Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Nov 2017 - 24 Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Nov 2017 - 24 Jan 2018
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:
29 July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
recommended by guideline
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: The Lab Animals Breeding Center “Pushchino”, Branch of Institute of Bioorganic Chemistry RAS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 11-13 weeks old at the initiation of dose administration (day 1)
- Weight at study initiation: males 336 ± 23 g, females 210 ± 14 g
- Housing: two animals in per sex in solid bottom polycarbonate cages (Type-4) with bedding, cages equipped with steel lids, steel separators for the food and steel label holders, all cages provided with environmental enrichment material. After mating, dams were housed alone to deliver and their litters were housed in these cages. Animals of satellite subgroup were housed 2-3 animals per cage in Type-4 cages.
- Diet: Laboratory Rodent Diet (SSNIFF V1534-300 autoclavable, Spezialdiaeten, GmbH), ad libitum
- Water: Filtered tap water, ad libitum
- Acclimation period: approx. 6 weeks

DETAILS OF FOOD AND WATER QUALITY:
Diet is analyzed by the manufacturer for nutritional components and environmental contaminants and routinely by BTL BIBC RAS for microbiological contaminants. Samples of water are analyzed routinely for microbiological contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 55
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: 16 Nov 2018 - 24 Jan 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared as a suspension of the neat test item in vehicle (corn oil). The required amount of the test item was weighed and suspended it in a mortar with the addition of vehicle (corn oil) followed by stirring with the calculated volume of vehicle. Test item formulations were prepared every three days and stored at room temperature and checked visually for homogeneity prior to initiation of dosing. The test item formulations were stirred continuously during dosing.

VEHICLE
- Justification for use and choice of vehicle: delivery vehicle for fat-soluble compounds, recommended by guideline
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: MKCC0462
- Quality Release Date: 05 Dec 2016
- Storage Conditions: Room temperature (20 ± 5 °C)
- Provider: Sigma-Aldrich CHEMIE GMBH, Germany
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test item in the vehicle was confirmed following storage for 7 days at room temperature during a method validation study. Analysis of formulations for homogeneity and concentration was conducted in the test facility using a validated method. For homogeneity analysis, quadruplicate samples (3 mL of each) were collected from the top, middle and bottom strata of each dosing formulation prepared during the study, and collected from the top and bottom strata of each resuspended formulation after 2 days of storage, at the beginning of in-life phase. Samples collected from the middle stratum for homogeneity analysis were used for concentration assessment.
Duration of treatment / exposure:
Males were administrated 14 daily doses prior to mating and continuing throughout the mating period for a total of 28 doses.
Females received 14 daily doses prior to pairing and were dosed through lactation day 13 for a total of 50 - 60 doses.
Frequency of treatment:
7 days/week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Main subgroup (control, 100, 300, 1000 mg/kg bw/day): 12 males, 12 females
Satellite subgroup (control, 1000 mg/kg bw/day): 5 males, 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for selecting satellite groups: 5 males and 5 females (vehicle and 1000 mg/kg bw/day) were not mated. Male satellite animals were dosed for a total of 28 consecuitve days, females for 55 days.
- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day
- Animals were observed once in the morning and once in the afternoon for morbidity and mortality.
- Animals were also observed for signs of toxicity approximately 1 h following dose administration. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week

BODY WEIGHT AND FOOD CONSUMPTION: Yes
- Time schedule for examinations: once per week
- Individual F0 male body weights were recorded during group assignment, on the first day of dose administration, and weekly thereafter throughout the study (at the same day as evaluation of food consumption), and prior to the scheduled euthanasia.
- Individual F0 female body weights were recorded during group assignment, on the first day of dose administration, and weekly thereafter until evidence of copulation was observed. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 h of parturition (day 0 or 1 post-partum), and at day 2 (for calculation of food consumption), day 4 and day 13 post-partum.
- Food consumption was assessed quantitatively by weighing of feeder (cage lid) at the beginning of the day and 24 h after.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: males: following 28 days of dose administration at day 29 (as a part of euthanasia); females: lactation day 13 (at day 14, as a part of euthanasia); animals of satellite groups: following two weeks post-treatment (as a part of euthanasia)
- Anaesthetic used for blood collection: Yes (mixture of Zoletil® and Xyla® (tiletamine+zolazepam+xylazine))
- Animals fasted: Yes, overnight (16 - 18 h)
- How many animals: six males and six females (randomly selected) of each group (main subgroups) and from all animals of satellite subgroups.
- Parameters examined: Red blood cells count (RBC), Haemoglobin (Hb), Haematocrit (HCT), White blood cells count (WBC), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Mean corpuscular volume (MCV), Red blood cell distribution width – coefficient of variation (RDW), Red blood cell distribution width – standard deviation (RDW-SD), Platelet count (PLT), Mean platelet volume (MPV), Plateletcrit (PCT), Platelet distribution width - coefficient of variation (PDW), Lymphocytes (LYM #), “Average-sized” cells (MON #), Granulocytes (GRA #)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: males: following 28 days of dose administration at day 29 (as a part of euthanasia); females: lactation day 13 (at day 14, as a part of euthanasia); animals of satellite groups: following two weeks post-treatment (as a part of euthanasia)
- Animals fasted: Yes, overnight (16 - 18 h)
- How many animals: remaining 6 males and 6 females of each group (main subgroups) and from all animals of satellite subgroups.
- Parameters examined: Total protein (TProt), Albumin (Alb), Globulin (G) (by calculation), Alb/G ratio (by calculation), Alkaline phosphatase (ALΡ), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total creatine kinase (CK-NAC), Glutamate Dehydrogenase, Gamma-Glutamyl Transferase, Creatinine (Crea), Urea (Urea), Total bilirubin (TBil), Total Cholesterol (Chol), Triglycerides (Trigs), Calcium (Сa), Inorganic phosphate (Phos), Chloride, Sodium, Potassium, Glucose, thyroxine (T4)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected in all F0 males before scheduled euthanasia (day 28-29, overnight) and in satellite subgroup males after 2 weeks post-treatment before scheduled euthanasia (day 42-43, overnight)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Appearance, Specific gravity, pH, Protein, Glucose, Blood cells, Ketones, Volume (overnight)

NEUROBEHAVIOURAL EXAMINATION / FUNCTIONAL OBSERVATIONAL BATTERY (FOB): Yes
- Time schedule for examinations: Males on day 28 of dosing and females on last day before euthanasia. Satellite subgroup animals were tested before euthanasia after 2 weeks post-treatment. Animals are tested at the same time on each day following approximately 1.5 h of dosing in a randomized order.
- Dose groups that were examined: 6 animals per dose group
- Battery of functions tested:
- cage side: posture, tremors, clonic convulsions, tonic convulsions, palpebral closure, feces consistency, piloerection, biting
- handling observations: ease of removal from cage, ease of handling animal in hand, lacrimation, chromodacryorrhea, salivation, piloerection, fur appearance, respiratory character, red deposits, crusty deposits, mucous membranes/eye/skin color, eye prominence, muscle tone
- open field observations: time to first step, rearing, mobilitiy, backing, grooming, gait character and score, arousal, bizarre/stereotypic behavior, urination, defecation
- sensory observations: approach response, touch response, startle response, tail pinch, olfactory orientation, pupil response, eyeblink response, forelimb extension, hindlimb extension, air righting reflex
- neuromuscular observations: hindlimb extensor strength, hindlimb foot splay, grip strength - hind- and forelimb
- physiological observations: catalepsy, body temperature, respiratory rate, body weight
- locomotor activity: distance travelled, resting time, ambulatory time, rearing amount
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Tissues and organs examined: Adrenals, Brain, Cowper’s glands, Epididymides, Heart, Glans penis, Kidneys, Levator ani & bulbocavernosus muscles (LABC) complex, Liver, Ovaries, Pituitary, Prostate, Seminal vesicles & coagulating glands, Spleen, Testes, Thymus, Thyroid (after fixation), Uterus (including cervix)
- Further details: A complete necropsy was conducted on all F0 animals. Necropsy included examination of the external surface of the body, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities including viscera. Organ weights were collected and tissues were preserved. For F0 females, the number of former implantation sites and corpora lutea were recorded. Vaginal smears were examined before necropsy to determine the stage of the oestrous cycle and allow correlation with histopathology of female reproductive organs.

HISTOPATHOLOGY: Yes
- Tissues and organs examined: Adrenals, Bone marrow (smear), Brain (cerebrum, cerebellum and pons), Esophagus, Eyes (including optic nerve), Gross lesions, Heart, Intestine (incl. Peyer's patches, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Kidneys, Lungs, Liver, Lymph nodes (Mesenteric, Submandibular), Pancreas, Peripheral nerve (sciatic), Pituitary, Salivary glands (mandibular), Skeletal muscle (biceps femoris), Skin with mammary gland, Spleen, Spinal cord (cervical), Sternum, Stomach, Thymus, Thyroids (with parathyroids), Trachea, Urinary bladder, Testes, Epididymides, Prostate, Seminal vesicles, Coagulating glands, Ovaries with oviducts, Uterus with cervix, Vagina
- Further details: Tissues listed above (excluding thyroids) from six randomly selected adult males and females in the control and high-dose groups (half of the animals in the main subgroup euthanized at the scheduled necropsy) were trimmed, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by light microscopy. Liver, adrenals, kidneys, epididymides, ovaries were examined in the low- and mid-dose groups as well as in satellite subgroup animals. In addition, seminal vesicles, prostate and pituitary were examined in males of the satellite subgroup. Microscopic examination of thyroid glands was done for all adult males and females and one male and female of day 13 pup from each litter in the control and high-dose groups.
Statistics:
Continuous data variables (mean body weights, body weight gain and food consumption data) were analyzed by multi-factor analysis of variance ANOVA-2, followed by the Duncan test, to determine inter-group differences. Oestrus cycle length, pre-coital intervals, gestation length, former implantation sites, corpora lutea, clinical pathology values and Functional Observational Battery data were analyzed by a parametric one-way analysis of variance (ANOVA). If the results of the ANOVA were significant (p<0.05), Dunnett's test was applied to the data to compare the treated groups to the control group. In addition to Dunnett's test, t-test was applied to compare absolute and relative organ weights in the high dose treated group and control group. Clinical pathology values and Functional Observational Battery values of satellite animals were analyzed by a t-test to compare the 1000 mg/kg/day group to the control group. Functional Observational Battery parameters which yield scalar or descriptive data were analyzed by Fisher's Exact Test. Gamma glutamyltransferase data was subjected to the Kruskal-Wallis nonparametric ANOVA test with Dunn’s test. Male copulation, female conception, male and female mating and fertility indices of the treated groups were compared to the control group using the Chi-square test.

Histopathological findings of each treated group were compared to those of the control group by the Fisher's Exact test. Organ weights (absolute and relative to body weights and relative to brain weights) were subjected to a parametric ANOVA test and Dunnett's test. In addition, the Student t-test was used to compare the organ weights of high dose group and the control group.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Red urine was observed for three days in one female (No.113) from the 1000 mg/kg bw/day group during daily handling, which was associated with severe renal tubular necrosis and supposed to be test substance-related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female in the 300 mg/kg bw/day group was euthanized on lactation day 8 due to poor clinical condition. The cause was determined to be trauma of esophagus and, therefore, was not attributed to test item administration. All other males and females at all dose levels survived until the scheduled necropsy.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decrease in food consumption was observed in all groups compared to the pre-treatment period. Since there were no corresponding effects on mean body weight gain and food consumption have increased by the end of the recovery period in animals of the satellite subgroup, the decrease in mean food consumption was attributed to the high caloric content of the vehicle.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related effects on hematology parameters in males. Effects observed in females of different dosing groups and the control were of small magnitude and were not considered toxicologically important.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following alterations in serum chemistry parameters were considered to be related to test item administration in the 1000 mg/kg bw/day group males: mean serum ALT (↑85.7%) and mean serum ALP (↑66.2%); these differences were significant (p<0.001 and p<0.01) when compared to the vehicle control group. The increase in ALP was slightly dose-related; the mean changes observed in the 100 and 300 mg/kg body weight/day group were higher (not significantly) of that in control group (↑20.4%, ↑17.2%, respectively). Additionally, mean serum globulins were significantly increased, and A/G ratio was reduced (p<0.05) in the 300 mg/kg bw/day male group (↑12.4, ↓9.5%, respectively).
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
An increase in absolute and relative weights of liver and kidneys is considered to be associated with administration of the test item. These changes were associated with histopathologic or/and clinical pathology changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At the scheduled F0 male and female necropsies, test item-related findings in kidneys (pale spots and deformation) were noted in the 300 mg/kg bw/day group (males and females) and 1000 mg/kg bw/day group (female). All other macroscopic findings noted in the test item-treated groups occurred infrequently, at similar frequencies in the vehicle control group, and/or in a manner that was not dose-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Necrosis of renal tubules and infiltration of pelvis observed in females at a dose of 300 mg/kg bw/day and in severe grade at a dose 1000 mg/kg bw/day is considered adverse.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS:
In the 300 mg/kg bw/day group, a single female (No. 86) was euthanized on lactation day 8 (dosing day 47) due to moribund condition. This female had clinical findings of hunched posture, dehydration, hypokinesia, dyspnoae and nasal haemorrhagic discharge. Upon macroscopic examination, the cause of moribund condition for this female was determined to be trauma of esophagus and, therefore, was not attributed to test item administration.
Daily clinical examination of females revealed low incidences of alopecia on the forelimbs, ventral inguinal and neck areas were observed for some females in the control group, medium and high dose group. Hair loss was noted as early as study day 33, generally continued throughout the study and was not considered to be toxicologically relevant.
Other findings noted in the test substance-treated groups occurred also infrequently and/or were observed in control group, and were not dose-related. Rales were noted for the one male in the 300 mg/kg body weight/day dose group and one female in the control group starting from dosing 21-22 and continuing throughout the study. Chromodacryorrhea in one male from 300 mg/kg body weight/day dose group and slight dehydration with reduced food intake in two females from 100 mg/kg bw/day dose group were noted as short-term findings which were not considered to be adverse.

HAEMATOLOGY:
There were no test item-related effects on hematology parameters in males. In females on lactation day 14, the hemoglobin concentration (MCHC) was lower (↓3.1%) and percentage reticulocyte count was higher (↑18.1%) in the 1000 mg/kg bw/day group when compared to the vehicle control group. The changes were significant (p<0.05), however were of small magnitude and were not considered toxicologically important. There were no significant differences in relative and absolute cell counts on lactation day 14. Nevertheless, the following changes were observed in 1000 mg/kg bw/day recovery subgroup: absolute white blood cell count (↑37.3%) and absolute monocyte count (↑33.3%). These findings were slight, observed only in post-treatment period in females and were not considered to be related to test item administration.
A significantly higher relative count in segmented neutrophils was observed in the 300 mg/kg bw/day group males (↑24.8%, p<0.05) and in the 1000 mg/kg bw/day males in post-treatment period (↑25.0%, p<0.05) when compared to the vehicle control group, that was not associated with changes in the absolute leucocyte differential counts in peripheral blood. In females, a slight decrease in the relative count of plasma cells on the bone marrow smear was observed in the 1000 mg/kg bw/day dose group (↓41.2%, p<0.05) that considered non-adverse.

CLINICAL CHEMISTRY:
There were no statistically significant alterations in serum chemistry parameters in females on lactation day 14. However, there was a clear tendency to increase the mean serum ALT (↑34.5) in the 1000 mg/kg bw/day group when compared to the control group using Dunnett's test. This increase was statistically significant (p<0.05) when compared to the control group with a t-test. After the 2-week post-treatment period, clinical chemistry parameters in 1000 mg/kg bw/day dose group were similar to the values in vehicle control group in both males and females.
There were no test item-related effects on a thyroxin (T4) level in serum of parental males. Mean thyroxin concentrations in the dose groups did not significantly differ from the values in the vehicle control group after 28 days of test item administration as well as after the 2-weeks of post-treatment period.

ORGAN WEIGHTS:
In satellite males, significant differences (p<0.01) in absolute and relative weights of prostate and seminal vesicles with coagulating glands were observed after the 1000 mg/kg bw/day post-treatment as compared with vehicle control group. The higher prostate and seminal vesicles weights were not associated with any gross or histopathologic changes; however, there were histologically revealed signs of an increased secretory activity of its glandular epithelium. In addition, the males after 1000 mg/kg bw/day post-treatment period had a slight but significant (p<0.05) increase in the absolute and relative weight of the pituitary gland. There were no changes in the weight of the pituitary gland during the period of test item administration; however, histological findings were observed in single animals in 300 and 1000 mg/kg bw/day dose groups, which suggest the relationship of changes in the weight of the pituitary gland with the administrated test item.
The higher weights of ovaries in the females of 1000 mg/kg bw/day recovery subgroup, epididymis (right) and adrenals in the males, and adrenals in the females of 1000 mg/kg bw/day dose group were not associated with any gross or histopathologic changes. However, for adrenals it may be due to a slight enhancement of secretory activity, not detectable histologically. These changes are not considered adverse or toxicologically important.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Atrophy of renal tubules, prominent hyaline casts, and nephropathy was observed in the kidneys of the 100, 300 and 1000 mg/kg bw/day group males and in one male from a control group. While hyaline droplets are considered a normal finding in untreated male rats, the incidence of hyaline casts and progressive nephropathy was higher in the test item-treated groups with a significant difference from the control group at a dose 100 mg/kg bw/day. In addition, in one male and one female from 300 and 100 mg/kg bw/day groups, respectively, a severe or moderate diffuse swelling of the renal tubular epithelium was revealed. The incidence of hyaline nephropathy was approximately the same to control group in the substance-treated group females. However, in the 300 and 1000 mg/kg bw/day group females, findings of renal tubules necrosis, infiltration of a cortex and renal pelvis were observed. The microscopic observations in kidneys correlated to the grossly observed discolored spots on kidneys and organ deformations, and the increased absolute and relative kidney weights. In one female No.113 in 1000 mg/kg bw/day group severe renal tubules necrosis correlated, in addition, to the in-life clinical observations (erythruria). The test item-related renal hyaline nephropathy is often found in rats, considered species-specific and is not toxicologically significant for humans. However, necrosis of the renal tubules and infiltration of pelvis observed in females at a dose of 300 mg/kg bw/day and in severe grade at a dose 1000 mg/kg bw/day is considered adverse. However, it must be taken into account that the females were in a different physiological condition (pregnancy and lactation) compared with the males, and the administrated period to the females was about twice as long.

Test item-related findings were found in the liver, kidneys and pituitary. Generalized or centrilobular hepatocellular hypertrophy was observed in all examined males in 1000 mg/kg bw/day group. In females, hepatocellular hypertrophy was noted in all groups, including the vehicle control group, but was not observed in the females of the satellite subgroup and can be due to the physiological state of lactation. However, in two females from the 300 and 1000 mg/kg bw/day groups hepatocellular hypertrophy was particularly marked. Hepatocellular hypertrophy correlated to the increased absolute and relative liver weights as well as the increased ALT and ALP serum level. Hepatocellular hypertrophy and higher liver weights are considered adaptive and hence non-adverse.

Cysts with pale eosinophilic proteinaceous fluid and vacuolar degeneration of the endocrine cells in pars distalis of pituitary were found in 300 and 1000 mg/kg bw/day males and in 1000 mg/kg bw/day female. The findings in the pituitary are supposed to be caused by an increase in secretion and are considered as non-adverse.

Findings of uncertain relationship to test item administration were noted in prostate and seminal vesicules of satellite males after post-treatment period. There were noticeable signs of increased secretion in both organs, visualized in the prostate by acinar dilatation as well as marked epithelial hypertrophy in the acini in some males. This correlated with an increase in the absolute and relative mass of the prostate and seminal vesicles, recorded in 1000 mg/kg bw/day males at post-treatment period. Probably, the activation of secretion is a compensatory rebound reaction of these organs at the post-treatment, considered toxicologically unimportant and non-adverse.

Cortical lymphoid depletion was observed in the thymus of one female from 1000 mg/kg bw/day group and two females from control group females. Thymic lymphoid depletion may be a result of the physiological state of pregnancy and lactation as well as stress condition in general and considered non-adverse.
Granulomatous in alveoli with peribronchial or interstitial infiltration was observed in one control female (No.119) and one female (No.78) from 100 mg/kg bw/day group. These findings correlated with macroscopic observations in lungs (white areas) and clinical observations during in-life phase (rales in female No.119). It is assumed that these changes are caused by aspiration of dose formulation during gavage and therefore have no toxicological significance.

One male from the 300 mg/kg bw/day group had severe crypt necrosis in the colon, which correlated with the gaseous distension of large intestine noted during necropsy. Such a finding was observed as an exceptional incident, possibly caused by an individual response of the intestinal microflora to the administered formulation and considered toxicologically unimportant.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no toxicologically relevant effects observed
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Tables summarising individual animal data are attached as pdf documents to the technical dossier.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
recommended by guideline
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: The Lab Animals Breeding Center “Pushchino”, Branch of Institute of Bioorganic Chemistry RAS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 11-13 weeks old at the initiation of dose administration (day 1)
- Weight at study initiation: males 336 ± 23 g, females 210 ± 14 g
- Housing: two animals in per sex in solid bottom polycarbonate cages (Type-4) with bedding, cages equipped with steel lids, steel separators for the food and steel label holders, all cages provided with environmental enrichment material. After mating, dams were housed alone to deliver and their litters were housed in these cages. Animals of satellite subgroup were housed 2-3 animals per cage in Type-4 cages.
- Diet: Laboratory Rodent Diet (SSNIFF V1534-300 autoclavable, Spezialdiaeten, GmbH), ad libitum
- Water: Filtered tap water, ad libitum
- Acclimation period: approx. 6 weeks

DETAILS OF FOOD AND WATER QUALITY:
Diet is analyzed by the manufacturer for nutritional components and environmental contaminants and routinely by BTL BIBC RAS for microbiological contaminants. Samples of water are analyzed routinely for microbiological contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 55
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: 16 Nov 2018 - 24 Jan 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared as a suspension of the neat test item in vehicle (corn oil). The required amount of the test item was weighed and suspended it in a mortar with the addition of vehicle (corn oil) followed by stirring with the calculated volume of vehicle. Test item formulations were prepared every three days and stored at room temperature and checked visually for homogeneity prior to initiation of dosing. The test item formulations were stirred continuously during dosing.

VEHICLE
- Justification for use and choice of vehicle: delivery vehicle for fat-soluble compounds, recommended by guideline
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: MKCC0462
- Quality Release Date: 05 Dec 2016
- Storage Conditions: Room temperature (20 ± 5 °C)
- Provider: Sigma-Aldrich CHEMIE GMBH, Germany
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation. If evidence of copulation was not detected after 8 days of pairing in presence of regular oestrus cycle, the female was housed with another male for which the mating was confirmed.
- Proof of pregnancy: presence of sperm following a vaginal lavage referred to as gestation day 0 (G0)
- After 8 days of unsuccessful pairing replacement of first male by another male with proven fertility.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test item in the vehicle was confirmed following storage for 7 days at room temperature during a method validation study. Analysis of formulations for homogeneity and concentration was conducted in the test facility using a validated method. For homogeneity analysis, quadruplicate samples (3 mL of each) were collected from the top, middle and bottom strata of each dosing formulation prepared during the study, and collected from the top and bottom strata of each resuspended formulation after 2 days of storage, at the beginning of in-life phase. Samples collected from the middle stratum for homogeneity analysis were used for concentration assessment.
Duration of treatment / exposure:
Males were administrated 14 daily doses prior to mating and continuing throughout the mating period for a total of 28 doses.
Females received 14 daily doses prior to pairing and were dosed through lactation day 13 for a total of 50 - 60 doses.
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Main subgroup (control, 100, 300, 1000 mg/kg bw/day): 12 males, 12 females
Satellite subgroup (control, 1000 mg/kg bw/day): 5 males, 5 females
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
During the period of expected parturition (starting with gestation day 19), females were observed twice daily for initiation and completion of parturition and for signs of dystocia.

DETAILED CLINICAL OBSERVATIONS: Yes:

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND: Yes

for further details see Section 7.5.1
Oestrous cyclicity (parental animals):
Oestrous cycles was monitored before start of the treatment to select the females with regular cyclicity (4-5 day cycles). Vaginal smears were also monitored daily for the last week during the pre-mating period with continued monitoring into the mating period until there was evidence of mating. Vaginal smears were examined before necropsy to determine the stage of the oestrous cycle and allow correlation with histopathology of female reproductive organs.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: Cowper’s glands, Epididymides, Glans penis, Levator ani & bulbocavernosus muscles (LABC) complex, Prostate, Seminal vesicles & coagulating glands, Testes
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Pups delivered, Liverborn, Stillborn, Unknown vital status, Runts, Pups found dead, Pups cannibalized, Survival relative to number born, Body weight, Anogenital distance normalized, Sex
- Additional information: Each litter was examined daily for survival and abnormalities and all deaths were recorded.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All F0 male animals were sacrificed following completion of the mating period and 28 days of dose administration.
- Maternal animals: All surviving F0 female animals were sacrificed on lactation day 14 after litters have been euthanized on PND 13.

GROSS NECROPSY
- Gross necropsy consisted of: refer to details under 7.5.1
The number of former implantation sites and corpora lutea (in total, corpora lutea (CL) of pregnancy and newly formed CL after the postpartum ovulation) was counted and recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Refer to details under 7.5.1
Postmortem examinations (offspring):
SACRIFICE
- Pups were sacrificed on post-natal day (PND) 13 post-partum.
- Dead pups and pups killed on PND 13 were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Blood samples from the day 13 pups were assessed for serum levels of thyroid hormone (T4).

GROSS NECROPSY
- Gross necropsy consisted of external examinations.
Statistics:
See Section 7.5.1
Reproductive indices:
The following parameters of reproductive performance were calculated for males and females:
- Pre-coital interval (days): Rats paired over a 12-hour dark cycle were considered to have been paired for 1 day
- Male Mating Index (%): (No. of males with evidence of mating / Total No. of males used for mating) X 100
- Female Mating Index (%): (No. of females with evidence of mating / Total No. of females used for mating) X 100
- Male Fertility Index (%): (No. of males siring a litter / Total No. of males used for mating) X 100
- Male Copulation Index (%): (No. of males siring a litter / No. of males with evidence of mating or females with confirmed pregnancy) X 100
- Female Fertility Index (%): (No. of females with confirmed pregnancy / Total No. of females used for mating) X 100
- Female Conception Index (%): (No. of females with confirmed pregnancy / No. of females with evidence of mating) X 100

The following parameters were evaluated for dams of each group:
- Duration of gestation (days): The time elapsed between confirmed mating and the day of first pup was delivered
- Gestation index (%): Number (N) of rats with live offspring / Number of pregnant rats
- Pre-natal loss of offspring: Implantations minus live births
- Post-natal loss of offspring: Live births minus alive at PND 13
Offspring viability indices:
The following parameters were evaluated for each litter:
- Dead pups (N, %): Including pups found dead, euthanazied in extremis and cannibalized. Missing pups were presumed cannibalized (PND 0-1, 1-4, 1-7, 1-13)
- Postnatal survival (N, % per litter): Number of live pups, % per litter (PND 0-1, PND 1-4, PND 1-7, PND 1-13)
- Viability index (N/N, %): Number of live pups / Number of liveborn pups on PND 0, PND 4, 7, 13

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Red urine was observed for three days in one female (No.113) from group 4 during daily handling, which was associated with the severe renal tubular necrosis and supposed to be test substance-related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female in the 300 mg/kg bw/day group was euthanized on lactation day 8 due to poor clinical condition. The cause was determined to be trauma of esophagus and, therefore, was not attributed to test item administration. All other males and females at all dose levels survived until the scheduled necropsy.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decrease in food consumption was observed in all groups compared to the pre-treatment period (study days 0-1). Since there were no corresponding effects on mean body weight gain and food consumption have increased by the end of the recovery period in satellite subgroup, the decrease in mean food consumption were attributed to the high caloric content of the vehicle.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related effects on hematology parameters in males. Effects observed in females of different dosing groups and the control were of small magnitude and were not considered toxicologically important.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following alterations in serum chemistry parameters were considered to be related to test item administration in the 1000 mg/kg bw/day group males: mean serum ALT (↑85.7%) and mean serum ALP (↑66.2%); these differences were significant (p<0.001 and p<0.01) when compared to the vehicle control group. The increase in ALP was slightly dose-related; the mean changes observed in the 100 and 300 mg/kg body weight/day group were higher (not significantly) of that in control group (↑20.4%, ↑17.2%, respectively). Additionally, mean serum globulins were significantly increased, and A/G ratio was reduced (p<0.05) in the 300 mg/kg bw/day male group (↑12.4, ↓9.5%, respectively).
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Necrosis of renal tubules and infiltration of pelvis observed in females at a dose of 300 mg/kg bw/day and in severe grade at a dose 1000 mg/kg bw/day is considered adverse.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

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

Details on results (P0)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test item-related effects on reproductive performance were observed at any dosage level. One male from 100 mg/kg bw/day group and one male from 1000 mg/kg bw/day group have no evidence of mating and did not sire a litter. The precoital interval was approximately the same in all groups. All females in groups were with evidence of mating. One gravid female from 300 mg/kg bw/day group did not deliver.

GESTATION LENGTH AND PARTURITION OBSERVATION
Mean gestation lengths were similar amongst all groups when the 100, 300, and 1000 mg/kg bw/day groups were compared to the vehicle control group. One female (No.93) from 300 mg/kg bw/day dose group did not deliver and had signs of post-implantation loss. Prolonged labor was observed in three females (No.139, No.128 and No.115 from the control group, 100 and 300 mg/kg bw/day groups, respectively) that was not attributed to the test item administration.

LOSS OF OFFSPRING
No statistically significant differences were noted between the vehicle control and test item-treated groups. One female (No.93) from 300 mg/kg bw/day dose group did not deliver in the presence of one implantation site. The other females from all dose groups delivered approximately the same number of live pups. The numbers of stillborn pups as well the values of pre-natal and post-natal loss of offspring were approximately the same between groups. Three dams from high dose group had abnormal pups identified as runts. However, there was no statistical difference from the control group, and this finding was considered to be substance unrelated. Also, one dam from the control group had pups with a visual abnormality of hindlimbs layout and brain hemorrhages in one of them. These pups have been identified as abnormals.

For further details refer to section 7.5.1

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse reproductive effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
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
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination of brain tissue collected from pup 139-8m (control group), and pup 115-8f (300 mg/kg body weight/day dose group) with signs of hemorrhage revealed moderate focal subarachnoid hemorrhage. The findings identified cannot be attributed to F0 parental test item administration.
Histopathological findings:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

LITTER VIABILITY ON PND0
There were no test item-related changes in the mean number of pups born, live litter size and survival between birth and PND 0 (relative to number born). Three pups from 1000 mg/kg bw/day group were identified as runts. However, there was no statistical difference from the control group in the percentage number, and this finding was considered to be test item unrelated.

SEX, BODY WEIGHT AND ANOGENITAL DISTANCE AT BIRTH
The mean number of females and males born, the percentage of males, body weights at birth, and absolute and relative anogenital distance in substance-treated groups did not differ significantly from the values in the control group. An increase in the mean number of females (↑24.1%) at birth in the 1000 mg/kg bw/day group were not significantly different compared to the vehicle control group and the percentage of males per litter was approximately similar to control value.

POST-NATAL SURVIVAL
The mean number of liveborn pups, live litter size and post-natal survival in the all dose-treated group did not significantly differ from the values in control group. The percentage of dead pups during postnatal period PND 1-13 was not increased in test item treated groups, and the viability index was similar to the control group value.

CLINICAL OBSERVATIONS
The general physical condition of all F1 pups in the 100, 300 and 1000 mg/kg bw/day groups were unaffected by test item administration. On the PND 0-1, several liveborn pups were found dead in groups 1, 2 and 3 (1, 1 and 2 pups respectively). Four, 1, 1 and 4 pups in the groups 1, 2, 3 and 4 were missing and presumed to have been cannibalized. One pup from control group 1 was euthanized in extremis on PND 0 in moribund condition. Litter 86 (group 3) and litter 131 (group 4) were euthanized in extremis due to starvation, which was associated with clinical condition of dams. The poor clinical condition of the female 86 was caused by a trauma of esophagus. The cause of emaciation of litter 131 was determined to be agalactia.

BODY WEIGHTS, BODY WEIGHT GAIN AND PERCENTAGE OF MALES
Mean body weights and body weight gains in male and female pups were unaffected by test item administration. No statistically significant differences from the vehicle control group were noted. The percentage of males in the litters during PND 0-13 was also approximately the same in all groups.

CLINICAL PATHOLOGY
There were no test item-related effects on a thyroxin level in serum of F1 offspring on postnatal day 13. Mean thyroxin concentrations in the 100, 300 and 1000 mg/kg bw/day dose groups did not significantly differ from the values in the vehicle control group.

NUMBER OF NIPPLES/AREOLAE
The test item did not increase nipples/areolae retention in males at PND 13. Areolae were observed in the following males, single in groups: 138-4m (Group 1, 6 areolae), 130-2m (Group 2, 4 areolae) and 125-2m (Group 3, 4 areolae), that was not treatment related. Female offspring exposed to 0, 100, 300 and 1000 mg/kg bw/day of the test item did not display decreased nipples at PND 13.

WEIGHTS OF THYROID GLANDS
There were no changes in the absolute and relative weights of the thyroid glands in F1 offspring males and females in all test item treatment groups compared with the control group.

MACROSCOPIC OBSERVATIONS
The number of pups necropsied on PND 0 as found dead or euthanized in extremis, was 13, 14, 11 and 6, respectively, in the control group, 100, 300 and 1000 mg/kg bw/day dose groups. No internal findings that could be attributed to F0 parental test item administration were noted at the necropsies of these pups. In the control group, pups no. 139-7 and no. 139-8 were suspected of an anomaly of the hind limbs, which was not confirmed. In addition, brain hemorrhage was revealed in the pup no. 139-8 as well as in the pup no. 115-8 from 300 mg/kg bw/day dose group. Aside from the presence or absence of milk in the stomach, no other internal findings were noted. Litter 86 (group 3) and litter 131 (group 4) were euthanized in extremis on PND 8 and PND 10, respectively, due to pups emaciation, which was associated with clinical condition of dams (trauma of esophagus in female No.86 and agalactia in female No. 131). There were no significant observations in all pups in the test item-treated groups and in the vehicle control group during scheduled necropsy on PND 13. No signs of demasculinization were found in any male in all groups.

MICROSCOPIC OBSERVATIONS
Microscopic examination of brain tissue collected from pup 139-8m (control group), and pup 115-8f (300 mg/kg bw/day dose group) with signs of hemorrhage revealed the following findings: moderate focal subarachnoid hemorrhage was observed in the posterior parietal cortex and in the thalamus of pup 139-8m, and marked focal hemorrhage was observed in the frontal and temporal cortex of pup 115-8f. The findings identified cannot be attributed to F0 parental test item administration. The processing of skeletons by Dawson's technique did not reveal any anomalies of ossification in pups 139-7f and 139-8m.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically relevant effects observed

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Tables summarising individual animal data are attached as pdf documents to the technical dossier.

Applicant's summary and conclusion