Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Daily administration of test item by oral gavage to Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) during the conditions of the

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD 422 (CitoxLab, 2016) did not result in adverse changes in neurological assessment, body weight, food consumption, clinical chemistry, haematology, coagulation or on urinalysis parameters. Additionally, no test item-related effects were indicated on male and female reproductive performance nor on the development of the F1 offspring.

At 1000 mg/kg/day test item caused some mortality in the adults. Total and postnatal mortality was significantly higher in the High dose (1000 mg/kg bw/day) group foetuses/pups, with fewer pups per litter but normal body size and development in all surviving pups. These findings could be explained as secondary effect due to observed maternal toxicity in the High dose group. Therefore, test item-related adverse effects in foetuses/pups were only noted at dose levels which are associated with marked maternal toxicity.

It is considered that the no observed adverse effect levels (NOAEL) in this study for the parental/adult and F1/pups generations were:

 

NOAELmaternal toxicity:                             300 mg/kg bw/day

NOAELpaternal toxicity:                              300 mg/kg bw/day

NOAELfoetal/pup toxicity:                           300 mg/kg bw/day

Additionally, no test item-related effects were indicated on male and female reproductive performance nor on the development of the F1 offspring.

 

NOAELreproduction:                                  > 1000 mg/kg/bw

NOAELdevelopmental toxicity:                   > 1000 mg/kg bw/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:
2015-08-26 to 2015-10-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany), from SPF colony
- Strain: Crl:WI Wistar rats
- Sex. male and female
- Age: Young adult rats, 10-11 weeks old at the start of the treatment and 12-13 weeks old at the start of mating
- Weight at study initiation: Males: 360-445 g, females: 220-265 g
- Housing:group-housed, with up to 4 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period
- Diet (e.g. ad libitum): 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)
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3 - 25.4 °C
- Humidity (%): 30 - 68 %
- Air changes (per hr): 15 - 20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the previous experimental work propylene glycol (abbreviated as PG in the raw data) was selected for vehicle of the study in agreement with the Sponsor. The same vehicle was used in the Dose Range Finding (DRF) study
- Concentration in vehicle: 0, 25, 75 and 250 mg/mL
- Dose volume: 4 mL/kg bw
- Lot/batch no. (if required): Propylene glycol (1,2-Propanediol, PG), Manufacturer: Sigma-Aldrich Co., Lot number: SZBD1280V / SZBE1770V

Details on mating procedure:
- Mating started after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male of the same dose group housed in a single cage.
- Females remained with the same male until copulation occurred or two weeks elapsed.
- 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
Remarks:
The samples were diluted into the calibrated range with dichloromethane then analysed by GC. Results see table "Any other information on results"
Details on analytical verification of doses or concentrations:
- Stability of the test item in the vehicle (PG) was assessed in the conditions employed on the study during the validation study (according to CiToxLAB study code 15/163-316AN, [4]).
- According to the results, the test item is stable in vehicle in concentration range of 1 mg/mL- 250 mg/mL for 7 days when stored at room temperature.
- Analysis of test item formulations for concentration was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Five samples were randomly taken and analysed from test item formulations and all concentrations.
- Sample analysis was performed on 3 occasions and all prepared formulations were analysed for concentration.
- One set was collected for analysis and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of test item.
Duration of treatment / exposure:
- Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
- Females were dosed for 14 days pre-mating, for up to 14 days of mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum). The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum. 54 days
Frequency of treatment:
daily on a 7 days/week basis
Details on study schedule:
- Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including post-partum/lactation Day PPD4.
- All F1 offspring were terminated on Day 4 post-partum.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
Twelve male and twelve female Wistar rats/group (with the exception of the High dose group, where 13 animals per sex was treated)
Control animals:
yes, concurrent vehicle
Details on study design:
- The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work including the results of a repeated dose range finding study in the rat, with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
- The oral route was selected, as it is one of the possible routes of human exposure.
Positive control:
not required
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS:
- 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.
- Animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality.
- More detailed examinations were made once prior to treatment (to allow for within-subject comparisons), then weekly in the morning.
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 was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT:
- 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 (GD) 0, 7, 10, 14, 17 and 20 and on postpartal days (PPD) 0 (within 24 hours after parturition) and PPD4 (before termination).

FOOD CONSUMPTION:
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g or 0.1 g at least weekly (on the days of body weight measurements).

NEUROTOXICITY:
- Assessment of any potential test item related neurotoxicity was performed during the last week of treatment (performed on Day 23 for males and PPD4 for females).

Observation of the delivery process, offspring and nursing instinct:
- Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. The duration of gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition.
- Dams were observed for signs of nest building with the bedding material and for covering their new-borns. Evidence of suckling was observed by the presence of milk in the pups' stomach.
- Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect the presence of gross abnormalities. Observations were reported individually for each adult animal.



Oestrous cyclicity (parental animals):
On gestation day (GD) 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).
Sperm parameters (parental animals):
ORGAN WEIGHTS
see below

HISTOPATHOLOGY
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structureParameters examined in [all/P/F1/F2] male parental generations:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, other:]
Litter observations:
- The pups (offspring) were monitored for any behavioural changes. Live pups were counted, sexed and weighed individually within 24 hours of parturition (PND0) and on PND4, with an accuracy of 0.01 g. All the litters were checked daily for the number of viable and dead pups.
- Pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination in order to identify the possible cause of death.
- All pups were culled on PND4.
Postmortem examinations (parental animals):
CLINICAL PATHOLOGY
Evaluation of clinical pathology blood and urine samples was conducted for 5 male and 5 female animals in each group and an additional male in the High dose group (the same animals selected for neurotoxicity assessment).

Haematology parameters examined (UNITS):
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 (blood clotting) parameters examined (UNITS):
APTT Activated Partial Thromboplastin Time (sec)
PT Prothrombin Time (sec)

Clinical chemistry (UNITS):
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
Urine samples were collected for 16 hours during an overnight period of food deprivation during the last week of the study (Day 27-28 for males and PPD4-5 for female animals, respectively) from each animal by placing the animals in metabolic cages. The evaluation of the urine samples were performed by observation (e.g. appearance, colour) and test strips.
PARAMETERS (UNITS):
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
Clarity/Appearance

SACRIFICE
Terminally, all surviving an one preterminally euthanized adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination.

GROSS NECROPSY
Gross necropsy was performed on all animals, irrespective of the date of death.
After exsanguination the external appearance was examined, 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 as applicable.


HISTOPATHOLOGY / ORGAN WEIGHTS
- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Paired organs were weighed together except testes and epididymides which were weighed individually. Individual and/or paired absolute organ weights were reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.

Tissue preservation and microscopic evaluation
The weighed organs and all organs showing macroscopic lesions were preserved. The eyes with the optic nerves were retained in modified Davidson’s fixative, the testes with epididymides in Bouin’s solution, and all other organs in 10% buffered formalin solution.
In addition, on completion of the macroscopic examination the following tissues and organs were retained from all animals:
Gross findings
Adrenal gland
Animal identification
Aorta
Brain
Epididymis
Eye with the optic nerve
Oesophagus
Femur with marrow
Heart
Kidney
Large intestine
Extraorbital lachrymal gland
Harderian gland
Liver
Lungs with bronchi
Lymph node
Ovary
Oviduct
Pancreas
Pituitary
Prostate
Salivary gland (including mandibular, sublingual and parotid glands)
Sciatic nerve
Seminal vesicle with coagulating gland
Skin, subcutis with mammary gland (inguinal)
Skeletal muscle (quadriceps)
Small intestine
Spinal cord
Spleen
Sternum with marrow
Stomach
Testis
Thymus
Thyroid with parathyroid gland7
Tongue
Trachea
Urinary bladder
Uterus
Vagina
The retained tissues and organs were embedded in paraffin wax; sections were cut at
4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscopy.

For the adult animals, detailed histological examination was performed first as follows:
• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
• additionally, due to the Study Director’s request on #4004 High dose male,
• all macroscopic findings (abnormalities), except of minor order from all animals,
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the control and high dose group and of all males that failed to sire and all females that failed to deliver healthy pups.

Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Based on the histopathology findings observed in the liver of the examined High dose group animals, slides of the liver samples of all Mid dose and Low dose animals, furthermore the rest of High dose group animals were processed and examined additionally.





Postmortem examinations (offspring):
SACRIFICE
All pups were culled on PND4.

GROSS NECROPSY
Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Statistics:
The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.
Reproductive indices:
Male Mating Index %
Female Mating Index %
Male Fertility Index %
Female Fertility Index %
Gestation Index %
Offspring viability indices:
Survival Index %
Pre-implantation mortality %
Intrauterine mortality %
Total mortality %
Sex ratio (% Females)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item related adverse effects were considered to appear in the High dose group (1000 mg/kg bw/day):
• Red discharge was detected for male #4006 on Day 9, noisy respiration and slightly decreased activity was also recorded for this animal which was later found dead on Day 12.
• Red discharge was also detected for two males (#4010 and #4011) on Day 3 and another male (#4004) on Days 18-20, other symptoms like slightly or moderately decreased activity, slight or moderate noisy respiration, piloerection and/or hunched back was also observed during the treatment period in those cases, however these animals survived until the scheduled necropsy.
´• For one female (#4504), piloerection and slightly decreased activity was observed on Day 2, then noisy respiration / laboured respiration / decreased respiratory rate and prostration were recorded, this animal was found dead on Day 3.
• Female (#4509) showed prostration and laboured respiration on Day 5, and from Day 36 tonic convulsions, fasciculation, discharge coloured, increased salivation, extremely decreased activity were observed, the animals was cold to touch and died on Day 39.

Female (#4502) was euthanized on Day 14 due to its general conditions (noisy respiration / laboured respiration, slightly / moderately decreased activity, distended abdomen, hunched back position, animals was also loosing weight).

Observations considered to be incidental and not directly test item related symptoms includes:
• In female #4509 a 4-cm wound, scar, fur thin were observed from Day 14.
• Minor signs in other animals in the High dose group: increased salivation was observed for three males on Day 2, a small scar was recorded for one male on Days 1-2 and one female on Days 18-26, noisy respiration was detected for five male animals on some days in the period of Days 3-27.

The treatment related clinical signs were restricted to the High dose group. The signs were generally non-specific indicators of toxicity, with several cases of laboured breathing which may be related to reflux of test item.

Scar (1-3 cm) was recorded for three female animals in the Control group in the period of Days 14-25. Fur thin (for three males in the period of Days 5-20) and slight noisy respiration (for one female animal between Days 19-21) were recorded in the Low dose group (100 mg/kg bw/day). Slight or medium noisy respiration was observed for two males and one female in the Mid dose group (300 mg/kg bw/day) on some days in the period of Days 2-24, fur thin was also detected for one female in the period of Days 6-20. These findings were considered to be unrelated to the test item.

Description (incidence and severity):
not applicable
Mortality:
mortality observed, treatment-related
Description (incidence):
One male in the High dose group (#4006) was found dead on Day 12, two females in the High dose group (#4504 and #4509) were found dead on Day 3 and Day 39, respectively. Furthermore one High dose female (#4502) was preterminally euthanized on Day 14. There was no mortality in the Control, Low and Mid dose groups during the study.
Notes:
1. With clinical signs of piloerection, hunched back, reduced activity, more than 20% weight loss over 3 days, laboured respiration, pallor on the visible mucosal membranes and an increased, soft palpable abdominal mass, High dose female #4502 was euthanized after consultation with the Clinical Veterinarian.
2. According to the macroscopic findings at necropsy, the most probable cause of death for female #4504 was treatment method and not test item related.
3. Female #4504 was replaced by female #4604, and male #4101 was added to the study as a pair for female #4604.

Test item related mortality (approximately 8% for males and 23% for males) and clinical adverse effects were observed in the High dose group animals (1000 mg/kg bw/day).

MORTALITY
One High Dose female (4502) was preterminally euthanized on Day 14 due to slightly laboured respiration, moderate decreased activity and hunched presence clinically observed. Additionally, two High Dose females (#4504 and #4509) and one High Dose male (#4006) were found dead.



Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No significant difference was measured in the body weight data in males or females during the experiment when compared to the Control group. However, body weight averages in both sexes were the lowest in the High dose group (1000 mg/kg bw/day) among the four examined groups.

Significantly (p < 0.05) lower body weight gain value in the period of Days 0-7 was observed in the High dose males (1000 mg/kg bw/day) when compared to the Control, although this effect lost the significance when observed on the whole treatment period.

Increased body weight gain was measured during GD7-GD14 in the Low (100 mg/kg bw/day) dose female group when compared to the Control (p < 0.01), this effect was considered to be incidental.

Decreased body weight gain was measured during the first two weeks and the whole gestation period (GD0-7, GD7-14 and GD0-20) in the High (1000 mg/kg bw/day) female group when compared to the Control (p < 0.05). Body weight gain decrease between GD20 and PP0 was smaller in the High (1000 mg/kg bw/day) female group when compared to the Control (p < 0.05), indicating a lower litter weight.

In summary, there were no statistically significances in body weight in any of the treated animals. High dose males showed what appeared to be an adverse effect on body weight gain, at the beginning of the study, which seems to have almost recovered by the study termination. Clear adverse effect on body weight gain was observed during the pregnancy of High dose female animals.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A slightly, but statistically significant lower mean food consumption (p < 0.05) was noted in the Mid and High dose (300 and 1000 mg/kg bw/day, respectively) males at the beginning of the treatment (D0-D7). During the later periods, food consumption was stabilized and was comparable to other groups.

No test item related adverse effect was present in the mean food consumption of the females; although in the second week significant values were recorded for the Mid and High dose groups (300 and 1000 mg/kg bw/day, respectively). As the actual food consumption slightly increased (Mid dose) or showed no difference (High dose), these differences were considered to be only statistical, and not biological differences, due to the individual variances.
It is considered that there were no biologically significant treatment related effects on food intake in either sex.

Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to the control means, the following main differences were observed during haematology examination.
• Statistically significant decrease (p<0.05) in the mean cell haemoglobin value in the males of the High dose group was observed, but the observed mean values in all groups were within the historical control range.
• Statistically significant decrease (p<0.05) in the relative monocyte ratio in the males of the High dose group was observed. In the female animals from the same dose group there was no effect. All values were within the normal historic range, indicating that the values are individual variations and not related to any test item effects.
• A dose-dependent, statistically significant (p < 0.05) decrease of relative large unclassified cell (LUC) ratio in all treated male groups (Low, Mid and High dose) was observed. There was no effect in female treated groups. Furthermore, the concurrent study control was high compared to the historical control database, the values in all groups were considered to be normal; the observed statistical differences were not considered to be an effect of test item.

In conclusion these findings, in the absence of consistent patterns or other correlates were regarded as incidental or individual findings, within the historical control range, and were considered to be unrelated to treatment and with no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to the control means, the following main differences were observed at the evaluation of the clinical chemistry data:
• A statistically significant increase (p<0.01) in the AST concentration was seen in High dose males. However, no similar observations were made in the female animal groups. Moreover, all values for both sexes were within the Historical Control range. The statistical difference is not considered to be an effect of test item.
• An apparently statistically significant increase (p<0.01) in the bile acid concentration in the Mid and High dose males. The Control group mean was below the range of the Historical Control, the other groups all had a relatively high standard deviation with no trend related to treatment (a single high dose male had a high value affecting the mean). The range of individual values in the female control group was similar to the high dose males. Taking account of the individual variability, it is considered that the statistical differences in the Mid and High dose males was not related to test item.
• Total protein concentration was significantly higher than control in the Low and High groups (p<0.01) and at p<0.05 for the Mid dose group. There was no clear dose response in males, female values were clearly unaffected and all groups were in the normal historical control range.
• Albumin concentration followed the same pattern as total protein, which is to be expected when all animals are normal and not affected by treatment.
• Calcium was significantly lower (p<0.05) in the High dose female animals when compared to the control, but values are well within the Historical Control range. No similar changes could be seen in the male animals.
• Phosphorus concentrations showed statistical significance (p < 0.05) in the High dose animals, but even this High dose value is lower than the female Control group mean. No similar change was seen in the female animals.
• Sodium concentrations showed a statistical significance (p < 0.05) in the High dose animals, but all values were within the normal biological and Historical Control ranges. No similar changes were seen in the female animals.

These findings, in the absence of consistent patterns or other correlates were regarded as incidental or individual findings, generally within the historical control range, and were probably unrelated to treatment and/or with no toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No statistically significant effects were noted in the evaluated urine parameters (volume, pH and specific gravity) of the test item treated male and female animals at any dose groups.
Urine sediment analysis in all groups showed similar results to the control.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the animal behaviour, general physical condition or in the reactions to different type of stimuli (Irwin test) in the control or treated groups in males and females (performed on Days 23 and PPD4, respectively).

The landing foot splay values were comparable with the control mean in all treated groups in males and females.

There were no statistical differences in the grip-strength values measured in the forelimbs or hind limbs in any dose groups.
The main objective of the automatic locomotor activity evaluation is to show that the normal behaviour of relatively high exploring activity is seen initially, with a reduced activity with time, to an approximate plateau at about 20-30 minutes. All groups in the study had a normal pattern of activity over the one hour observation period, showing a lack of effect of the test item.

Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic Findings
Test item-related microscopic findings were noted in the liver (300 and 1000 mg/kg bw/day) and kidney (1000 mg/kg bw/day).
The liver changes were characterized as microvesicular vacuolation of centrilobular/periportal hepatocytes. The hepatocytes were filled by numerous small vacuoles (microvesicular form). The nuclei of hepatocytes were not displaced to the periphery. In the males, minimal severity was noted in 2/12 Mid Dose males and minimal to mild intensity was seen in 6/12 High Dose males.

Minimal to moderate vacuolation was present in 7/12 Mid Dose and 9/10 High Dose females. Minimal degree was observed at the Mid Dose level versus minimal to moderate change in the High Dose females.

No test item-related changes were seen in the liver at the dose level of 100 mg/kg bw/day.

Test item-related changes were observed in the cortical tubules of the kidneys. In the males only, minimal to mild bilateral diffuse accumulation of the eosinophilic droplets was recorded in 4/6 males. These rounded droplets with varying size appeared within the cytoplasm of the tubule cells or within the lumen of the tubules. In the females, minimal to mild bilateral vacuolation of the cortical tubules was observed in 4/5 animals. Microvesicular forms of intracytoplasmic clear vacuoles with variable size were microscopically detected.

All other changes in examined tissues and organs were incidental or regarded as common background observations in this rat strain.

Non pregnant females
Due to failed in delivery of healthy pups, detailed histopathological examination was performed on retained reproductive organs in following animals: 2504, 2505 and 4501. No test item-related changes were recorded by light microscopy in these females.

Histopathology Conclusion
In conclusion, the treatment produced test item-related microscopic changes in the liver (300 and 1000 mg/kg bw/day) and kidney (1000 mg/kg bw/day). These changes were considered to be non-adverse.
Minimal to moderate microvesicular vacuolation of centrilobular/periportal hepatocytes was observed in H&E/phloxine stained liver. In the males, minimal severity was noted in Mid Dose males and minimal to mild intensity was seen in High Dose males. In the females, minimal degree was observed at the Mid Dose while minimal to moderate vacuolation was recorded in the High Dose females. Higher incidence was observed in the female dosing groups comparing with treated males.

There were no meaningful differences in incidence or severity between treated males and females. In the kidneys stained with H&E/phloxine, test item-related microscopic findings were observed in the cortical tubules of High dose animals in both sexes. In the males only, minimal to mild bilateral diffuse accumulation of the eosinophilic droplets was observed. The rounded droplets with varying size appeared within the cytoplasm of the tubule cells or within the lumen of the cortical tubules. In the females, minimal to mild bilateral vacuolation of the cortical tubules was detected. Under the conditions of this study, test item did not cause an increased α2µ-globulin levels in male rats when immunohistochemically examined. In contrast, the values obtained by image analysis using previously a monoclonal antibody were generally lower as compared with control animals (statistically significant). The presence of α2µ-globulin in the kidneys of male Wistar rats of this strain is deemed to reflect the normal physiological situation. The differences in the measured values reflect the normal variation and hence, are incidental.

There was one High Dose female preterminally euthanized on Day 14 due to slightly laboured respiration, moderate decreased activity and rapid weight loss. Test item-related minimal microvesicular hepatocellular vacuolation of the liver (with centrilobular/periportal zonation) was microscopically noted in this animal. Two females and one male from the High Dose groups were found dead. Test item-related mild to moderate microvesicular centrilobular/periportal vacuolation of the liver was present in two unscheduled deaths. Test item-related mild bilateral vacuolation of the cortical tubules altered the kidneys of one found dead female.

In addition, there were two Low Dose and one High Dose females with failed delivery of healthy pups, therefore detailed histopathological examination was performed on retained reproductive organs. No test item-related changes were recorded by light microscopy in these females.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the reproductive parameters of males and females in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day) when compared to the control.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 6 days of pairing (cohabitation). Mean duration of mating was 3.1 days in Control group and 2.6, 3.2 and 2.7 days in the Low, Mid and High dose groups, respectively. While all mating period length in the treated groups showed statistically difference, no consistent direction of the change was present, and the small differences did not indicate any test item effect.
see tables below
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
The mean duration of pregnancy was similar in the control and test item treated groups, and all the parturitions were normal.

Compared to control, the number of corpora lutea and number of implantation were similar in all treatment groups.

No significant differences were noted in preimplantation, intrauterine, and postnatal mortality in any test item treated groups, but in the High dose group higher values were recorded for these parameters. Total mortality was significantly higher in the High dose group (p<0.05).

The number of pups born as well as the number of live born pups was significantly lower in the High dose group (p<0.05). Test item-related adverse effects in foetuses/pups were only noted at dose levels which are associated with marked maternal toxicity.

These effects in the High dose group are probably due to the test item.

see tables below
In summary, daily administration of test item by oral gavage to Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) during the conditions of this study did not result in adverse changes in clinical signs, neurological assessment, body weight, food consumption, clinical chemistry, haematology, coagulation or on urinalysis parameters.

Adults/Parental animals
Test item related mortality (approximately 8% for males and 23% for males) and clinical adverse effects (red discharge, noisy respiration, slightly or moderately decreased activity, slight or moderate noisy respiration (or in some cases laboured respiration or decreased respiratory rate), prostration, tonic convulsion, increased salivation, decreased body temperature, piloerection and/or hunched back) were observed in the High dose group animals (1000 mg/kg bw/day).

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period in any groups.

Test item-related liver weight and microscopic changes (microvesicular vacuolation of centrilobular/periportal hepatocytes) were present in the liver of Mid and High dose animals in both sexes. The relatively low severity of these changes and in the absence of hepatic degenerative hepatic changes, these observations were considered to be non-adverse.

In the kidney cortical tubules of High dose animals, minimal to mild bilateral vacuolation in females and minimal to mild bilateral diffuse accumulation of the eosinophilic droplets in males, were observed. There was no effect on α2µ-globulin levels. Taking into account the lack of degenerative changes, and the type and severity of the changes, the observations are not considered to be adverse.

There were no test item-related microscopic changes in reproductive organs. All other changes in examined tissues and organs in any groups were incidental or regarded as common background observations in the applied rat strain.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: mortility/clinical effects (see above)
Organ:
other: clinical effect/mortility
Treatment related:
yes
Dose response relationship:
yes
Description (incidence and severity):
not applicable
Body weight and weight changes:
no effects observed
Description (incidence and severity):
not applicable
Description (incidence and severity):
not applicable
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Summary of clinical observation of the pups
Parameters Groups/Concentration (mg/kg bw/day)
Control Low dose (100) Mid dose (300) High dose (1000)

Number of pups born 167 147 171 96* CH
Number of live born pups 166 147 171 95 NS

Postnatal Day 0 (PND 0)
Number of dead pups 0 1 0 2 NS
Number of viable pups 166 146 171 93 NS
Survival index (%) 99.40 99.44 100.0 97.41 NS

Postnatal Day 4 (PND 4)
Number of viable pups 165 143 168 90 NS
Number of dead pups
( born alive) 1 4 3 5* CH
Survival index (%) 98.88 97.37 98.31 94.76 NS
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Due to the reasons detailed in the previous section, number of pups born was significantly lower in the High dose (1000 mg/kg bw/day) group (p < 0.05). In addition, the number of dead pups during the PND 0-4 period was significantly higher in the High dose group.

Number of male pups was significantly lower in the High dose group when compared to the Control on PND 0 and consequently also on PND 4 (p < 0.05).

The number and ratio of male and female pups was similar in the control and test item treated dose groups, although a dose dependent, but not statistically significant increase in the percentage of female pups was present in the treated groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the offspring body weight on PND0 or PND4, and the body weight gain during this period. Mean total litter weights on PND 0 and PND 4 were lower (-26.1% and -25.1%, respectively) in the High dose group compared to the control, due to the smaller litter size in this dose group, with no effect on the weight or growth of the individual pups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
A total of eight dead pups were observed (1 Control, 2 Low Dose, 2 Mid Dose and 3 High Dose). Negative floating test was seen in one Control and one High Dose F1 offspring. Milk in the stomach in one High Dose pup was noted. In addition, one Low Dose and one High Dose pups were not sucked.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental and reproduction toxicity
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: adverse effects in foetuses/pups were only noted at dose levels which are associated with marked maternal toxicity.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Summary of reproductive parameters (males)

Parameters

Groups/Concentration (mg/kg bw/day)

Control

Low dose (100)

Mid dose (300)

High dose (1000)

Number of treated animals

12

12

12

12

Number of pre-terminal death (found dead)

0

0

0

1

Number of mated animals

12

12

12

11

Number of infertile animals

0

2

0

1

Male mating index (%)

100

100

100

100

Male fertility index (%)

100

83

100

92

Summary of reproductive parameters (females)

Parameters

Groups/Concentration (mg/kg bw/day)

Control

Low dose (100)

Mid dose (300)

High dose (1000)

Number of treated animals

12

12

12

13

Number of pre-terminal death

0

0

0

3

Number of mated animals

12

12

12

11#

Number of sperm positive females

12

12

12

11

Number of surviving sperm positive females

12

12

12

10

Number of non-pregnant females

0

2

0

1

Number of pregnant females

12

10

12

9##

Number of pregnant females with live born(s)

12

10

12

9

Female mating index (%)

100

100

100

100

Female fertility index (%)

100

83

100

90

Female gestation index (%)

100

100

100

100

#Animal No. 4502 was paired, but at the same day preterminally euthanized – no mating occurred

##Animal No. 4509 was found dead on GD19. The developmental stage of foetuses correlated to their age.

DOSE FORMULATION ANALYSIS

The concentration of the test item was determined by a validated GC method on three analytical occasions during the study. The measured concentrations of test item evaluated for each test item-dose group varied between 90% and 103% of the nominal contents.

No test item was detected in the Control samples.

Nominal concentrations

Measured concentrations on 26 August 2015 (Day 0)

mg/mL

Percentage of the nominal

Control

not detectable

-

25 mg/mL

23.7 ± 2.99

95

75 mg/mL

68.5 ± 1.68

91

250 mg/mL

247 ± 13.1

99

Nominal concentrations

Measured concentrations on 23 September 2015 (Day 28)

mg/mL

Percentage of the nominal

Control

not detectable

-

25 mg/mL

23.1 ± 0.83

92

75 mg/mL

67.8 ± 1.49

90

250 mg/mL

239 ± 21.9

95

Nominal concentrations

Measured concentrations on 07 October 2015 (Day 42)

mg/mL

Percentage of the nominal

Control

not detectable

-

25 mg/mL

25.7 ± 0.79

103

75 mg/mL

69.2 ± 1.49

92

250 mg/mL

254 ± 14.7

102

Conclusions:
In conclusion, under the conditions of this study, at 1000 mg/kg/day test item caused some mortality in the adults and in foetuses/pups, but all surviving pups were normal in size and development. Minor changes in the liver in the Mid and High dose groups of the parental generation, as well as changes in kidneys in the High dose group were not considered to be adverse.

Therefore, test item-related adverse effects in foetuses/pups were only noted at dose levels which are associated with marked maternal toxicity.
 
It is considered that the no observed adverse effect levels (NOAEL) in this study for the parental/adult and F1/pups generations were:
 
NOAELmaternal toxicity:                             300 mg/kg bw/day
NOAELpaternal toxicity:                              300 mg/kg bw/day
NOAELfoetal/pup toxicity:                           300 mg/kg bw/day

Additionally, notest item-related effects were indicated on male and female reproductive performance nor on the development of the F1 offspring.
 
NOAELreproduction:                                  > 1000 mg/kg/bw
NOAELdevelopmental toxicity:                   > 1000 mg/kg bw/day
 
Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was to obtain information on the toxicity of the test item following repeated daily administration by oral gavage to Wistar rats. The study 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, parturition and also on the development of the F1 offspring from conception to Day 4 post-partum.

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including post-partum/lactation Day PPD4.

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

For the adult animals, histopathological examination was performed on the selected list of retained organs in the Control and High dose groups, and in the liver and kidney samples in the Control and all treated groups. Furthermore, additional renal immunohistochemistry analyses were performed to detect alpha-microglobulin.

Results

In summary, daily administration of test item by oral gavage to Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) during the conditions of this study did not result in adverse changes in neurological assessment, body weight, food consumption, clinical chemistry, haematology, coagulation or on urinalysis parameters.

Additionally, no test item-related effects were indicated on male and female reproductive performance nor on the development of the F1 offspring.

Adults/Parental animals

Test item related mortality (approximately 8% for males and 23% for females) and clinical adverse effects (red discharge, noisy respiration, slightly or moderately decreased activity, slight or moderate noisy respiration (or in some cases laboured respiration or decreased respiratory rate), prostration, tonic convulsion, increased salivation, decreased body temperature, piloerection and/or hunched back) were observed in the High dose group animals (1000 mg/kg bw/day).

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period in any groups.

Test item-related liver weight and microscopic changes (microvesicular vacuolation of centrilobular/periportal hepatocytes) were present in the liver of Mid dose (300 mg/kg bw/day) and High dose (1000 mg/kg bw/day) animals in both sexes. The relatively low severity of these changes and in the absence of hepatic degenerative hepatic changes, these observations were considered to be non-adverse.

In the kidney cortical tubules of High dose (1000 mg/kg bw/day) animals,minimal to mild bilateral vacuolation in females and minimal to mild bilateral diffuse accumulation of the eosinophilic droplets in males, were observed. There was no effect on α2µ-globulin levels. Taking into account the lack of degenerative changes, and the type and severity of the changes, the observations are not considered to be adverse.

There were no test item-related microscopic changes in reproductive organs. All other changes in examined tissues and organs in any groups were incidental or regarded as common background observations in the applied rat strain.

Foetuses/pups

Total and postnatal mortality was significantly higher in the High dose (1000 mg/kg bw/day) group foetuses/pups, with fewer pups per litter but normal body size and development in all surviving pups. These findings could be explained as secondary effect due to observed maternal toxicity in the High dose group.

Conclusion

 

In conclusion, under the conditions of this study, at 1000 mg/kg/day test item caused some mortality in the adults and in foetuses/pups, but all surviving pups were normal in size and development. Minor changes in the liver in the Mid and High dose groups of the parental generation, as well as changes in kidneys in the High dose group were not considered to be adverse.

Therefore, test item-related adverse effects in foetuses/pups were only noted at dose levels which are associated with marked maternal toxicity.

 

It is considered that the no observed adverse effect levels (NOAEL) in this study for the parental/adult and F1/pups generations were:

 

NOAELmaternal toxicity:                             300 mg/kg bw/day

NOAELpaternal toxicity:                              300 mg/kg bw/day

NOAELfoetal/pup toxicity:                           300 mg/kg bw/day

Additionally, no test item-related effects were indicated on male and female reproductive performance nor on the development of the F1 offspring.

 

NOAELreproduction:                                   > 1000 mg/kg/bw

NOAELdevelopmental toxicity:                      > 1000 mg/kg bw/day

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
The study is a guideline study with Klimisch score 1 (reliable without restrictions).
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

No studies have been performed to explicitily address the question of developmental toxicity in animals caused by the test item.

However, daily administration of test item by oral gavage to Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) during the conditions of the

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD 422 (CitoxLab, 2016; IUCLID chapter 7.8.1.) did not result in any test item-related effects on the development of the F1 offspring.

At 1000 mg/kg/day test item caused some mortality in the adults.Total and postnatal mortality was significantly higher in the High dose (1000 mg/kg bw/day) group foetuses/pups, with fewer pups per litter but normal body size and development in all surviving pups. These findings could be explained as secondary effect due to observed maternal toxicity in the High dose group.Therefore, test item-related adverse effects in foetuses/pups were only noted at dose levels which are associated with marked maternal toxicity.

Therefore, no test item-related effects were indicated on male and female reproductive performance nor on the development of the F1 offspring.

 

NOAELreproduction:                                  > 1000 mg/kg/bw

NOAELdevelopmental toxicity:                   > 1000 mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
The study is a guideline study with Klimisch score 1 (reliable without restrictions).
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available studies the test item is not classified regarding toxicity to reproduction according to the criteria of EC Directive 67/548/EECand EC Regulation 1272/2008.