 – 19 hours.
- How many animals: 5 adult males and 5 adult females from each group (0, 1, 2, 3, 0 SAT, and 3 SAT). Additionally 5 other males (from all groups) and 7 other females (from group 0, 1, 2, 3) or 5 females (from 0 SAT and 3 SAT group) were subjected to hematological (without bone marrow) biochemical and enzymatic examinations.. The level of thyroid hormone, i.e. total T4 (TT4) in serum was determined in all adult males after termination.
- Parameters examined: - total protein, albumin, globulin (albumin subtracted from total protein, total cholesterol, urea nitrogen, creatinine, glucose, bilirubin, bile
acids, sodium, potassium, chlorides and calcium. Also aspartate aminotransferase (AST), alanine a minotransferase (ALT) and alkaline phosphatase (AP).

URINALYSIS: Yes
- Time schedule for collection of urine: On the last day of the experiment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined.: colour, volume (mL), specific gravity, pH, protein (g/L), glucose (mmol/L), ketone bodies (mmol/L), bilirubin (qualitative test), blood (Ery/μL), urobilinogen (μmol/L), leukocytes (leu/μL), urine sediment, squamous and columnar epithelium, leukocytes, erythrocytes, and crystals were determined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Males from group 0, 1, 2 and 3 were examined at the end of the administration period (1-2 days before euthanasia). Females from group 0, 1, 2 and 3 were examined during the last week of lactation (1 day before euthanasia). The behavioural studies on the males and females from satellite groups were performed at the end of the administration period (measurement 1) and then before the end of the additional observation period (measurement 2).
- Dose groups that were examined: 5 adult males and 5 adult females from each group were subjected to the studies.
- Battery of functions tested: observations of animal behaviour in an open field, responses to stimuli, locomotor activity, and forehand hindlimb grip strength.

IMMUNOLOGY: Yes
- Time schedule for examinations:
- How many animals: 5 adult males and 5 adult females
- Dose groups that were examined: from each group (0, 1, 2, 3, 0 SAT, and 3 SAT).
- Parameters examined.: The evaluation of the immune system was based on the results of clinical-chemical examinations and post-mortem examinations (gross and histopathological examinations) of the spleen, thymus, and lymph nodes, as well as the statistical analysis of the absolute and relative weights of the spleen and thymus. The clinical-chemical parameters included blood morphology with a picture of peripheral blood and bone-marrow smears, the concentrations of albumin, total protein, the albumin/globulin ratio, creatinine, urea nitrogen, cholesterol, total bilirubin, and the activity of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase.

OTHER - HORMONE TESTING:
The level of thyroid hormone, i.e. total T4 (TT4) in serum were determined. The Mouse/Rat Thyroxine (T4) ELISA kit produced by Calbiotech were used.
Blood samples were collected from:
- all dams,
- all adult males after termination.
Blood samples from adult males were measured for TT4. There was no statistically significant changes in the hormone level, absolute/relative weight of the thyroid and histopathological changes of the thyroid connected with the test item, therefore further measurements of TT4 (dams) were not made.
Oestrous cyclicity (parental animals):
Females were screened for normal ostreus cycles in a 2-week pre-treatment period. Ostreus cycles were monitored before treatment starts to select for the study females with regular cyclicity. Females that fail to exhibit typical 4-5-day cycles were not included in the study. Vaginal smears were monitored daily during the pre-treatment period and then from the beginning of the treatment period until evidence of mating. Vaginal smears were examined on the day of necropsy to determine the stage of the ostreus cycle and allow correlation with histopathology of female reproductive organs.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
Spermatogonia, spermatocytes and spermatides were evaluated as well as the accuracy of the particular layers of the sex epithelium structure. The lumen of the seminiferous tubules was examined to exclude the presence of the immature forms of spermatogenetic cells or giant cells. In epididymides the presence of sperm content was evaluated.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Day 0: clinical observations, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities,
Day 4: AGD (anogenital distance) of each pup, body weight
Day 13: presence of nipples/areolae in male pups

Body weights of pups in litter were measured on day 0, 4, 7 and 13 post-partum

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Dead pups and the ones euthanized on day 4 and 13 post-partum were carefully examined for gross abnormalities. The examinations involved observations of the external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with all organs and their content. Particular attention in pups was paid to the external reproductive genitals which were examined for signs of altered development.

Additionally the thyroid gland was collected from 1 male and 1 female from each litter in the control and treated groups for histopathological examination, there was an exception, because of insufficient number of live males and females per litter thyroid gland was collected from 2 females (litter from female no.13 of group 0) .

OTHER - HORMONE TESTING:
The level of thyroid hormone, i.e. total T4 (TT4) in serum were determined. The Mouse/Rat Thyroxine (T4) ELISA kit produced by Calbiotech were used.
Blood samples were collected from:
- two pups per litter on day 4 after birth,
- two pups per litter on day 13 after birth,
Blood samples from 13-day pups were measured for TT4. There was no statistically significant changes in the hormone level, absolute/relative weight of the thyroid and histopathological changes of the thyroid connected with the test item, therefore further measurements of TT4 (4 – days pups) were not made.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after at least 28 days of administration including pre-mating and mating periods
- Maternal animals: All surviving animals after about 51- 59 days of administration including pre-mating, mating, gestation, and 13 days of lactation.
For post-mortem examinations all animals were euthanized with a mixture of xylazine and ketamine at the doses of 20 mg xylazine/kg b.w. and 200 mg ketamine/kg b.w.

GROSS NECROPSY
- Gross necropsy consisted of detailed observations of external body surface, all natural apertures, the cranial, thoracic and abdominal cavities with all organs and their contents. During the necropsy in all parental females the numbers of corpora lutea (separately in the left and the right ovary) and implantation sites (separately in the left and the right horn of the uterus) were determined. Special attention was paid to the organs of the reproductive system in all adult animals.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination and weighed, respectively.
Weights of organs of the animals used in the study were measured. Absolute and relative weights of brain, pituitary gland, thymus, thyroid gland, heart, liver, spleen, kidneys and adrenals from all adult rats from each group were recorded. Additionally, absolute and relative weights of testes, epididymides, levator ani, bulbocavernosus muscle complex, Cowper’s glands and glans penis, prostate with seminal vesicles and coagulating glands of all adult males as well as ovaries and uterus with cervix of all adult females were determined. Relative weights of internal organs were calculated on the basis of absolute weights, with reference to body weights of living animals.

The following organs and tissues of animals were collected for histopathological examinations from all adult males and females (groups 0, 1, 2 ,3, 0 SAT and 3 SAT): all gross lesions, brain (cerebrum, cerebellum and pons), spinal cord, eye with optic nerve, pituitary gland, thyroid with parathyroids, stomach, small and large intestines including Peyer’s patches (duodenum, jejunum, ileum, cecum, colon), pancreas, liver, kidneys, ureters, adrenals, spleen, heart, aorta, thymus, trachea, lungs (preserved by inflation with fixative and then immersion), testes, ovaries with oviducts, uterus with cervix, epididymides, accessory sex organs (prostate, seminal vesicles and coagulating glands), vagina, skin, mammary gland, urinary bladder, lymph nodes, skeletal muscle with tibial nerve and femur with knee joint. Organs with gross lesions i.e. lesion in liver (7mm long, yellowish, grainy, on the visceral side of the left lobe) of the female no. 46, group 0 SAT; lesion in subcutaneous tissue of cervical area above the salivary gland (yellowish, elastic, in the size of 5 x 2 mm) of the male no. 31, group 3 SAT; lesion in fat tissue in left ovary area (yellowish, 5 mm long) of the female no. 41, group 3 SAT, lesion located in fat tissue in pelvis cavity inlet area (yellowish, 5 mm long) of the female no. 46, group 3 SAT were also collected for evaluation.
As for the gross lesions, thyroid, testicles, epididymides and prostate with accessory sex organs i.e. vesicular and coagulating glands as well as the ovaries with oviducts, they were examined in adult animals of all experimental groups (0, 0 SAT, 1, 2, 3, 3 SAT).
As for the organs and tissues of the reproductive system, despite of routinely proceeded evaluation, detailed histopathological evaluation of the testicles and ovaries for the prospective gametogenesis disorders were carried out. In the testicles spermatogonia, spermatocytes and spermatides were evaluated as well as the accuracy of the particular layers of the sex epithelium structure. The lumen of the seminiferous tubules was examined to exclude the presence of the immature forms of spermatogenetic cells or giant cells. In epididymides the presence of sperm content was evaluated. In the ovaries there were ovarian follicles at various stages of maturation and corpora lutea organisation evaluated. During the histopathological examination special attention was paid to the tissues and organs which could have been the indicators of endocrine disruption influenced by the test item (thyroid, pituitary, adrenals, mammary gland and organs of the reproductive system of tested animals).
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
Dead pups and the ones euthanized on day 4 and 13 post-partum were carefully examined for gross abnormalities.

GROSS NECROPSY
- Gross necropsy consisted of the examinations involved observations of the external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with all organs and their content. Particular attention in pups was paid to the external reproductive genitals which were examined for signs of altered development.


HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid gland was collected from 1 male and 1 female from each litter in the control and treated groups for histopathological examination, there was an exception, because of insufficient number of live males and females per litter thyroid gland was collected from 2 females (litter from female no.13 of group 0)
Statistics:
The treated groups, i.e. groups 1, 2, and 3 were compared to the control group. The treated satellite group, i.e. 3 SAT was compared to group 0 SAT. Each sex was analyzed separately.
The clinical results were statistically analyzed using the one-way analysis of variance and the Dunnet’s test (group 0, 1, 2 and 3) and Student’s t-test (group 0 SAT, 3 SAT), (p ≤ 0.05).

Clinical-chemical examinations results: The results obtained in the groups 0, 1, 2 and 3 were statistically analysed using the one-way analysis of variance or Kruskal-Wallis test (in the absence of normal distribution or heterogeneous variances) and then Dunnet’s test (p ≤ 0.05). The results of the clinical-chemical examinations in satellite group were statistically analyzed using Student’s t test (p ≤ 0.05) or U Mann-Whitney test (in the absence of normal distribution).
Some results of the urinalysis such as bilirubin and urine sediment were statistically analyzed using nonparametric statistics test - Kruskal-Wallis and then Dunnet’s test (p ≤ 0.05) (group 0, 1, 2, 3) and U Mann-Whitney test (group 0 SAT, 3 SAT), (p ≤ 0.05).

Post-mortem examinations results
The results, i.e. absolute and relative weights of internal organs as well as the numbers of implantation sites and corpora lutea were statistically evaluated using Dunnett’s test for groups 0, 1, 2, 3 and student’s T-test for satellite groups 0 SAT and 3 SAT (at p≤0.05).

Statistical analyses were conducted using the Microsoft Excel 2010 and the Statistica 10 programmes.
Reproductive indices:
Mating index for males, mating index for females, fertility index for males, fertility index for females, pregnancy index, the average number of days required for copulation, the length of gestation.
Offspring viability indices:
Index of live births, index of 4-day survival, index for 13-day survival,
Clinical signs:
no effects observed
Description (incidence and severity):
There were no differences in physical appearance and behaviour between the treated and the control groups. The animals didn’t show any clinical signs during the entire experiment. The only exception was the thinning of hair all over the body in one female from group 1 but this change is not considered to be treatment-related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
During the experiment, no mortality of adults animal was observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in body weights between parental animals from treated and control groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no statistically significant differences in food intake by males and also by females from the treated (1, 2, 3, 3 SAT) and the control groups (0, 0 SAT).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmic examinations did not reveal any pathological changes.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological examinations: In case of peripheral blood examinations of animals from groups 1, 2 and 3, statistically significant changes in females of group 1 were stated (statistically significant: decrease in the hemoglobin concentration, decrease in the hematocrit value and decrease in the number of erythrocytes). Mentioned above changes were considered as accidental due to no confirmation in higher dosage i.e. group 2 and 3. There were no changes in males of group 1, 2, and 3. In case of peripheral blood examinations of males and females from group 3 SAT, statistically significant increase in the number of thrombocytes was stated compared to the control group (0 SAT). However, number of thrombocytes in group 3 SAT compared to the group 0 was at a similar level, so this change should not be connected with the test item administration. Statistically significant increase in the MCHC value in females of group 3 SAT was considered as accidental because this change was isolated, and no other deviations in red blood cells parameters were stated. In bone marrow examinnations, statistically significant decrease in the number of proerythroblasts was stated in males of group 3 SAT. This change was regarded as random because there were no changes on the next stages of erythroblast development. Statistically significant decrease in the number of filamented eosinophils (bone marrow examination) in males of group 3SAT was also considered as accidental.

Coaglulological examinations: The treatment with the test item at three different dose levels did not affected coagulological parameters.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Biochemical examinations: In case of biochemical parameters, statistically significant increase in the chloride concentration in males of group 1, 2 and 3 were observed. Increase in chloride concentration was small, by about 2% compared to the control group and did not increase with increasing dose. For this reason statistically significant change which was observed may be considered as not treatmentrelated. These change was not observed in the satellite group, after 14-days post-treatment. In males of group 3 stattistically significant increase in the calcium concentration was stated, it can be also considered as random due to low intensity. Statistically significant increase in the creatinine concentration was stated in females of group 3. This change was considered as accidental because the histopathological examination of the kidneys revealed no changes considered as treatment related. Statistically significant increase in the total protein concentration and decrease in the glucose concentration was stated in males of group 3 SAT compared to the control group (0 SAT) and those changes could be random. In case of females of group 3 SAT, statistically significant decrease in the urea nitrogen concentration and increase in the sodium concentration were stated and those changes were considered as accidental due to change in sodium was small (increase by about 0.9% compared to the group 0 SAT) and change in urea nitrogen is in the range of variability of the control group.

Enzymatic examinations: All changes observed in the males and females of group 1 and 2 (statistically significant decrease in the AP activity in males of group 1 and 2 and statistically significant decrease in the AST activity in females of group 2) were considered as random due to no confirmation in the higher dosage group. Statistically significant decrease in AST activity in males of group 3 SAT was consider as accidental, because in case of toxic impact on the liver function, an increase in activity of AST is observed.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decrease in the protein level was stated in females of group 1, 2 and 3 but this is not an adverse change so it will not be taken into account. Statistically significant increase in the level of urine protein in males of group 3SAT was stated and this change was considered as accidental.
Statistically significant decrease in the level of urine bacteria in males of group 3SAT is not an adverse change so it will not be taken into account.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The behavioural studies showed that the nervous system functioned properly at dose levels of 33.3, 100, and 300 mg/kg b.w./day.
The detailed clinical observations made during and after the treatment, and the open field observations did not reveal any muscarinic symptoms (weakness, lacrimation, salivation, and anxiety), nicotinic symptoms (diarrhea and motor coordination difficulties), or central symptoms caused by the test item. Delayed neurotoxicity, i.e. of paralysis of the hindlimbs, clumsiness, sensory disturbances, and general paralysis were not observed. During the open field observations, a few cases of increased and decreased arousal were found. Changes in arousal was observed both in treated and control groups so it should not be connected with the administered material.
A statistically significant increase of horizontal locomotor activity was noticed in males and females from group 2 compared to the control group. This change may be considered as accidental because there was not appear in other treated groups. There were statistically significant higher average of the number of urine pools left by males from group 3 SAT compared to control group (0 SAT) at the end of the 14-day period of additional observation (measurement 2). However, no similar changes were found in males from other treated groups and also in males from group 3 SAT at the end of the treatment (measurement 1), so it can be assumed as fortuitous change and not related to the administration of the test item. It was also discovered that the test item did not affect responses to stimuli. The latency of pain responses in females in the satellite group 3 SAT at the end of the treatment (measurement 1) was significantly shorter compared to the group 0 SAT. However, similar changes were not observed in females in group 3 (measurements of females from group 3 and 3 SAT was conducted at the same stage of the experiment - at the end of the treatment). Hence, it can be concluded that this change was accidental.

The administration of the test item did not influence fore- and hindlimb grip strength in the treated groups. Only exception was statistically significant increase of forelimb grip strength observed in females from group 3 SAT at the end of the additional observation period (measurement 2) compared to the group 0
SAT. It can be assumed that this is an accidental change.
Immunological findings:
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
On the basis of the results of the histopathological examination of organs and tissues it may be concluded that all of the revealed lesions were not associated with the test item.
Histopathological, there were observed cases of congestion (thymus, heart, lungs, liver, spleen, mandibular and mesenteric lymph nodes, glomeruli of kidneys, islets of pancreas, prostate, uterus, ovaries), erythrocytorrhagia (heart, lungs, thymus, kidneys, mandibular lymph nodes), edema (lungs), effusion (thymus, liver, mandibular lymph nodes, uterus), inflammatory lesions such as lymphocyte infiltration (lungs, colon, kidneys, prostate, urinary bladder), foamy cells infiltrations (lungs), leukocyte infiltrations (heart), lymphatic follicle reactive hyperplasia (cecum, colon), regressive lesions such as lymphocytic tissue depletion (thymus), emphysema (lungs), foci of fibrosis (heart), foci of atrophy of lobules with fibrosis (pancreas), left gland atrophy with medulla microcyst (adrenals), glomeruli atrophy, Bowman’s capsule extension and retention microcysts (kidneys), hemosiderin deposits in spleen and progressive lesions such as mesenteric lymph nodes hyperplasia, left ventricular myocardium hypertrophy (heart), islets of Langerhans hypertrophy and ducts proliferation (pancreas), glomeruli hypertrophy (kidneys).
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were also observed individual cases of small benign tumors such as lipomas of the adipose tissue and fibroids of the uterus. In the subcutaneous tissue of region of cervical area above the salivary gland (male in the group 3 SAT) and region of the abdominopelvic cavity (two females in the group 3 SAT) the small lipomas were occurred. As for the uterus (two females in the group 1 and one female in the group 2) the fibroids were observed. The occurrence of them was clearly incidental to the treatment and should not be linked with the test item.
Both, the lipomas and fibroids were considered as spontaneous lesions, consistent with background findings commonly observed in rats. The occurrence of the left adrenal gland atrophy with medulla microcyst and right adrenal gland hyperplasia in one of the females in the group 3 is also accidental. It seems to be a kind of a malformation.
Other effects:
no effects observed
Description (incidence and severity):
Hormonal examinations: Statistically significant changes in the TT4 level, absolute/relative weight of the thyroid and histopathological changes of the thyroid related with the test item in all males were not observed, so the determination of TT4 levels were not extended to females.
There were found no adverse effects of the test item on an endocrine system. There were no significant histopathological lesions in the thyroid of adults in all experimental group of animals.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The detailed histopathological examination of the ovaries has proven that the test item did not affect the gametogenesis process in animals exposed at all dose levels. No lesions or disorders in the gonads (ovaries) were observed. One of the females from the group 2 was non-pregnant (no corpora lutea in ovaries and no implantation sites in uterus were found)
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The detailed histopathological examination of testicles and epididymides has proven that the test item did not affect the gametogenesis process in animals exposed at all dose levels. No lesions or disorders in the gonads (ovaries) were observed. As for the testicles particular layers of the sex epithelium were correct. In the epididymides a sperm reserve content was preserved. There were also no pathological lesions in the seminal vesicles and coagulating glands. Cases of lymphocyte infiltration in the prostate in males exposed at all dose levels and the control were observed. These changes can be considered as the background lesions, not associated with the test item.
Reproductive performance:
no effects observed
Description (incidence and severity):
The indices relating to fertility of the parental animals, including mating indices for males and females, fertility indices for males and females, and pregnancy indices in the treated and the control groups were similar, which suggests that the test item at the dose levels of 33.3, 100, and 300 mg/kg b.w./day did not affect fertility of the parental rats.
However, one female (no. 11 from group 2) was not pregnant, although sperm was found. Histopatological examination revealed no corpora lutea in ovaries and no implantation sites. Pathomorphological (gross and histopathological examinations) results of the male which mounted the non-pregnant female were correct, therefore it is difficult to identify the reason for the female not being pregnant. Two females in control group were also not pregnant (although sperm was found), so the lack of pregnancy in one treated female from group 2 can not be associated with the test item.
The test item had no impact on numbers of corpora lutea in the ovaries and implantation sites in the uterus.
As for the adrenals, pituitary, mammary gland, gonads (ovaries and testes), accessory sex organs (uterus with the cervix, vagina, epididymides, seminal vesicles with coagulating glands, and prostate) there were also found no adverse effects of the test item.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item adverse effects observed at the highest dose tested.
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Hematoma on the snout in one pup from group 0 and hematoma on the head in one pup from group 2 were transient. In a few pups thinning of hair all over the body, thinning of hair on the back and smaller body were also noticed. These changes occurred both in treated and control groups. Hence, they should not be associated with the administered test item. Hematomas in pups after birth were also observed in pups, including control ones in other combined repeated dose toxicity studies with the reproduction/developmental toxicity screening test performed at the same laboratory [OECD 422; unpublished reports].
The analysis of the AGD and number of nipples/areolae showed that the test item did not cause any significant changes in development of pups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
There were 541 pups in the study, whereof: 34 pups were born dead, 29 animals were eaten by mother, 4 pups was found dead at the first day of life. The remaining animals survived the 13-day observation period. The test item did not influence the total numbers of pups/litters, the numbers of live births and the percentages of males and females in litters in group 1, 2 and 3. number of stillbirths in group 1 (2.8% of total number of pups in group), group 2 (11.8% of total number of pups in group) and group 3 (8.8% of total number of pups in group) were higher compared to the control group (0.9% of total number of pups in group). Moreover, mortality (number of death pups during the study period) was also higher in treated groups compared to the control group. In group 1 it was 2.8%, in group 2 - 15.0% and in group 3 - 5.6% of total number of pups in group, while in control group it was 0.9% of total number of pups in group. Based on this results, it may be concluded that the test item in group 2 and 3 affected pups survival.
The analysis of the indices relating to pups survival from the treated parental animals (indices of live births, 4-day survival and 13-day survival of offspring) also confirmed a negative influence of the test item in group 2 and 3.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight determinations: It may be concluded that the test item at the dose levels of 33.3, 100 and 300 mg/kg b.w./day (males and females) had no adverse effects.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food intake measurements: It may be concluded that the test item at the dose levels of 33.3, 100 and 300 mg/kg b.w./day (males and females) had no adverse effects.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Statistically significant changes in the TT4 level, absolute/relative weight of the thyroid and histopathological changes of the thyroid related with the test item in 13 days pups were not observed, so the determination of TT4 levels were not extended to 4-days pups.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
no effects observed
Description (incidence and severity):
Histopathological examination of the thyroid of the 13-day pups revealed no lesions.
Other effects:
no effects observed
Description (incidence and severity):
Hormonal examinations: Statistically significant changes in the TT4 level, absolute/relative weight of the thyroid and histopathological changes of the thyroid related with the test item in 13 days pups were not observed, so the determination of TT4 levels were not extended to 4-days pups. Histopathological examination of the thyroid of the 13-day pups revealed no lesions.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 33.3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
ca. 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Key result
Critical effects observed:
not specified
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Table 5: Main study. Clinical signs - adult animals

Clinical signs

Group / sex / number of animals / computer numbers of animals

0

1

2

3

males

females

males

females

males

females

males

females

Test animals

10

12

10

12

10

12

10

12

Animals with clinical signs

0

0

0

1

0

0

0

0

Animals with ophthalmic

changes

0

0

0

0

0

0

0

0

Death of animals

-

-

-

-

-

-

-

-

SKIN AND COAT

Thinning of hair all over the body

-

-

-

1 [16]

-

-

-

-

[ ] computer numbers of animals

Table 6- VL3. Main study. Clinical signs - offsring

Clinical signs

Group / sex / number of animals / computer numbers of animals

0

1

2

3

males

females

males

females

males

females

males

females

Number of live births pups

52

57

64

76

56

56

88

58

Number of pups with clinical

signs

9

10

0

0

5

7

1

0

Hematoma on the snout

1 [13]1

-

-

-

-

-

-

-

Hematoma on the head

-

-

-

-

1 [20]1

-

-

-

Thinning of hair all over the

body

2 [15]

9 [15]

-

-

4 [15]

4 [15]

-

-

Thinning of hair on the back

6 [21]

1 [21]

-

-

-

-

-

-

Smaller body

-

-

-

-

-

1 [15]

1 [20]

1 [21]1

1 [21]

 

-

Stillbirth

-

1 [13]

-

1 [11]

3 [20]

1 [12]

3 [17]

2 [19]

1 [17]

4 [19]

1 [20]

4 [22]

1 [13]

1 [19]

4 [22]

Death of animal

1 [13]2

-

2 [20]3

2 [20]3

4 [16]3

3 [17]3

1 [18]3

1 [19]3

1 [16]2

2 [16]3

6 [17]3

1 [18]3

1 [12]3

2 [18]3

1 [19]3

1 [22]3

1 [14]3

2 [22]2

1 [22]3

Undefined sex stillbirth*

-

-

3 [19]

1[13]

3[22]

[ ] – computer numbers of mothers of animals in which clinical signs were observed

1 – temporary change 2 – animal died at the first day of life 3 – pups eaten by mother * - pups sex could not be identified because pup were bitten by mother.

Table 7. VL3. Main study. Open field observations – females

Parameter

Group

0

n = 5

1

n = 5

2

n = 5

3

n = 5

involuntary clonic

movement

no changes

no changes

no changes

no changes

nvoluntary tonic

movement

no changes

no changes

no changes

no changes

gait

no changes

no changes

no changes

no changes

arousal

no changes

no changes

[12]4, [15]4, [16]4

[11]1, [12]4, [14]4

stereotypies

no changes

no changes

no changes

no changes

number of defecations

2.80±2.17

1.80±2.49

2.20±2.05

1.00±1.73

number of

urinations

2.60±1.52

4.20±0.84

4.00±1.73

2.20±1.64

distance 0-3 min [m]

109.44±18.48

106.48±12.37

162.06±40.87*

125.40±39.00

number of rearings

0-3 min

13.60±4.22

12.80±2.77

18.60±8.20

19.20±7.09

x± SD

nnumber of test animals

*-statistically significant difference at p ≤ 0.05-Dunnett’s test

[ ] - computer numbers of animals with changes

Classification of changes:

1 - slight decrease (a rat sometimes does not move, but exploratory activity is observed)

2 - moderate decrease (a rat is slightly numb; only the head moves)

3 - strong decrease (paralysis, coma)

4 - slight increase (a rat is slightly excited or tense; it suddenly moves forward and stays without motion)

5 - strong increase (a rat is very vigilant; it runs and suddenly moves from its place

Table 8. VL3. Main study. Mating of parental animals – list of results

Parameter

Group

0

1

2

3

Number of males to

mating

10

10

10

10

Number of females to

mating

12

12

12

12

Number of males which

copulated

10

10

10

10

Number of females

which were mated

12

12

12

12

Average number of

days to copulation

1.0

1.2

1.0

1.1

Number of fertilized

females

12

12

12

12

Number of pregnant

females

10

12

11

12

Number of females

which delivered

10

12

11

12

Number of not

pregnant females

2

0

1

0

Number of females

which delivered

offspring

10

12

11

12

Number of females

which delivered live

offspring

10

12

11

12

Table 9. VL3. Main study. Indices concerning fertility of parental animals

Parameter

Group

0

1

2

3

Mating index for males

100.0

100.0

100.0

100.0

Mating index for females

100.0

100.0

100.0

100.0

Fertility index for males

90.0

100.0

100.0

100.0

Fertility index for females

83.3

100.0

91.7

100.0

Pregnancy index

100.0

100.0

100.0

100.0

Length of gestation

22.5 ± 0.5

22.5 ± 0.5

22.5 ± 0.7

22.5 ± 0.7

Table 10. VL3. Main study. Average number of pups in litter

Parameter

Group

0

1

2

3

Total number of pups

in litter

11.00 ± 2.75

12.00 ± 3.05

11.55 ± 2.50

13.33 ± 2.90

Number of live births

in litter

10.90 ± 3.03

11.67 ± 3.03

10.18 ± 3.22

12.17 ± 4.22

Number of pups in

litter, min.max.

4 - 13

4 - 17

7 - 15

7 - 16

Percentage of males

in litter

47.27

44.44

50.00

58.97

Percentage of

females in litter

52.73

55.56

50.00

41.03

Table 11. VL3. Main study. Indices connected with survival of offspring

Parameter

Group

0

1

2

3

Index of live

births

99.09

97.22

88.19

91.25

Index of 4-day

Survival

99.08

97.14

83.04

93.84

Index of 13-day

survival

98.90

96.61

80.21

92.74

Mortality [%]

0.9

2.8

15.0

5.6

Table 12. VL3. Main study. Average AGD [mm] in male pups in litter on 4th day of life 

Day 4

Group

0

n= 10

1

n = 12

2

 n = 11

3

n = 12

Male

5.93±0.38

5.90±0.30

5.78±0.29

5.83±0.51

Female

3.92±0.32

3.90±0.30

3.76±0.93

3.74±0.22

x± SD

n - number of litters in group

 

Table 13. VL3. Main study. Average number of nipples / areolae in male pups in litter on 13th day of

Life

Day

Group

0

n= 10

1

n = 12

2

 n = 11

3

n = 12

13

0.0±0.0

0.0±0.0

0.0±0.0

0.0±0.0

Table 14. VL3. Main study. Results of hormonal examination - pups 13 day after birth

Parameter

MALES

GROUP

0

1

 

 

2

3

n=9

n=12

 

 

n=9

n=11

TT4

5.31 ± 0.73

5.54 ± 0.91

5.36 ±0.79

5.200.93

 

FEMALES

GROUP

0

1

 

2

3

n=11

n=12

 

n=9

n=10

TT4

5.40 ±0.90

5.74 ± 0.87

5.37 ±0.72

5.030.51

Table15. VL3. Main study. Offspring subjected to pathomorphological examination.

Group

Sex / number of animals / computer numbers of parental females

Males

Females

Undefined sex

Number of animals per group

Dead

animals

Stillbirth

Euthanized animals at day 4

Euthanized animals at day 13

Number of animals per group

Dead

animals

Stillbirth

Euthanized animals at day 4

Euthanized animals at day 13

Number

of

animals

Dead

animals

Stillbirth

0

52

1

 

8

43

58

 

1

10

47

 

 

 

 

 

[13]

 

 

 

 

 

[13]

 

 

 

 

 

 

 

 

 

 

 

 

 

1[11]

 

 

 

 

 

1

62

-

-

11

51

78

-

3[20]

11

63

-

-

-

 

 

 

1[12]

 

 

 

 

1[17]

 

 

 

 

 

2

53

-

3[17]

8

39

53

1[16]

4[19]

8

38

3

-

3[19]

 

 

 

2[19]

 

 

 

 

1[20]

 

 

 

 

 

 

 

 

 

 

 

 

 

1[13]

 

 

 

 

 

3

87

-

4[22]

10

73

62

2[22]

1[19]

12

42

4

-

1[13]

 

 

 

 

 

 

 

 

4[22]

 

 

 

 

3[22]

[]-computer numbers of parental females

 

Table 16. VL3. Main study. Gross lesions: euthanized, dead and stillborn pups.

Gross lesions

Group / number of animals / computer numbers of parental females

0

1

2

3

Animals euthanized at day 4

18

22

16

22

Animals with gross lesions

-

-

-

-

Animals euthanized at day 13

90

114

77

115

Animals with gross lesions

-

-

-

-

Dead animals

1

-

1

2

Partial autolysis of internal organs

-

-

-

2 [22]

Autolysis of internal organs

-

-

1[16]

-

Partially eaten by mother

1[13]

-

-

-

Stillbirth

1

4

15

14

Partial autolysis of internal organs

1[13]

3[20]

4[17]

8[22]

Autolysis of internal organs

-

-

6[19]

1[13]

Partially eaten by mother

-

-

3[19]

1[13]

Partial autolysis of internal organs, partially eaten by mother

-

-

-

3[22]

No lesions

-

1[11]

1[12], 1[20]

1[19]

 

Table 17. VL3. Main study. Number of corpora lutea and implantation sites

 

Group

Number of

0

1

2

3

 

n=10*

n=12

n=11*

n=12

Corpa lutea

15.200          2.530

16.500          2.541

20.000          7.266

17.334                  3.576

Implantation sites

13.200          2.936

13.417          2.644

14.273                 1.348

14.667                  1.723

xSD

n - number of tested animal

*number of tested animals excluding non-pregnant female

Table 18. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0f

1m

2m

3f

organ / tissue

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Pituitary

microcyst

2

[1,4]

-

-

-

-

-

-

Thyroid

congestion

-

-

-

-

-

-

1

[9]

parathyroid

hypertrophy

-

1

P2]

-

-

-

-

-

 

congestion

1

[2]

-

-

1

[18]

-

-

2

[3,9]

Thymus

erythrocytorrhagia

-

-

-

-

-

-

1

[3]

effusion

-

1

[22]

-

1

[17]

 

 

-

 

lymphocytic tissue depletion

-

1

[17]

-

-

-

1

[13]

-

 

erythrocytorrhagia

3

[1,2,10]

-

-

1

[18]

-

-

1

[8]

 

leukocyte infiltration

1

[10]

-

-

 

-

-

1

[10]

 

Heart

foci of fibrosis

1

[10]

-

-

-

-

-

-

 

foci of fatty degeneration

1

[10]

-

-

 

-

-

-

 

left ventricular myocardium hypertrophy

-

-

-

1

[18]

-

1

[18]

-

 

Table 18 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0f

1m

2m

3f

organ / tissue

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Heart

left and right ventricular myocardium hypertrophy

1

[10]

-

-

-

-

-

-

 

congestion

1

[6]

-

3

[3,6,9]

2

[19,20]

1

[2]

2

[14,22]

2

[1,5]

2

[11,13]

 

erythrocytorrhagia

5

P6,8,9,10]

9

[12,13,14,

15,16,18,

20,21,22]

4

[3,6,7,9]

10

[11,14,15,

16,17,18,

19,20,21,

22]

3

P,3,4]

9

[13,14,15,

16,17,19,

20,21,22]

6

[1,2,4,5,7,

10]

8

[11,12,13,

14,15,17,

18,22]

 

edema

3

P6,10]

4

[13,14,18,

21]

3

[3,6,9]

2

[11,17]

-

2

[17,22]

2

[5,10]

1

[18]

Lungs

emphysema

4

[2,4,8,10]

9

[11,13,14,

15,17,19,

20,21,22]

-

10

[11,12,14,

15,16,18,

19,20,21,

22]

1

[2]

3

[13,14,21]

5

[1,4,5,6,10]

3

[11,18,22]

 

foamy cell infiltration

2

[7,8]

7

[11,15,17,

18,20,21,

22]

-

3

[11,16,17]

-

4

[13,19,20,

21]

1

[6]

1

[18]

 

lymphocyte

infiltration

1

[8]

-

-

-

-

-

1

[10]

 

leukocyte infiltration

1

[10]

-

-

-

-

-

-

 

Table 18 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0f

1m

2m

3f

organ / tissue

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

 

congestion

1

[10]

2

[15,17]

-

-

-

-

1

[9]

 

effusion

1

[10]

-

-

-

-

-

-

Liver

bile ducts proliferation

1

[10]

-

-

-

-

-

-

 

Browicz-Kupffer cell proliferation

1

[10]

-

-

-

-

-

-

 

hepatocyte fine- droplet fatty degeneration

1

[3]

-

-

-

-

-

-

Spleen

congestion

-

1

[18]

-

-

-

-

-

depletion of the white pulp

-

2

[17,18]

-

-

-

-

-

Pancreas

islets of Langerhans hypertrophy

1

1

P1]

-

-

-

-

3

[4,6,8]

ducts proliferation

-

1

P1]

-

-

-

-

-

Jejunum

lymphatic follicle reactive hyperplasia

1

[5]

-

-

-

-

-

-

Ileum

lymphocyte

infiltration

-

1

[11]

-

-

-

-

-

 

Table 18 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0f

1m

2m

3f

organ / tissue

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Cecum

lymphatic follicle reactive hyperplasia

1

[7]

-

-

-

-

-

-

1

[14]

Colon

lymphatic follicle reactive hyperplasia

1

[4]

-

-

-

-

-

2

P,4]

1

[14]

 

erythrocytorrhagia

1

[8]

1

[15]

-

-

-

-

1

[2]

 

glomeruli congestion

6

[1,2,3,5a

7]

4

[12,17,20,

22]

-

-

-

-

10

[1,2,3,4,5,6,7

8,9,10]

6

[13,14,15,

17,20,22]

 

lymphocyte

infiltration

-

2

[12,21]

-

-

-

-

1

[8]

Kidneys

glomeruli

hypertrophy

9

[1,2,3,5,6,7,8

,9,10]

7

[12,15,17,

19,20,21,

22]

-

-

-

-

9

[1,3,4,5,6,7,8

9,10]

9

[12,13,14,

15,17,19,

20,21,22]

glomeruli

atrophy

4

[1,8,9,10]

5

[15,17,18,

20,21]

-

-

-

-

10

[1,2,3,4,5,6,7

8,9,10]

8

[12,13,14,

15,19,20,

21,22]

 

Bowman's capsule extension

1

[9]

2

[18,21]

-

-

-

-

6

[2,3,5,6,8,9]

 

retention microcysts

-

2

[12,15]

-

-

-

-

1

[8]

2

[14,21]

 

hyaline casts

-

1

[12]

-

-

-

-

-

 

 Table 18 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

Examined organ / tissue

Type of change

GROUP / sex / number of animals / computer numbers of animals

0f

1m

2m

3f

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Adrenals

left adrenal gland atrophy with medulla microcyst; right adrenal gland hyperplasia

-

-

-

-

-

-

-

1

[11]

Urinary bladder

lymphocyte

infiltration

-

-

-

-

-

-

5

[1,3,5,6,7]

Prostate

congestion

2

[1,9]

 

-

-

-

-

2

[2,8]

 

edema

2

[1,9]

-

-

-

-

-

-

lymphocyte

infiltration

5

[1,3,7,9,10]

-

3

P,4,9]

-

6

[2,4,5,7,8,9]

-

6

[2,3,5,8,9,

10]

Uterus

congestion

-

-

-

1

[16]

-

2

[16,20]

-

effusion

-

-

-

2

[12,16]

-

2

[20,21]

-

fibroid

-

-

-

2

[12,16]

-

1

[20]

-

Mandibular lymph nodes

congestion

1

[6]

-

-

-

-

-

-

hyperplasia

1

[6]

-

-

-

-

-

-

lymphocytic tissue depletion

-

1

[17]

-

-

-

-

-

 

Table 18 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

Examined organ / tissue

Type of change

GROUP / sex / number of animals / computer numbers of animals

0f

1m

2m

3f

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Mesenteric lymph nodes

congestion

-

-

-

-

-

-

1

[6]

hyperplasia

1

[6]

-

-

-

-

-

1

[6]

lymphocytic tissue depletion

-

1

[17]

-

-

-

-

-

[]-computer numbers of animals in which the histopathological lesions were found n - number of tested animals f - full histopathology

m - histopathology of thyroid, testicles, ovaries with oviducts, epididymides, prostate, seminal vesicles, coagulating glands and gross lesions were conducted

Table 19. VL3. Main study. Histopathological lesions: thyroid of 13-day offspring.

Histopathological examinations of thyroid

Group / sex / number of animals / computer numbers of parental females

0

1

2

3

males

females

males

females

males

females

males

females

13-day offspring

9

11

12

12

9

9

11

11

Animals with histopathological lesions

-

-

-

-

-

-

-

-

 

Table 20. VL3. List of effects on reproduction/development

OBSERVATIONS

VALUES

Dosage (mg/kg b.w./day)

0

33.3

100

300

Pairs started (N)

12

12

12

12

Ostreus cycle (at least mean length and frequency of irregular cycles)

4.1

0/12

(0%)

4.0

0/12

(0%)

4.0

0/12

(0%)

4.0

0/12

(0%)

Females showing evidence of copulation (N)

12

12

12

12

Females achieving pregnancy (N)

10

12

11

12

Conceiving days 1-5 (N)

10

12

11

12

Conceiving days 6 -N)

0

0

0

0

Pregnancy < 21 days (N)

0

0

0

0

Pregnancy = 22 days (N)

5

6

6

7

Pregnancy > 23 days (N)

5

6

5

5

Dams with live young born (N)

9 + 1*

10 + 2*

7 + 4*

9 + 3*

Dams with live young at day 4 pp (N)

9 + 1**

11+1**

9 + 2**

7 + 5**

Implants/dam (mean)

13.2

13.4

14.3

14.7

Live pups/dam at birth (mean)

10.9

11.7

10.2

12.2

Live pups/dam at day 4 (mean)

10.8

11.3

8.5

11.4

Sex ratio (m/f) at birth (mean)

1.1

0.9

1.1

1.5

Sex ratio (m/f) at day 4 (mean)

1.1

1.1

1.0

1.5

Litter weight at birth (mean) [g]

66.27

70.63

60.65

69.90

Litter weight at day 4 (mean) [g]

116.02

118.93

102.61

127.49

Pup weight at birth (mean) [g]

6.14

6.15

5.96

5.70

Pup weight at the time of AGD measurement (mean males, mean females, PND 4)

(M) 10.93

(F) 10.54

(M) 10.86

(F) 10.65

(M) 10.28

(F) 9.86

(M) 10.81

(F) 10.35

Pup AGD on the same postnatal day, birth­day 4 (mean males, mean females, PND

4)

(M) 5.93

(F) 3.92

(M) 5.90

(F) 3.90

(M) 5.78

(F) 3.76

(M) 5.83

(F) 3.74

Pup weight at day 4 (mean) [g]

10.74

10.75

10.07

10.58

Pup weight at day 13 (mean) [g]

28.51

28.61

28.57

28.20

Male pup nipple retention at day 13 (mean)

0.00

0.00

0.00

0.00

ABNORMAL PUPS

Dams with 0

7

12

7

11

Dams with 1

1

0

1

1

Dams with >2

2

0

3

0

LOSS OF OFFSPRING

 

Pre-natal (implantations minus births)

 

Females with 0

2

2

1

5

Females with 1

3

3

2

2

Females with 2

0

4

3

0

Females with > 3

5

3

5

5

Post-natal (live births minus alive at postnatal day13)***

 

 

 

 

Females with 0

9

11

7

7

Females with 1

1

0

1

3

Females with 2

0

0

1

1

Females with > 3

0

1

2

1

(1) last day of the mating period * number of dams with at least 1 dead young born ** number of dams with at least 1 dead young to day 4

*** pups taken at day 4 after birth to blood examination, were not taken into account

(M) male (F) female

Conclusions:
Based on number of stillbirths and mortality in group 2 and 3, it may be concluded that the test item affected pups survival. Therefore, on the grounds of the used doses, the no observed adverse effect level (NOAEL) for reproduction/developmental toxicity for pups can be determined on 33.3 mg/kg b.w./day.
Executive summary:

A Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted in rats according to the OECD Guideline No. 422 (2016) with GLPs. The study involved the determination of the effects of the test item on body weights, food intake, behaviour, clinical, haematological, coagulation, biochemical, enzymatic, urine, and hormonal parameters and the evaluation of gross and histopathological lesions in tissues and internal organs. Two dose range finding tests and the main study were conducted. The first Dose range finding stated that 7-day exposure of rats to VL3 at the doses of 250, 500 and 1000 mg/kg b.w./ day cause one treatment-related change, i.e. decrease in number of leukocytes (males from group 1 and females from group 1 and 3).The second Dose-range finding test revealed that 7-day exposure of rats to VL3 at the doses of 31.3, 62.5 and 125.0 mg/kg b.w./day did not cause treatment-related changes. The main study was performed on 60 males and 68 females of Wistar rats treated with vehicle control or with 33.3, 100, 300 mg/kg b.w./day of test item. The animals received the test item/vehicle control by gavage once a day (7 days/week). After two weeks of initial treatment, the animals were mated. The test item/vehicle control was administered to males for 28 days (2 weeks prior to mating, during the mating and post-mating period). Females were dosed throughout the study. This included 2 weeks prior to mating, the variable time to conception, the duration of pregnancy, and at least 13 days after delivery (about 51-59 days, depending on the duration of the mating and pregnancy period). Furthermore, two satellite groups were used;: a control group and one treated group which was given VL3 at the dose of 300 mg/kg b.w/day. The satellite groups were not be mated. Males from groups 0 SAT and 3 SAT were treated for 28 days. Females from groups 0 SAT and 3 SAT were treated for 53 days (on average as long as females from groups 0, 1, 2 and 3). After that, males and females from satellite groups were observed for 14 days to evaluate the reversibility, stability, or delay in the onset of possible harmful effects. Offspring was examined for survival, body weights, clinical signs and hormonal parameters. Reproduction parameters such as oestrous cycle, sperm parameters and reproductive organs were also examined on parental animals. During the experiment, no mortality of the adults was observed. There were no differences in physical appearance and behaviour of adult animals between the treated and the control groups. No treatment-related clinical signs, ophthalmic alterations, T4 levels altered, or significant differences in body weights and food intake were observed. Statistical analysis of the clinical-chemical examinations results obtained in males and females of group 1, 2 and 3 and satellite groups revealed some statistically significant changes however all of them were considered as not related with the administration of the test item. Fertility parameters on parental animals did not reveal significant changes. There were gross changes observed in control group 0, treated groups 1, 2 and 3 and satellite groups 0 SAT and 3 SAT. However, these changes occurred regardless of the degree of exposure to the test item (this changes were observed both in control and treated groups). Statistical analysis of absolute and relative weights of internal organs revealed statistically significant changes but these organ weight disorders seem to be accidental. The macroscopic and the histological examinations of the rats treated with the test item at the doses of 33.3, 100, and 300 mg/kg b.w./day did not reveal any toxic effects. Additionally, based on results, it was concluded that the test item is not immunotoxic for rats. As for offspring, 34 pups were born dead, the remaining offspring survived the 13-day observation period, except for 4 pups which died at the first day of life. Number of stillbirths in group 2 and 3 were higher compared to the control group. Moreover, mortality was also higher in group 2 and 3 with comparison to the control group. Based on this results, it may be concluded that the test item at dose 100 and 300 mg/kg b.w./day affected pups survival. Nor significant clinical signs were observed among pups neither body weight changes. In conclusion, the treatment of adult (male and female) rats with the test item at the dose levels of 33.3, 100 and 300 mg/kg bw/day by the repeated oral administration, revealed no adverse effects. Based on the results, the no observed adverse effect level (NOAEL) of the test item for systemic toxicity was determined to be 300 mg/kg b.w./day. As for pups, based on number of stillbirths and mortality in group 2 and 3, it may be concluded that the test item affected pups survival. Therefore, on the grounds of the used doses, the no observed adverse effect level (NOAEL) for reproduction/developmental toxicity for pups can be determined on 33.3 mg/kg b.w./day.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study has a Klimisch score of 1.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted in rats according to the OECD Guideline No. 422 (2016) with GLPs. Based on preliminary works, the main study was performed on 60 males and 68 females of Wistar rats treated with vehicle control or with 33.3, 100, 300 mg/kg b.w./day of test item. During the experiment, no mortality of the adults was observed. There were no differences in physical appearance and behaviour of adult animals between the treated and the control groups. No treatment-related clinical signs, ophthalmic alterations, T4 levels altered, or significant differences in body weights and food intake were observed. Statistical analysis of the clinical-chemical examinations results obtained in males and females of group 1, 2 and 3 and satellite groups revealed some statistically significant changes however all of them were considered as not related with the administration of the test item. Fertility parameters on parental animals did not reveal significant changes. There were gross changes observed in control group 0, treated groups 1, 2 and 3 and satellite groups 0 SAT and 3 SAT. However, these changes occurred regardless of the degree of exposure to the test item (this changes were observed both in control and treated groups). Statistical analysis of absolute and relative weights of internal organs revealed statistically significant changes but these organ weight disorders seem to be accidental. The macroscopic and the histological examinations of the rats treated with the test item at the doses of 33.3, 100, and 300 mg/kg b.w./day did not reveal any toxic effects. Additionally, based on results, it was concluded that the test item is not immunotoxic for rats. As for offspring, 34 pups were born dead, the remaining offspring survived the 13-day observation period, except for 4 pups which died at the first day of life. Number of stillbirths in group 2 and 3 were higher compared to the control group. Moreover, mortality was also higher in group 2 and 3 with comparison to the control group. Based on this results, it may be concluded that the test item at dose 100 and 300 mg/kg b.w./day affected pups survival. Nor significant clinical signs were observed among pups neither body weight changes. In conclusion, the treatment of adult (male and female) rats with the test item at the dose levels of 33.3, 100 and 300 mg/kg bw/day by the repeated oral administration, revealed no adverse effects. Based on the results, the no observed adverse effect level (NOAEL) of the test item for systemic toxicity was determined to be 300 mg/kg b.w./day. As for pups, based on number of stillbirths and mortality in group 2 and 3, it may be concluded that the test item affected pups survival. Therefore, on the grounds of the used doses, the no observed adverse effect level (NOAEL) for reproduction/developmental toxicity for pups can be determined on 33.3 mg/kg b.w./day.

Effects on developmental toxicity

Description of key information

Key study: According to OECD 422. GLP study. The no observed adverse effect level (NOAEL) of the test item for systemic toxicity was determined to be 300 mg/kg b.w./day (highest dose tested). As for pups, based on number of stillbirths and mortality in group 2 and 3, it may be concluded that the test item affected pups survival. Therefore, on the grounds of the used doses, the no observed adverse effect level (NOAEL) for reproduction/developmental toxicity for pups can be determined on 33.3 mg/kg b.w./day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study planned
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out : tri{[(2S)-1-ethoxy-1-oxopropan-2-yl]oxy}(vinyl)silane.

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION
- Available GLP studies : Not available.
- Available non-GLP studies : Not available.
- Historical human data : Not available.
- (Q)SAR : Not available.
- In vitro methods : Not available.
- Weight of evidence : No data available.
- Grouping and read-across : No data on analogues.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- The test item is not considered to be genotoxic carcinogen nor germ cell mutagen. Moreover, it can't be proven that there is no systemic absorption and there is significant human exposure.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Species:
rat
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
33.3 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study has a Klimisch score of 1.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted in rats according to the OECD Guideline No. 422 (2016) with GLPs. Based on preliminary works, the main study was performed on 60 males and 68 females of Wistar rats treated with vehicle control or with 33.3, 100, 300 mg/kg b.w./day of test item. During the experiment, no mortality of the adults was observed. There were no differences in physical appearance and behaviour of adult animals between the treated and the control groups. No treatment-related clinical signs, ophthalmic alterations, T4 levels altered, or significant differences in body weights and food intake were observed. Statistical analysis of the clinical-chemical examinations results obtained in males and females of group 1, 2 and 3 and satellite groups revealed some statistically significant changes however all of them were considered as not related with the administration of the test item. Fertility parameters on parental animals did not reveal significant changes. There were gross changes observed in control group 0, treated groups 1, 2 and 3 and satellite groups 0 SAT and 3 SAT. However, these changes occurred regardless of the degree of exposure to the test item (this changes were observed both in control and treated groups). Statistical analysis of absolute and relative weights of internal organs revealed statistically significant changes but these organ weight disorders seem to be accidental. The macroscopic and the histological examinations of the rats treated with the test item at the doses of 33.3, 100, and 300 mg/kg b.w./day did not reveal any toxic effects. Additionally, based on results, it was concluded that the test item is not immunotoxic for rats. As for offspring, 34 pups were born dead, the remaining offspring survived the 13-day observation period, except for 4 pups which died at the first day of life. Number of stillbirths in group 2 and 3 were higher compared to the control group. Moreover, mortality was also higher in group 2 and 3 with comparison to the control group. Based on this results, it may be concluded that the test item at dose 100 and 300 mg/kg b.w./day affected pups survival. Nor significant clinical signs were observed among pups neither body weight changes. In conclusion, the treatment of adult (male and female) rats with the test item at the dose levels of 33.3, 100 and 300 mg/kg bw/day by the repeated oral administration, revealed no adverse effects. Based on the results, the no observed adverse effect level (NOAEL) of the test item for systemic toxicity was determined to be 300 mg/kg b.w./day. As for pups, based on number of stillbirths and mortality in group 2 and 3, it may be concluded that the test item affected pups survival. Therefore, on the grounds of the used doses, the no observed adverse effect level (NOAEL) for reproduction/developmental toxicity for pups can be determined on 33.3 mg/kg b.w./day.

Justification for classification or non-classification