[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19. Jun 2019 - 03. Feb. 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

- Residual solvent: 43.1 % w/w

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 6-08697-43
- Expiration date of the lot/batch: 27.09.2022
- Purity: 52.4 % in product solution
Sum of impurities: 4.5 % w/w in product solution
Solvent (water): 43.1 % w/w
Total solids content: 56.9 % w/w

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (20 ± 5 °C)
- Stability under storage conditions: assumed stable
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: assumed stable
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): assumed stable
Stability of the test item in the vehicle prepared at concentrations of 20 and 200 mg/mL was confirmed following storage for 7 days at room temperature (the temperature range, 20 – 25 °C) during method validation study.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

standard species according to guideline

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Lab Animals Breeding Center “Pushchino”, Branch of Institute of Bioorganic Chemistry RAS:
Nauki 6, Puschino, Moscow region, Russia 142290
(www.spf-animals.ru)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 8-9 weeks old on day of pre-mating oestrous cycle evaluation;
approximately 10-11 weeks old at the initiation of dose administration (day 1);
approximately 12-13 weeks old when paired on study day 14.
- Weight at study initiation: Males: 323 ± 21 g, N = 58
Females: 200 ± 14 g, N = 58
- Fasting period before study: no
- Housing: Following group forming and until mating, all F0 females and males were housed for two animals in solid bottom polycarbonate cages
- Diet ad libitum
- Water ad libitum
- Acclimation period: The animals were received from Lab Animals Breeding Center “Pushchino” at the age of 3-4 weeks 06.06.2019 by separate litters to avoid of sibling mating. Each animal was examined on the day of receipt.

DETAILS OF FOOD AND WATER QUALITY:
The animals were fed Laboratory Rodent Diet (SSNIFF V1534-300 autoclavable)
Filtered tap water was provided

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 40 - 70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The solution of the test item in the vehicle (water) was administered once daily orally by gavage, via an appropriately sized stainless-steel ball-tipped dosing cannula connected with a syringe. A separate cannula for each group was used. The dosage volume for all groups was 5 mL/kg body weight. Individual dosages were based on the last body weight value to provide the correct mg/kg bw/day dose. All animals were dosed at approximately the same time each day (09:00 – 13:00).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Method
The validated High-performance liquid chromatography (HPLC) method was used for detection of the test item concentration in vehicle formulations ranging from 20 to 200 mg/mL. The validation of the analytical method was performed before the first analysis of formulations, where linearity, accuracy, precision/repeatability, specificity/selectivity, sensitivity (LLOQ), and formulation samples stability were assessed.

Duration of treatment / exposure:

The males were dosed during study days 1-28 (14 days prior to pairing and continuing throughout the mating period) for a total of 28 days. Males of satellite subgroup (not mated) were dosed for a total of 28 days with following two weeks recovery period.
The females were dosed during study days 1 through the day prior to euthanasia (14 days before pairing through lactation day 13) for a total of 49-54 days. Females that failed to deliver were dosed through the day prior to euthanasia for a total of 55 days. Females of satellite subgroup (not mated) were dosed for a total of 55 days with following two weeks recovery period.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 + 5 Satellite subgroup (control and highest dose group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dosage levels are based on a dose-range-finding study (BTL BIBC Study No. 672/19) with treatment of male and female animals for 14 days at dose of 1000 mg/kg bw/day. Results from this study as well as published toxicity data from structurally closely related substances (Monosodium tartrate, CAS No. 526-94-3, Potassium dihydrogenorthophosphate, CAS No. 7778-77-0, and Disodium succinate hexahydrate, CAS No. 6106-21-4) were considered for dose selection.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

Oestrus Cycle Evaluation
Oestrous cycle was monitored for two weeks before start of the treatment to select females with regular cyclicity (4-5 day cycles). Vaginal smears were also monitored daily for two weeks 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.

Cage Side and Clinical Observations (F0)
All rats were observed twice daily, once in the morning and once in the afternoon, for morbidity and mortality. Each F0 male and female were also observed for signs of toxicity approximately 5-20 min following dose administration. In addition, the presence of findings at the time of dose administration was recorded for individual animals. Females expected to deliver were also observed twice daily during the period of expected parturition (from the day 19 of gestation) and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties. Detailed physical examinations were recorded for all parental animals before first dosing and regularly on a weekly basis throughout the study using following score system:

Body Weights (F0)
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 hours of parturition (day 0 or 1 post-partum), and at day 2 (for calculation of food consumption), day 4 and day 13 post-partum.
Furthermore, body weights were recorded during Functional Observation Battery (FOB) measurements (animals allocated to these components of the study only.
Mean weekly body weights (g) and body weight changes (%) are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating period (males and females) and for the entire F0 treatment period (males only). Mean gestation body weights and corresponding mean body weight changes are presented for all intervals and for the overall gestation interval (days 0-20); mean lactation body weight changes are presented for lactation days 1-13. Weekly body weights for female which did not deliver are presented in the individual report table until necropsy.

Food Consumption
Food consumption was assessed quantitatively by weighing of feeder (cage lid) at the beginning of the day and 24 hours after.
Food consumption was recorded for each cage prior to the initiation of dose administration (day 0-1), and weekly until cohabitation. Food consumption was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0-1, 6-7, 13-14, and 19-20 and on lactation days 1-2, 4-5, and 12-13. Weekly food consumption for female with evidence of mating but without signs of gestation and which did not deliver are presented in the individual report table until necropsy. Food consumption are reported as g/kg bw/day.


Functional Observation Battery Assessment
FOB assessments and locomotor activity were recorded for six F0 males and six F0 females, randomly selected from each group on day 28 of dose administration for males, and on lactating day 13 for females. Satellite subgroup animals were tested before euthanasia after 2 weeks post-treatment. Animals were tested at the same time on each day following approximately 40 min of dosing in a randomized order.
All animals were observed for the following FOB parameters using score system as described in SOP Neu/7 of BTL BIBC RAS:
Cage Side Observation
Posture (Score, 1-8) Palpebral closure (Score, 1-4)
Tremors (Score, 1-5) Feces Consistency (Score, 1-4)
Convulsions: Clonic (Score, 1-7) Piloerection (Score, 1-3)
Convulsions: Tonic (Score, 1-5) Biting (Score, 1-3)
Handling Observation
Ease of removal from cage (Score, 1-6) Respiratory character (Score, 1-5)
Ease of handling animal in hand (Score, 1-4) Red deposits (Score, 1-2)
Lacrimation (Score, 1-3) Crusty deposits (Score, 1-2)
Chromodacryorrhea (Score, 1-3) Mucous membranes/eye/skin color (Score, 1-5)
Salivation (Score, 1-3) Eye prominence (Score, 1-3)
Piloerection (Score, 1-3) Muscle tone (Score, 1-3)
Fur appearance (Score, 1-3)
Open Field Observations (after locomotor activity evaluation, 8.9.1.)
Time to first step (seconds) Convulsions: Clonic (Score, 1-7)
Rearing (counts) Convulsions: Tonic (Score, 1-5)
Mobility (Score, 1-4) Tremors (Score, 1-5)
Backing (Counts) Arousal (Score, 1-5)
Grooming (Counts) Bizarre/Stereotypic behavior (Score, 1-16)
Gait Character (Score, 1-7) Urination (Counts)
Gait Score (Score, 1-5) Defecation (Counts)
Sensory Observations
Approach response (Score, 1-5) Pupil response (Score, 1-2)
Touch response (Score, 1-5) Eyeblink response (Score, 1-2)
Startle response (Score, 1-5) Forelimb extension (Score, 1-2)
Tail pinch (Score, 1-5) Hindlimb extension (Score, 1-2)
Olfactory orientation (Score, 1-2) Air righting reflex (Score, 1-4)
Neuromuscular Observations
Hindlimb extensor strength (Score, 1-3) Grip strength hind- and forelimb (a)
Hindlimb foot splay (mm)
Physiological Observations
Catalepsy (seconds) Respiratory rate (bpm, Score 1-3) (c)
Body Temperature (°C) (b) Body Weight (g)
(a) Measurement of grip strength was performed using the Grip Strength Meter, Columbus Instruments, following of FOB measurements. The tension force of the dynamometer in kg (Chatillon DFIS-10, AMETEK, Inc/Columbus Instruments) was used to record the muscle strength of forelimbs and hindlimbs.
(b) Body temperature was measured after Open Field Observations rectally using digital thermometer WT-04 Standart B.Well (United Kingdom).
(c) Respiratory rate was measured after Open Field Observations using PowerLab 8/35 and Spirometer computerized system (ADInstrument Inc.) in counts per minute.

Locomotor Activity
Locomotor activity was measured after FOB handling observation automatically using a personal computer-controlled system OPTO-VARIMEX with Auto-Trek software v. 4.2 (Columbus Instruments) utilizes a series of infrared photobeams surrounding a clear plastic, rectangular cage to quantify each animal’s motor activity. Each animal was tested separately in a randomized order for 6-minute period to measure following parameters:
Distance traveled, cm Ambulatory Time, seconds
Resting Time, seconds Rearing amount (manually)

Sacrifice and pathology:

Diuresis and Urinalysis
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) using metabolic cages (Tecniplast s.p.a). Overnight urine volume was measured and diuresis was calculated (mL/kg body weight/24 h).
The following urinalysis determinations were performed in first portion of urine using strips Aution Sticks 10EA (ARKRAY Factory, Inc) and analyzer Pocket Chem PU-4010:
Blood Collection
Blood samples for clinical pathology evaluations (hematology, coagulation and serum chemistry) were collected from F0 animals at the scheduled necropsies: following 28 days of dose administration, males (at day 29, as a part of euthanasia) and lactation day 13, females (at day 14, as a part of euthanasia), following two weeks post-treatment from satellite animals (as a part of euthanasia), and from female that failed to deliver (as a part of euthanasia). The parameters of clinical pathology were determined in half of the animals randomly selected in the group.
The F0 males were fasted overnight (16-18 hours) prior to blood collection being in metabolic cages. Dams were fasted overnight (16-18 hours) from lactation days 13-14 after the pups were removed and euthanized on PND 13.
The blood was collected terminally following anesthesia by mixture Zoletil® and Xyla® (tiletamine+zolazepam+xylazine) from the inferior vena cava. For assessing the level of glucose, a drop of blood was obtained by tail-tip amputation prior to anesthesia (animals randomly selected for clinical chemistry).
The blood for coagulation and hematology analysis was collected from six males and six females (randomly selected) of each group (main subgroups) and from all animals of satellite subgroups. For coagulation analysis, the blood samples (~0.9 mL) were placed in tubes with 3.2 % sodium citrate using the standard whole-blood-to-anticoagulant ratio (9:1 vol/vol whole blood/3.2 % citrate). Plasma for coagulation analysis was obtained after centrifugation of citrate samples for 15 min at 1500 g and 22 °C. Plasma samples were analyzed immediately after being taken.
For hematology analysis (including blood smear and reticulocyte count), the blood samples (~0.5 mL) were placed in Microvette® tubes containing K3EDTA. For reticulocyte counting, ~50 μL of blood was placed in a tube with 1 % cresyl blue dye for supra-vital staining followed by blood smear. For differential leucocyte count, the blood smear was prepared followed by Pappenheim staining.
For clinical chemistry analysis, the blood samples were collected from the remaining 6 males and 6 females of each group (main subgroups) and from all animals of satellite subgroups. Data from females which has not delivered are presented in individual tables. The blood sample was placed in tube without anticoagulant. The blood was allowed to clot for 50 min and centrifuged (1600 x g, 4 °C, 15 min) for serum separation. Serum from each animal were divided into 3 aliquots (for serum biochemistry, ion-selective, and T4 analysis) and immediately frozen at –20 °C until assayed.
If possible, the remaining blood from all animals of each group was collected in tube without anticoagulant. These serum samples were frozen at –20 °C and stored as a backup samples.
Coagulation
The fresh plasma was analyzed for the following parameters with a 4-channel semi-automatic coagulometer CL 4 (BehnkElektronik, Germany) using the reagents for human clinical trials (Renam, Russia).
Activated partial thromboplastin time (APTT)
Prothrombine time (PT)
Fibrinogen (Fg)
Hematology
The blood samples in EDTA were analyzed using Mythic 18 automated cell counter with the veterinary software (C2 DIAGNOSTICS S.A., France) for the following parameters:
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 #)
Reticulocyte Count was evaluated by manually counting the reticulocytes present in 1000 red blood cells stained with 1 % cresyl blue dye.
White Cell Differential (neutrophils, eosinophils, basophils, lymphocytes, monocytes) was evaluated in the control and high dose treated female groups by manually counting 100 white cells in blood smear stained with Papendheim.
Serum Chemistry
The serum was analyzed with a SAPPHIRE 400 (Prestige 24i) automatic biochemical analyzer (Tokyo Boeki, Japan) using reagents from Randox Laboratories Ltd. for the following parameters:
Total protein (TProt) Creatinine (Crea)
Albumin (Alb) Urea : Creatinine ratio *1000 (by calculation)
Globulin (G) (by calculation) Total bilirubin (TBil)
Alb/G ratio (A/G ratio, by calculation) Total Cholesterol (Chol)
Alkaline phosphatase (ALΡ) Triglycerides (Trigs)
Aspartate aminotransferase (AST) Calcium (Сa)
Alanine aminotransferase (ALT) Inorganic phosphate (Phos)
Total creatine kinase (CK-NAC, Total CK) Chloride (Cl-) (a)
Glutamate Dehydrogenase (GLDG) Sodium (Na+) (a)
Gamma-Glutamyl Transferase (GGT) Potassium (K+) (a)
Urea (Urea) Glucose (b)

(a) Chloride, sodium and potassium ions were measured by ion-selective electrodes using analyzer EX-D (JOKOH Co, Ltd).
(b) Glucose level was estimated by glucose oxidase electrochemical method using Sattelite Express® glucometer (ELTA, Russia) routinely controlled with control strip. Glucose was determined in a drop of whole blood taken from the tip of the tail just before the animal was anesthetized for euthanasia.
T4 Assay
The serum was analyzed by competitive inhibition enzyme immunoassay technique using Rat thyroxine (T4) ELISA Kit (Cusabio) and Multiskan™GO Microplate Spectrophotometer (Termo Scientific) and according to standard procedure of manufacturer and SOP of BTL BIBC RAS. Specification information from manufacturer is the following:
Product Type ELISA Kit
Code Cusabio: CSB-E05082r
Protein Biological Process Thyroid function
Species Rat
Detect Range: 20 ng/mL - 320 ng/mL
Sensitivity: 20 ng/mL
Assay Time: 1-5 h
Sample Volume: 50-100uL
Detection Wavelengh: 450 nm
Lot: CO640040660
Expiration Date: 28 Oct 2019
According to the study plan, samples of 6 animals/sex/group randomly selected for clinical serum pathology, and samples of satellite animals were analyzed. After a change in thyroxin level was observed in the male Group No.3, an assay was done for all back-up samples of male serum (N=11 in total/per group). All collected samples of serum assayed for F1 pups on a postnatal day 4 and postnatal day 13.
Anatomic Pathology (F0)

Termination Procedures for F0 Males
Following completion of the mating period and 28 days of dose administration, the F0 males were euthanized by anesthesia (Zoletil® / Xyla®) followed by terminal blood sampling from vena cava for clinical pathology, subjected to a gross necropsy, organ weight collection and tissue preservation as described below.Termination Procedures for F0 Females
The F0 females that delivered were euthanized on lactation day 14 after litters have been euthanized on PND 13. Dams were fasted overnight (lactation day 13-14) and euthanized by anesthesia (Zoletil® / Xyla®) followed by terminal blood sampling and exsanguination. A gross necropsy was performed, organ weights were collected, and tissues were preserved as described in Section 8.11.7. The number of former implantation sites was counted and recorded.
The F0 females, which have not delivered were euthanized after 55 days of dosing by anesthesia (Zoletil® / Xyla®) followed by terminal blood sampling, exsanguination and subjected to gross necropsy. The uterus horns were placed in 10 % ammonium sulphide solution for detection of early implantation loss. Organ weights were collected, and tissues were preserved as described in Sections 8.11.6-8.11.7.
Terminal Procedures for Satellite Animals
Not mated males and females of satellite subgroup were subjected to euthanasia at the end of two weeks post-treatment period. Animals were anesthetized (Zoletil® / Xyla®) and euthanized following terminal blood sampling for clinical pathology, and exsanguination, subjected to a gross necropsy, organ weight collection and tissue preservation as described below.
Macroscopic Examination F0 Animals
A complete necropsy was conducted on all F0 animals euthanized in extremis or at scheduled termination. 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 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.
Organ Weights
The following organs were weighed from all F0 animals (sex corresponding). Paired organs excluding testes and epididymides were weighed together. Organ-to-body weight and organ-to-brain weight ratios in percentages were calculated.
Adrenals Ovaries
Brain Pituitary
Cowper’s glands Prostate
Epididymides Seminal vesicles & coagulating glands
Heart Spleen
Glans penis Testes
Kidneys Thymus
Levator ani & bulbocavernosus muscles (LABC) complex Thyroid (after fixation)
Liver Uterus (including cervix)
Tissue Collection
At the time of necropsy, the following tissues and organs from F0 animals were collected and placed in 10% neutral-buffered formalin (except as noted):
Adrenals Lungs (b) Thymus
Bone marrow (smear) Liver Thyroids (with parathyroids) (d)
Brain (cerebrum, cerebellum and pons) Lymph nodes Trachea
Esophagus Mesenteric Urinary bladder
Eyes (including optic nerve) (a) Submandibular Males:
Gross lesions Pancreas Testes (c)
Heart Peripheral nerve (sciatic) Epididymides (c)
Intestine (incl. Peyer's patches): Pituitary Prostate
Duodenum Salivary glands (mandibular) Seminal vesicles
Jejunum Skeletal muscle (biceps femoris) Coagulating glands
Ileum Skin with mammary gland Females:
Cecum Spleen Ovaries with oviducts
Colon Spinal cord (cervical) Uterus with cervix
Rectum Sternum Vagina
Kidneys Stomach
(a) were placed in Davidson’s fixative;
(b) preserved by inflation with fixative and then immersion;
(c) were placed in modified Davidson’s fixative.
Bone Marrow Smear
Six randomly selected animals/sex from all F0 groups have been chosen for bone marrow smear. Bone marrow was collected by aspiration from the right femur into a centrifuge tube and smear was prepared for hematological analysis. Two slides per animal were prepared (one is a back-up for re-counting) for following cell counting:
All erythroid cells (E) (%) Monocytes (%)
Myeloid/granulocytic cell line (M) (different. calc, %) Lymphoid cells (%)
M:E ratio
Microscopic Examination
Tissues listed in Section 8.11.7. (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, kidneys, heart, pituitary, and spleen were examined in the low- and mid-dose groups as well as in satellite subgroup animals based on the data of clinical pathology or/and organ weights.
Microscopic examination of thyroid glands was done for all adult males and females in all groups including satellite subgroups, and one male and female of day 13 pup from each litter in control and high dose groups.
For testes, additional transverse section of each of the pair was done for Periodic acid-Schiff’s–hematoxylin (PAS-H) staining. A detailed qualitative examination of the testes was done with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.

Other examinations:

Examinations on F1 Generation and reproductive parameters are described in IUCLID section 7.8.

Statistics:

see below

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No significant test item - related clinical findings were observed in males and females.
In two males from 100 and 300 mg/kg bw/day dose groups and one female from 100 mg/kg bw/day dose group, red-brown nasal discharges were noted during the daily post-dose examination. They were slight, transient, and can be caused by the incidental aspiration of the formulation.
Daily clinical examination of females revealed few incidences of alopecia on the forelimbs or thorax were observed for females in the control group, medium, and high dose group. Hair loss was noted as early as study day 26, generally continued throughout the study, and was not considered to be toxicologically relevant.
In female No. 92 from 1000 mg/kg bw/day dose group, the changes in breathing like wheezing were noted on gestation day 20 (study day 35), followed by early prolonged labor. This female was daily examined for additional delivery and was euthanized on day 41 due loss of all four delivered pups.
In one female (No. 125) from 100 mg/kg bw/day dose group, blood in the vagina was observed on post-coital day 12. This finding was transient and was not associated with the gross urogenital observations during necropsy. Female No. 125 was not pregnant, had no implantation sites, and postimplantation loss. Two females (No. 87 and No. 136) from the 300 and 1000 mg/kg bw/day dose groups had white vaginal discharge on gestation days 16-18.
This finding was not associated with the observations during delivery and gross findings during necropsy.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no morbidity and mortality of animals caused by the test item administration. One female (No. 92) from 1000 mg/kg bw/day dose group was euthanized in extremis after the early prolonged labor and offspring loss. This incident is considered to be as not test item related. Other males and females in all dose level groups survived until the scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and body weight gains in the 100, 300, and 1000 mg/kg bw/day male groups were unaffected by the test item administration during the pre-mating (study days 1-14) and overall treatment (study days 1-28) periods.
In the 1000 mg/kg bw/day recovery subgroup males, mean body weights and body weight gains were also similar to the vehicle control group
Mean body weights and body weight gains in the 100, 300, and 1000 mg/kg bw/day female groups were unaffected by the test item administration during the pre-mating, gestation and lactation periods.
In recovery subgroup of non-mated females, body weights and body weight gains were also similar between the control group and the 1000 mg/kg bw/day dose group for all study periods.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption in the 100, 300 and 1000 mg/kg bw/day group was similar to that in the vehicle control group during all study days. In the satellite 1000 mg/kg bw/day male subgroup, the mean food consumption values were the same as in the control group for any treatment periods.
The mean values of food consumption in all dose treated groups did not significantly differ from the values in the vehicle control group during the pre-mating, gestation, and lactation periods. In the 300 mg/kg bw/day dose group, the mean value of food consumption was decreased (not significant) compared to other groups with an increased value of standard deviation due to the low consumption in dam No.138 with only one pup in the litter.
Food consumption in the satellite (not mated) female subgroup was approximately the same at a dose of 1000 mg/kg bw/day and in the control group for any treatment periods

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):

The values of activated partial thromboplastin time (APTT), prothrombin time (PT), and fibrinogen (Fg) in the 100, 300 and 1000 mg/kg bw/day dose male and female main groups were not statistically changed when compared with the vehicle control group.
In the 1000 mg/kg bw/day male recovery subgroup, the decrease in APTT value was observed. This change was slight (by 4.5 % compared value in the control recovery subgroup) and considered to be not test item related.
In the 1000 mg/kg bw/day dose group, the increase in the absolute granulocyte count was observed in males and females (by 45 % and 54.5 % compared to the control value) with a tendency to an increase in relative granulocyte count. Besides, in females, the increase in absolute lymphocytes (by 33.3 %) and total white blood cell count (by 40.3 %) was registered. The increase in granulocytes count was due to the rise in segmented neutrophils absolute count confirmed in blood smear in females (by 44.8 % for absolute count, p < 0.05). Changes in the blood of the 1000 mg/kg bw/day administered males and females correlated to the findings in the spleen in females of this group , but not to the findings in the bone marrow smear. They were slight, significant compared to the control vehicle group only using t-test (p < 0.05), not observed in recovery subgroups, and considered as non-adverse.

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 300 and 1000 mg/kg bw/day dose group males, concentration of urea was higher (by 20 %, p < 0.05 and 22 %, p < 0.01) as compared to the control vehicle group. In females of 1000 mg/kg bw/day dose group, the urea was also increased (by 16.5 %, p < 0.05). Urea and serum creatinine are routinely used to measure kidney damage. Serum creatinine concentration was not changed in all dose male and female groups resulting in an increase in urea/creatinine ratio statistically significant in the 100 mg/kg bw/day dose female and 1000 mg/kg bw/day dose male and female groups. An increase in urea and in urea/creatinine ratio correlated to increase in kidneys weight (males, 1000 mg/kg bw/day) and heart weight (females, 1000 mg/kg bw/day), and to the microscopic findings in kidneys (males and females) and heart (females); however, it was reversible, not observed in recovery subgroups, and considered as non-adverse.
In all dose-treated male and female groups, the total bilirubin level was increased. Despite the absence of a statistical difference from the control groups, this change was notable (15.4 – 42 % in males and 36.4 – 48.5 % in females). After the 2-week post-treatment period in 1000 mg/kg bw/day male group, bilirubin concentration was decreased compared with the control group, probably due to a recovery rebound effect. Increase in total bilirubin was not correlated to the any signs of hemolysis or hemorrhage and to the histological changes in the liver, and can be caused by the conjugation disorder due to the enhanced proteinuria. Although this assumption is speculative, the increase in bilirubin level is considered to be test item related, but non-adverse.
In females of all dose test item treated groups, the mean value of GLDH was slightly increased. GLDH is used as a biomarker of liver injury and a mechanistic biomarker for mitochondrial dysfunction (Jaeschke H., and McGill M.R. (2013). Serum glutamate dehydrogenase - biomarker for liver cell death or mitochondrial dysfunction. Toxicol. Sci. 134, 221–222; McGill M.R., et al. (2012). The mechanism underlying acetaminophen-induced hepatotoxicity in humans and mice involves mitochondrial damage and nuclear DNA fragmentation. J. Clin. Invest. 122, 1574–1583). The increase in GLDH in females was not dose-depended with statistical significance in the 100 mg/kg bw/day dose group when compared using t-test, not correlated to the change in liver weight and microscopic findings in liver in females. So, an increase of GLDH considered as test item related, but non-adverse.
The increase in the total cholesterol was registered in the 300 mg/kg bw/day male group. There was no such change in the high dose group, and a slight increase in cholesterol in the medium dose-treated group is non-adverse, and considered to be of unclear relationship with the test item.
Besides, in males of the 1000 mg/kg bw/day dose group, the level of glucose was statistically lower as compared to the control value (p < 0.01). This finding is regarded as treatment-related, can be associated with changes in metabolism due to impaired phosphate exchange, but considered as not toxicologically significant. The associated increase in the mean value of total creatine kinase (CK) in all dose-treated male and female groups was slight, variable, and non-significant. The decrease in albumin level in the 1000 mg/kg bw/day dose recovery males is considered to be a post-treatment effect without toxicological significance.

A decrease in thyroxin level was observed in the 300 mg/kg bw/day male group. This change was slight and significant only using paired t-test (11.4 % compared to the control vehicle group), was not dose depended, and was within the historical control range of thyroxin (Min-Max, 33.81 - 68.38 ng/mL, N = 61, Appendix 35). The decrease in thyroxin level in the medium dose-treated males correlated to the decrease in thyroids weight in this group, so this finding can be test item related but non-adverse. In females at the lactation day 13 (main subgroups), the mean thyroxine level was slightly decreased in the 1000 mg/kg bw/day dose group. This change by 9.6 % was not significant compared to the control vehicle group; however, in the 1000 mg/kg bw/day post-treatment females, the decrease in thyroxin level was more pronounced (13.5 %, p < 0.05). The decrease in thyroxin level in females did not correlate to the changes in thyroids weight and histological alterations in thyroid glands and therefore considered as test item related but non-adverse.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Diuresis measured as mL/kg body weight/24 hours in the test item-treated male groups did not significantly differ from the control group at the end of the 28-day administration period. In the 1000 mg/kg bw/day post-treatment group, the urine volume, and diuresis value were significantly lower compared to the control vehicle post-treatment group. The decrease in diuresis in recovery males was not correlated to the microscopic findings in kidneys in these males; however, it can be as a result of changes in the kidneys found in the 1000 mg/kg bw/day main group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home cage, handling, open field, sensory, neuromuscular, and physiological parameters evaluated during Functional Observation Battery testing were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the vehicle control group on study day 28 (males) or lactation day 13 (females). After a two-week recovery period, the FOB parameters in 1000 mg/kg bw/day dose group were also comparable with the control group.
Locomotor activity patterns (distance traveled for 6 minutes, ambulatory and resting time, and rearing counts) in F0 animals were unaffected by test item administration at all doses when evaluated on day 28 of dosing (males) and lactation day 13 (females). After a two-week post-treatment period, the parameters of locomotor activity in the 1000 mg/kg bw/day dose group were comparable with the control group in both males and females

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The weight of kidneys was increased in the 1000 mg/kg bw/day male group. The increase was significant as compared to the control group for absolute value (10.5 %, p < 0.01) as well as for relative to body weight value (p < 0.001), and for relative to brain weight value (p < 0.05). In females of a high dose group, the increase in the kidneys weight was not notable with a non-significant change of absolute value by 4.8 %. The increase in weight of kidneys in 1000 mg/kg bw/day male group was correlated to the elevated concentration of serum urea and urine protein and considered to be test item-related. In recovery subgroup males, the kidneys weight did not differ from the control group.
The weight of the liver (absolute and relative to body weight) was increased in the 1000 mg/kg bw/day dose male (10.2 %, p < 0.05). This change was associated with a slight increase in total bilirubin and considered to be test item-related. An increase in liver weight was slight, was not correlated to any histological alteration in the liver, and so considered as non-adverse. After the 1000 mg/kg bw/day dose post-treatment period, the absolute and relative liver weight in males did not significantly differ from the values in the control group.
The weight of the heart (absolute and relative to body weight) was increased in females treated with the 1000 mg/kg bw/day dose. This change was slight but significant compared to the control vehicle group using a t-test (by 12.2 % for absolute value, p < 0.05). Increase in weight of heart correlated to the increased values urea/creatinine ratio and slight non-significant elevated total creatine kinase in all dose-treated females, microscopic changes in the heart (females, 1000 mg/ kg bw/day), and considered to be test item related. In the recovery subgroup females, the weight of the heart was approximately similar in the control and high dose groups.
In females of the 1000 mg/kg bw/day dose group, the weight of pituitary (absolute and relative to brain weight) was decreased relative control vehicle group (by 13.0 % for absolute value, p < 0.05). This change was not associated with the microscopic changes in pituitary tissue, and therefore is regarded as change with an unclear relationship with the test item administration.
In males of the 300 mg/kg bw/day dose group, the weight of thyroids was decreased (absolute and relative) compared to the control vehicle group using the paired t-test (by 11.3 % for absolute value, p < 0.05). Decrease in thyroids weight was not dose-dependent; however it correlated to the decrease in thyroxine level (Section 11.2.10.5) in this male group.
In the 1000 mg/kg bw/day recovery subgroup males, an increase in relative weights of testes was observed (p < 0.05 for left testi). There were no alterations in the weight and histology of the testes or other organs of the male reproductive system in the treatment period. Therefore, the slight increase in the relative weight of testes in recovery males is regarded as a change with an unclear relationship with the test item administration.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross findings related to the test item administration.
One male (No. 2) in the 100 mg/kg bw/day dose group had atrophy of the left kidney and deformation of the left seminal vesicle. This finding was considered incidental and not related to the test item administration.
Macroscopic findings in the thymus, lymph nodes, thyroids, and stomach noted in the test item-treated groups occurred infrequently, in a manner that was not dose-related and did not correlate to the changes in organ weight, clinical pathology or any microscopic observations, and so is not test item-related.
Female No. 92 in the 1000 mg/kg bw/day group was euthanized in extremis due to the total litter loss. A single small mass on the one uterine horn caused by the purulent inflammation was found during the necropsy of this females without any other gross observation.
For non-delivered female No. 70 (1000 mg/kg bw/day group), the one implantation site was revealed. For non-delivered females No. 82 (0 mg/kg bw/day), No.76 and No.105 (100 mg/kg bw/day), the absence of implantation sites was confirmed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In female No. 92 from 1000 mg/kg bw/day dose group, which was euthanized in extremis due to the total litter loss, no test item related microscopic findings were found. Histological alterations in the liver, spleen, and thymus of this female are supposed to be associated with prolonged labor and a total litter loss. In the uterine myometrium of one horn, the focus of purulent inflammation was observed with an unclear relation to the test item administration.
Test item-related histological alterations were observed in kidneys, heart, and spleen.
In the kidneys of males, the signs of chronic progressive nephropathy (CPN) of minimal to slight grade (tubule basophilia, tubule dilation and atrophy, and hyaline casts) were observed in approximately similar frequency including the control group. CPN is one of the most common spontaneous lesions and the single most important renal disease in rats, and male rats are more severely affected than female rats. In males, the incidence and severity of CPN have not been exacerbated by the test item administration. Whereas in females, CPN findings (hyaline casts in the dilated renal tubules and tubular basophilia) were observed only in the test item treated groups with a moderate severity in the 1000 mg/kg bw/day dose group. In males of the 1000 mg/kg bw/day dose group, an initial sign of renal tubule degeneration as epithelium swelling was found in 2 of 6 animals. Microscopic findings in kidneys of 1000 mg/kg bw/day administered males and females correlated to the clinical pathology changes, increase in kidneys weight in males of this group, considered as test item related, and potentially adverse. In one female from 1000 mg/kg bw/day recovery subgroup, nephroblastematosis of the moderate grade was observed. Nephroblastematosis can be a spontaneous lesion in rats, and just one case in post-treatment females had unclear relation with the test item administration.
In the heart of females of the 1000 mg/kg bw/day dose group, the hypertrophy of the left or left and right ventricle was found in two of six animals with hypertrophy of cardiomyocytes of left ventricle microscopically observed in one female. Myocardial hypertrophy was correlated to the increase in heart weight in 1000 mg/kg bw/day female group, clinical pathology findings, and histological alteration in kidneys. It can be caused by cardiovascular changes due to the disturbance of potassium-sodium exchange and impaired renal function. In males, no such changes in the myocardium were detected, probably due to the shorter administration period of the test item. Although the changes in the heart in the 1000 mg/kg bw/day treated females were functional, they can be recognized as adverse. Besides, in one female and three males from the 1000 mg/kg bw/day dose group, the mononuclear infiltration of the myocardium of the left or/and right ventricle was observed. One incident of mononuclear infiltration was also found in the control male group. Cardiomyopathy is a temporally progressive lesion occurred more commonly in the male rats and characterized at the early stage by focal to multifocal necrosis of individual or small numbers of cardiomyocytes with a mixed inflammatory cell that progresses to mixed mononuclear inflammatory cells only. The findings of mononuclear infiltration were focal and of minimum grade without cardiomyocytes necrosis, were observed with a rare frequency, but with a slightly high grade in the high dose group, so considered as treatment-related and non-adverse.
In the spleen, the signs of increased extramedullary hematopoiesis (EMH) was observed in 4 of 6 females in each of 300 and 1000 mg/kg bw/day dose groups. EMH is commonly observed in rodents as a regular component of the splenic red pulp. In the control group, increased EMH was noted for two males and not for females. Microscopic observations in the spleen of the medium and high dose treated females were slight, but the incidence was significant (p < 0.05, Fisher Exact test) and correlated with the hematological changes in the 1000 mg/kg bw/day dose group, so considered as test item-related, but non-adverse. Additionally, in five and four females from 300 and 1000 mg/kg bw/day dose groups, the increased brown pigment (presumable hemosiderin) was observed in red pulp. The presence of a small amount of intracytoplasmic pigment within the splenic red pulp macrophages is a common background finding in rodents and was noted in three males and one female of the control group. The higher frequency of pigment found in the 300 and 1000 mg/kg bw/day dose female groups may be associated with treatment-induced increased hematopoietic cell proliferation. The spleen congestion observed in all six evaluated females of the 1000 mg/kg bw/day dose group, but also three control females and one control male had unclear relation with the test item administration.
The findings of an unclear relationship with the test item were observed in submandibular lymph nodes, trachea, glandular stomach, and pancreas. In submandibular lymph nodes, lymphocyte follicular hyperplasia was found in four males and two females from 1000 mg/kg bw/day dose group and in two males from the control group. The grade of lymphoid hyperplasia in males from the higher dose group was slightly increased, so the possible relation of this alteration with the test item cannot be denied. However, the change was slight, with a frequency not significantly increased, and therefore considered not to be adverse. In the trachea, mononuclear infiltration of mucosa of the slight grade was noted in 4 of 6 females in the 1000 mg/kg bw/day dose group (p < 0.05, Fisher Exact test). This change can be caused by probable aspiration of the test item formulation during the gavage. However, one incident of cell infiltration in the trachea was observed in the control male group. In the glandular stomach, the neutrophil and lymphocyte infiltration of the submucosa of the minimum and slight grade was found in two males from 1000 mg/kg bw/day dose group. It can be speculated that cell infiltration in the trachea and stomach is associated with the allergic potential of the test substance (SDS on the test item: Corrsave 100, vers. 1.0, 2019-01-24). However, findings in submandibular lymph nodes, trachea, and glandular stomach were slight and non-adverse. In the pancreas, the increased adipocytes accumulation was noted in two males and one female from 1000 mg/kg bw/day dose group. Males in this group also showed a decrease in blood glucose discussed above (Section 11.2.10.4). It can be assumed that the test item stimulates glucose uptake in adipocytes. However, the metabolic effects of the test item remain as speculation; the accumulation of adipocytes in the pancreas was slight and toxicologically insignificant.
There were no test item-related histological alterations observed in the liver. The slight increase in liver weight in males of the 1000 mg/kg bw/day group did not correlate with any microscopic findings and can be due to an adaptive increase in synthetic function. Hepatocellular generalized hypertrophy was found in all female groups with the same frequency and severity, including the control females, that can be due to the physiological state of pregnancy and lactation. Besides, cytoplasmic alteration (glycogen accumulation) of minimal to moderate grade was found in a few examined females, including control.
No histological changes in pituitary were revealed in females that correlated to the decrease in the pituitary weight in 1000 mg/kg bw/day dose-treated females. Dilated Rathke’s cleft contained eosinophilic colloid-like proteinaceous material is relatively common in rats, and it was observed in the approximately same frequency in all male and female groups, including control. In some males and females, the cyst in the pars distalis or pars intermedia was found, which is a frequent incidental finding in rats and not test item related.
In thyroid glands, there were no test item-related microscopic findings, and the increase in thyroids weight in the 300 mg/kg bw/day dose males was of unclear cause. Follicular degeneration of minimal to slight grade with epithelium desquamation was observed with the same low frequency in all groups. The ectopic lymphoid tissue is a developmental abnormality that can occasionally be found in rats and mice and is generally unrelated to treatment.
Remaining histological changes not discussed above were considered to be incidental findings or related to some aspect of physiological condition and variability, experimental manipulation other than the administration of the test item. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue changes.
No test item-related histological findings in organs of the male and female reproductive systems were revealed. During qualitative examination of the testes in the 1000 mg/kg bw/day group with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure (see Methods, Section 8.11.9), no test item related spermatogenic disturbance, tubular vacuolation, contraction, dilatation, necrosis, dilated rete, nor Leydig cell atrophy, hypertrophy, hyperplasia, adenoma were revealed. In the prostate of males from 1000 mg/kg bw/day dose group, two incidents of mononuclear infiltration of the ventral lobe were observed. Prostatitis is a common microscopic observation in rodents. This finding was unfrequent and slight and considered as not treatment-related.
In ovaries and oviducts of the females, there were no significant microscopic changes that could be associated with the test item. Findings in the uterus, observed with a similar frequency in the control group and 1000 mg/kg bw/day group, are assumed to be related to parturition (presented in individual microscopic data).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Bone Marrow Smear
There were no significant changes in the bone marrow cells count in the 1000 mg/kg bw/day dose group correlated to the hematological changes in the blood. The decrease in the relative count of monocytes in males and an increase in the total erythrocaryocytes relative count in females were observed compared to the values in the control vehicle group. However, these changes were slight, non-adverse, and toxicologically non-relevant.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Under the conditions of this screening study, the no-observed-adverse-effect-level (NOAEL) was 300 mg/kg bw/day for F0 (males and females) systemic toxicity