[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19. Jun - 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 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 confirm
ed 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 evaluatio
n;
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” a
t the age of 3-4 weeks 06.06.2019 by separate litters to avoid of sibling mating. Each animal was e
xamined 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 exposure:

The solution of the test item in the vehicle (water) was administered once daily orally by gavage, v
ia an appropriately sized stainless-steel ball-tipped dosing cannula connected with a syringe. A se
parate 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).

Details on mating procedure:

The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for the F0 males and females avoiding sibling mating (animals were received with an indication of litters). Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of sperm following a vaginal lavage. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0 (G0).

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, p
recision/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

Details on study schedule:

according to Guideline

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:

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 wel
l 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

Parental animals: Observations and examinations:

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 fo
llowing score system:

Oestrous cyclicity (parental animals):

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.

Sperm parameters (parental animals):

During Necropsy (microscopic examination):
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.

Litter observations:

Pups Identification
The day of birth (when parturition was judged to be complete) was defined as day 0 post-partum (PND 0). Upon completion of parturition (PND0 or PND1 for some litters), all pups were individually identified by tattoo marking applied to the digits. Each tattoo was as an individual set of dye points applied subcutaneously on 1-4 toes of the forelimbs.
Litter Standardization
On day 4 after birth (PND 4), the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, five pups per sex per litter. Randomization was done using a standard procedure by generating random numbers in Excel sheet. The body weight and anogenital distance of pups were not used as a criterion. Randomization records are attached to the raw data file. No pups were eliminated when litter size dropped below the culling target (< 10 pups). Blood samples were collected from extra pups euthanized during litter adjustment (see Section 8.12.6). Pups were euthanized by decapitation after CO2- inhalation.
Parameters on Postnatal Day 0
After birth, pups were examined for gross malformations, sexed, individually identified, and the numbers of stillborn and live pups were recorded. The following parameters for each litter were evaluated:
Pups delivered (N)
Liverborn (N, %)
Stillborn (N, %)
Unknown vital status (N, %)
Runts (N, %)
Pups found dead (N, %)
Pups cannibalized (N, %)
Survival relative to number born (%)
Body weight
Anogenital distance normalized (mm divided to the cube root of the body weight)
Sex: females (N), males (N, %), with unknown sex (N/N)
Postnatal Survival
Each litter was examined daily for survival and abnormalities, and all deaths were recorded. A daily record of litter size was maintained and the following parameters were evaluated for each litter and reported:
Parameters Calculation / Note
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
Clinical Observations of Pups
Each litter was examined daily in the cage for survival and abnormalities. On postnatal days 4, 7 and 13, a more detailed manual clinical examination of pups with weighting was carried out.
Body Weights and Sex Determination
Each pup was weighted and sexed after parturition completion (PND 0 or PND 1 for some litter), as well as on postnatal days 4, 7 and 13. The following values for litter are reported:
Body weight (separately for males and females) per litter (g) PND 0, 4, 7, 13
Body weight gain (separately for males and females) per litter (g, %) PND 0-13
% of males per litter PND 0, 4, 7, 13

Postmortem examinations (parental animals):

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 d
iuresis was calculated (mL/kg body weight/24 h).
The following urinalysis determinations were performed in first portion of urine using strips Aution St
icks 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 administratio
n, 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 blo
od 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 us
ing 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 d
ifferential 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 fem
ales which has not delivered are presented in individual tables. The blood sample was placed in tub
e 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 bioche
mistry, 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 anticoa
gulant. 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 c
oagulometer 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 veterina
ry 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 #)

Postmortem examinations (offspring):

Blood Sample Collection in Pups
Blood samples were collected from two of the surplus pups per litter euthanized during litter adjusting on PND 4, and from two of the pups per litter at termination on PND 13. If there was only one pup available above the culling target, only one pup was eliminated and used for blood collection. Sample collection occurred at approximately the same time of the day (approximately from 10:00 to 12:00).
Pups on PND 4 were decapitated for blood collection after short-term inhalation of CO2, pups on PND 13 were anesthetized by mixture Zoletil® / Xyla® and blood was collected from inferior vena cava. Blood was collected into tubes without anticoagulant, allowed to clot for 50 min and centrifuged (1600 x g, 4 °C, 15 min) for serum separation. Serum samples from pups of the same litter were pooled for using these “litter means” as statistical unit. The cumulative sample was divided into two aliquots (if the sample volume was >200 uL) and immediately frozen at –20 °C until assayed.
For each pup found dead after birth (PND 0 or 1, pups not cannibalized or too autolyzed to examine), the lungs were removed and placed in a saline-filled jar. If the lungs sank to the bottom of the jar, the pup was considered stillborn (for found dead on PND 0). If the lungs floated, and/or there was the presence of milk in the stomach, the pup was reported as born alive unless the pup had previously been noted as alive. Tissues were not preserved.
Stillborn pups and pups found dead were necropsied using a fresh dissection technique for macroscopic observations. Tissues were not preserved. Necropsy of partially cannibalized newborns was made, if possible, to confirm the vital status (stillborn or liveborn).
At scheduled euthanasia on PND 13, one male and one female from each litter were used for thyroid evaluation. These pups were anaesthetized (Zoletil® / Xyla®) and euthanized by terminally blood collection from inferior vena cava, subjected to a gross necropsy with examination for gross abnormalities and particular attention to the external reproductive genitals; thyroid glands were preserved and weighted (after fixation). All other pups from litter were euthanized on PND 13 by CO2 inhalation, necropsied with examination for gross abnormalities and particular attention to the external reproductive genitals.
Number of Nipples/Areolae
Before scheduled euthanasia and necropsy on PND 13, the number of nipples / areolae was counted in all males and females described as a dark focal area (with or without a nipple bud) and no distinction was made between the retention of an areola or a nipple.
Macroscopic Observations
All surviving pups were subjected to a gross necropsy with examination for gross abnormalities and particular attention to the external reproductive genitals; thyroid glands were preserved and weighted (after fixation) in one male and one female from each litter.
The external genitalia were inspected in all males on PND 13 before necropsy. The following criteria were used to investigate whether male external genitals were demasculinized:
Score 0, no effect: normal genital tubercle, the urethral opening is found at the tip of the genital tubercle and the preputial skin is intact. In the perineal area, thick fur extends caudally from the base of the genital tubercle and half the distance to the anus. A furless area circumscribes the anus.
Score 1, mild dysgenesis of the external genitalia: a small cavity on the caudal surface of the genital tubercle or a minor cleft in the preputial opening is observed. The furless area around anus expands towards the base of the genital tubercle, but thick fur is still present at the base of the genital tubercle.
Score 2, moderate dysgenesis of the external genitalia: the preputial cleft is larger. The urethral opening is situated half way down the inferior side of the genital tubercle (hypospadia). Partly furless, e.g. thin fur is noted in the perineal area ranging from the base of the genital tubercle and caudally to the furless area circumscribing the anus.
Score 3, severe dysgenesis of the external genitalia: The preputial cleft is large. The urethral opening is situated further than half way down the inferior side of the genital tubercle to the base of the genital tubercle. At the base of the genital tubercle a groove extending laterally is observed (similar to control females at PND 13). The rat is totally furless in the whole perineal area.
Thyroid glands were weighted in one male and one female from each F1 litter on PND 13 after approximately 24 hours fixation.

Statistics:

see below

Reproductive indices:

Parameters Calculation / Note
Pregnancy < 21 days (N)
Pregnancy = 22 days (N)
Pregnancy > 23 days (N)
Assigned to natural delivery (N)
Pregnant (N, %)
Delivered a litter (N, %)
Duration of gestation, days The time elapsed between confirmed mating and the day of first pup was delivered
Pre-delivery findings
Gestation index (%, N/N) Number (N) of rats with live offspring / Number of pregnant rats
Significant findings during parturition
Dams with live pups at 4 postpartum day (N, %)
Dams with live pups at 13 postpartum day (N, %)
Dams with stillborn pups (N, %)
Dams with no liveborn pups (N, %)
Dams with 0 abnormal pups
Dams with 1 abnormal pups
Dams with > 2 abnormal pups
Implantation sites (a) per delivered litter (a) During necropsy
Pre-natal loss of offspring Implantations minus live births
Dams with 0
Dams with 1
Dams with 2
Dams with > 3
Post-natal loss of offspring Live births minus alive at PND 13
Dams with 0
Dams with 1
Dams with 2
Dams with > 3

Offspring viability indices:

Pups delivered (N)
Liverborn (N, %)
Stillborn (N, %)
Unknown vital status (N, %)
Runts (N, %)
Pups found dead (N, %)
Pups cannibalized (N, %)
Survival relative to number born (%)
Body weight
Anogenital distance normalized (mm divided to the cube root of the body weight)
Sex: females (N), males (N, %), with unknown sex (N/N)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 a
s early as study day 26, generally continued throughout the study, and was not considered to be tox
icologically 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 p
ost-coital day 12. This finding was transient and was not associated with the gross urogenital obse
rvations during necropsy. Female No. 125 was not pregnant, had no implantation sites, and postimpla
ntation 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 f
emales 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 v
alues 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) com
pared to other groups with an increased value of standard deviation due to the low consumption in d
am 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 ch
anged 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 granulocy
tes 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.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The following alterations in serum chemistry parameters were considered to be related to test item a
dministration:
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 k
idneys (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 a
bsence 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 hemoly
sis or hemorrhage and to the histological changes in the liver, and can be caused by the conjugatio
n 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 dysfuncti
on (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 mech
anism 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 as
sociated 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 eff
ect 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.

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 r
ecovery 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

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 o
bserved 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 a
nd 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 findin
gs in kidneys of 1000 mg/kg bw/day administered males and females correlated to the clinical pathol
ogy 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 m
icroscopically observed in one female. Myocardial hypertrophy was correlated to the increase in hea
rt 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. Bes
ides, in one female and three males from the 1000 mg/kg bw/day dose group, the mononuclear infiltra
tion 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 mul
tifocal 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 freque
ncy, 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 d
ose 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 macr
ophages 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 admini
stration.
The findings of an unclear relationship with the test item were observed in submandibular lymph n
odes, 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 su
bstance (SDS on the test item: Corrsave 100, vers. 1.0, 2019-01-24). However, findings in submandib
ular 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 (Secti
on 11.2.10.4). It can be assumed that the test item stimulates glucose uptake in adipocytes. Howeve
r, 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 eo
sinophilic 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 findi
ng 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 min
imal to slight grade with epithelium desquamation was observed with the same low frequency in all g
roups. 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, sever
ity, 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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The duration of the estrous cycle, being evaluated during the administration period before the onset of pregnancy, was approximately the same in all groups. Two females (No.126 and No.127) in the 1000 mg/kg bw/day dose group had an irregular estrous cycle that nevertheless did not affect the onset of pregnancy. The irregularity in the oestrus cycle was not statistically increased compared to the control group, and these two incidents are supposed to be not treatment-related.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

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.

Reproductive performance:

no effects observed

Description (incidence and severity):

The test item did not affect the reproductive performance of males and females at any dosage level.
In the control vehicle group, one pair was without a confirmed date of mating. Female No.82 having a regular oestrus cycle, was cohabitated with male No. 1 during oestrus. After this, the oestrus cycle stages in this female were not observed due to possible coitally induced pseudopregnancy. As a result, this female was recorded as not mated, non-gravid, and the male as not sired a litter.
All males in the 100, 300, and 1000 mg/kg bw/day dose groups were with evidence of mating and with pre-coital intervals that did not differ from the value in the control group. In the 100 mg/kg bw/day dose group, two females (No. 76 and No. 105) were not pregnant, and two males (respectively, No. 2 and No. 54) did not sire a litter. In 300 and 100 mg/kg bw/day dose groups, all males sired a litter, and indices of fertility and copulation were 100.0 %.
All females in all test item groups were with evidence of mating. In the 100 mg/kg bw/day dose group, 83.3 % of females were gravid and delivered (excluding two females discussed above). However, fertility and conception indices (83.3%) were not significantly differed from the values in the control group and historical control data (Appendix 31). One female (No.70) in the high dose group did not deliver, but had one implantation site revealed during necropsy. Indices of mating, fertility, and conception in the 300 and 1000 mg/kg bw/day dose groups were 100.0 %.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item-related mortality of offspring as well as test-item related clinical observations at the post-natal period.
The main finding of the external examination of newborns was subcutaneous hemorrhage. Subcutaneous hemorrhage was observed in the same frequency in all groups, can be found with low frequency in newborn pups, and has unclear toxicological significance. Some pups in all groups have a remaining umbilical cord. This finding is associated with the postpartum activity of the female, but its toxicological significance is also unclear. Two pups from the control group, two pups from the medium-dose group, and four pups from the high dose group (including litter No. 92 of early parturition) were small of a size. Small and runt newborns can be found in the normal rat population with a low frequency, and these newborns are not related to the test item. In general, all external findings were incidental and considered to be not treatment-related. A number of litters with noted alterations were approximately the same among groups.

Mortality / viability:

no mortality observed

Description (incidence and severity):

The mean number of delivered and liveborn pups in the test item treated groups did not differ significantly from the values of the control vehicle group and were within the range of historical control data (Appendix 31).
Two pups in each of 300 and 1000 mg/kg bw/day dose group were identified as stillborns. There were no stillborns in the control vehicle group; however, the frequency of stillborn was lower and was observed previously as single findings in the control rats. High mortality in the litter No. 92 from the 1000 mg/kg bw/day dose group is presumably associated with the poor state of the female and the early prolonged labor. There were no malformations or variations found during the external examination of newborns. Some alterations of pups were subcutaneous hemorrhages and small size of pup (see Section 11.3.3). The frequency of external alterations of newborns in the test item administered groups did not significantly differ compared to the control vehicle group.
The mean values of offspring survival on a postnatal day 0 were not changed by the test item administration.
The test item in all administrated doses did not affect the survival of the F1 offspring in the postnatal period. The mean number of liveborn pups, live litter size, and postnatal survival in all dose-treated groups did not significantly differ from the values in the control group. Three pups (one from the control group and two from each of medium and high dose group) died at the first day after birth. In the subsequent postnatal days, there were no dead, missing, or euthanized in extremis pups. On PND 4, the percent of postnatal survival decreased equally in all groups due to adjustment of litters. Viability indices were approximately similar in all groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight and body weight gain of male and female offspring in groups administered by the test item did not significantly differ in the postnatal period compared to the control vehicle group.

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

Description (incidence and severity):

The mean serum thyroxin values in PND 4 and PND 13 offspring was not significantly changed in all dose test item administrated groups compared the values in the control vehicle group and were within the range of historical data (Min – Max for PND 4, 25.21 – 34.25 ng/mL, Min – Max for PND 13, 30.22 – 67.73 ng/mL).

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The percentage of males in all dosage groups did not significantly differ from the value in the control vehicle group.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The male and female amount, body weight of newborns, absolute and normalized anogenital distance and the percentage of males in test item treated groups did not differ significantly from the values in the control vehicle group.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The test item did not cause areoles retention in male pups. On a postnatal day 13, there were no males with areolae/nipples in all groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No statistically significant changes were found in the absolute weight of the thyroid glands in male and female offspring in all dose administrated groups. In 1000 mg/kg bw/day dose group, the relative value of thyroids weight was slightly increased compared to the control vehicle group. The statistical significance of this change was observed when comparing by the paired t-test, was not dose-dependent, not correlated with the changes in thyroxin level in F1 offspring and / or in parental females. An increase in the relative value of thyroids weight can be due to the lower mean value of male final body weight in 1000 mg/kg bw/day dose group. However, the body weight value, as well as body weight gain, was not significantly altered compared to the control group. So, the increase in the relative weight of thyroid glands in PND 13 male pups of the high dose group is considered as not test item related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The visceral examination of all stillborn and died pups did not indicate any test item-related effects on the morphological development of the offspring.
The number of newborn pups necropsied on PND 0-1 as found dead was 1, 0, 4, and 4, respectively, in the control group, 100, 300 and 1000 mg/kg bw/day dose groups. Two pups from the 300 mg/kg bw/day dose group and two pups from the 1000 mg/kg bw/day dose group were stillborn with non-floating lungs and absence of milk in the stomach. Some of them had cyanosis and signs of autolysis in the abdomen. Two of four pups found dead from litter 92 (1000 mg/kg bw/day dose group) were liveborn; however, they were non-viable; dehydration and absence of milk on the stomach were noted for these pups. No gross findings were revealed during the necropsy of these pups as well as for one liveborn pup found dead from the 300 mg/kg bw/day dose group.
There were no test item-related gross observations in all pups during a scheduled necropsy on PND 13. No signs of demasculinization were found in any male in all groups.

Histopathological findings:

no effects observed

Description (incidence and severity):

No test item-related histological changes were observed in thyroid glands of male and female pups examined in postnatal day 13. In one female pup from the control group, ectopic lymphoid tissue (thymic) was observed within thyroid tissue. Ectopic thymus is a developmental abnormality that can occasionally be found in rats and mice and is generally unrelated to treatment.

Other effects:

no effects observed

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL for F0 reproductive toxicity and F1 developmental toxicity was considered to be 1000 mg/kg bw/day