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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Remarks:
90 day oral toxicity study with 21 days recovery with rats
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 APR 2020 - 10 MAR 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid
EC Number:
278-934-5
EC Name:
6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid
Cas Number:
78521-39-8
Molecular formula:
C13H19NO4S
IUPAC Name:
6-(4-methylbenzenesulfonamido)hexanoic acid
Test material form:
solid: particulate/powder
Details on test material:
white powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Species: Rattus spp.
Strain: Sprague-Dawley (SD), outbreed
Outbred Sprague Dawley (SD) rats recognized as an appropriate animal model for toxicity studies were used as the test system in this study and the prenatal toxicity study for the ASCplus®.
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)
- Age at study initiation: Approximately 6-7 weeks old
- Weight at study initiation: Body weight at first day of dosing, g (MEAN ± SD): Males: 147 ± 12, N = 52; Females: 129 ± 10, N = 52
- Housing:
Husbandry practice meets the standards defined by the Directive 2010/63/EU on the protection of animals used for scientific purposes. Suitable ranges of environmental parameters are chosen in agreement with The Guide for Care and Use of Laboratory Animals (National Academy Press, Washington D.C., 2011).
Cages: All animals were housed in solid bottom polycarbonate cages (Type IV, 598 х 380 х 200 mm (LxWxH), 2272 cm sq., Tecniplast s.p.a.) with bedding. Cages are equipped with steel lids, steel separators for the food and steel label holders. The environmental enrichment material Lignocel Nesting Ball (JRS Germany) and red transparent polycarbonate igloo was provided in all cage.
During adaptation, the animals were housed by groups. After the assigning to the experimental groups, the animals were kept two in a cage.
Bedding: Commercial autoclaved woodchip bedding was used (LIGNOCEL BK 8/15, JRS, GmbH). A document from the manufacturer on the composition and quality of the bedding is placed in the study file as raw data. BTL BIBC RAS routinely tests the bedding for microbiological contamination. Results of analyses are kept in an archive at the Test Facility. The copy of the latest check is placed in the study file as raw data.
- Diet (e.g. ad libitum): The animals were fed Laboratory Rodent Diet (SSNIFF V1534-300 autoclavable, Spezialdiaten GmbH, Ferdinand-Gabriel-Weg 16, DE-59494 Soest, Germany) ad libitum. This diet is analyzed by the manufacturer for nutritional components and environmental contaminants and routinely by BTL BIBC RAS for microbiological contaminants. Results of analyses are kept in archive at the Test Facility. The copy of the latest check is placed in the study file as raw data.
- Water (e.g. ad libitum): Filtered by MilliRO tap water was provided ad libitum in standard water bottles. Samples of water are analyzed routinely for microbiological contaminants. Results of analyses are kept in archive at the Test Facility. The copy of the latest check is placed in the study file as raw data.
- Acclimation period: The animals were received from Lab Animals Breeding Center “Pushchino” (Nauki 6, Puschino, Moscow region, Russia 142290 (www.spf-animals.ru) at the age of 4 weeks 14.04.2020. The general clinical examination of each animal was done on the day of receipt.
During adaptation/acclimatization, animals were kept in groups (by sex) in the barrier zone 1 of facility and animal’s condition was evaluated daily by cageside observation.
- Health and physiological status: Just weaned animals of 4 weeks old with defined microbiological health status. The animal health monitoring is performed by breeder under FELASA-guidelines quarterly in AnLab, s.r.o. (Czech Republic). The result of the last check is attached to the raw data.

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Actual mean temperature ranged from 20 °C to 24 °C.
- Humidity (%): Mean relative humidity ranged from 30 % to 70 % There were the deviations from indicated humidity on some dates, which did not negatively influence the animal condition.
- Air changes (per hr): Renewal of the room air at least 12 times hourly
- Photoperiod (hrs dark / hrs light): Automatic change of day and nighttime (08:00-20:00 - "Day", 20:00-08:00 - "Night")

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The vehicle and test item were administered orally by gavage, via an appropriately sized stainless steel ball-tipped dosing cannula connected with a syringe once daily during 90 days. A separate cannula for each group was used. The dosage volume for all groups was 10 mL/kg bw. Individual dosages were based on the last value body weights to provide the correct mg/kg bw/day dose. The total volume of the formulation was constantly stirred during sampling for dosing. All animals were dosed at approximately the same time each day in the first half of the day (09:00 – 13:00).
Vehicle:
water
Remarks:
Control Item (Vehicle) Name in the study: Vehicle Composition: Distilled water Manufacturer/Provider: Tap water filtered in BTL using MilliRO (Millipore) system Storage conditions: room temperature (15-25°C)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in a required volume of the vehicle (distilled water) in order to achieve the homogenous suspensions. Formulation suspensions were thoroughly homogenized using a magnetic stirrer and sonication.
Test item formulations were prepared every four days, aliquoted to the required volumes of days of the administration and stored in tightly closed glass jars at room temperature in the dark. For the control group, the required volume per day of distilled water was placed in a labeled jar.
On the day of dosing, the aliquot of each formulation was mixed thoroughly and transferred to the barrier zone.

VEHICLE
- Justification for use and choice of vehicle: The test item was suspended in water as in recommended vehicle be considered first. This vehicle was also used in the previously conducted screening toxicity study of the test item according to OECD guideline 422.
- Concentration in vehicle: 10, 40, and 160 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
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 the detection of the test item concentration in-vehicle formulations. The validation of the analytical method was performed before the first analysis of dose formulations, where linearity, accuracy, precision/repeatability, specificity/selectivity, sensitivity (LLOQ) were established, and stability of formulation samples was assessed for 7 days of storage. The method validation report is attached to this study report.

Stability, Homogeneity and Concentration:
The stability of the test item in the vehicle (water) prepared at concentrations of 10 and 160 mg/mL was confirmed following storage for 7 days at room temperature (the temperature range, 20 – 25 °C) during method validation study. Besides, homogeneity analysis was performed for formulations of 10 and 160 mg/mL after 3 days of storage after re-mixing.
Analysis of formulations for homogeneity and concentration during the dosing period was conducted in the test facility (BTL BIBC RAS) approximately every two weeks using a validated method.
For homogeneity analysis, quadruplicate samples (approximately 0.1 mL of each) were collected from the top, middle, and bottom strata of each dosing formulation.
For concentration analysis, quadruplicate samples were collected from the middle stratum of each dosing formulation (including the vehicle control group). Samples collected from the mean stratum for homogeneity analysis used for this purpose.
A pair of quadruplicate samples from each stratum was used for analysis, and the other pair was stored as back-up samples at room temperature in tightly closed flasks, analyzed if necessary based on primary assays to verify concentration and were discarded after the study director's approval of the analytical results.
On some date on analysis, 3-4 samples were taken from each level of 160 mg/mL formulation due to the problematic homogenization of this high concentration and variability of the analyzed concentration between samples.
Acceptance criteria for the formulations analysis are based on the test item in vehicle composition as a suspension. The actual concentration of analyzed samples collected from the mean stratum of formulations should be within the range of 85% - 115% of the target concentration. The acceptance criteria for homogeneity are RSD<15% (for suspensions), with the mean concentration within 85% to 115% of the target concentration.
Duration of treatment / exposure:
once daily during 90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
21 day recovery group with 6 males and 6 females
Dose / conc.:
1 600 mg/kg bw/day (actual dose received)
Remarks:
21 days recovery group with 6 males and 6 females
Dose / conc.:
1 600 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
No. of animals per sex per dose:
10 sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels 100, 400, and 1600 mg/kg bw/day with 4-fold optimal interval were selected for the current study based on data for conducted screening toxicity study of the test item according to OECD guideline 422.
- Rationale for animal assignment (if not random): For assignment to the experimental groups, all animals were weighed, and clinical observations were recorded. Animals without clinical signs of health abnormalities were allocated to the experimental groups, according to a stratification procedure, so that the average body weight of each group did not statistically differ. Animals were transferred to the experimental room of the barrier zone 2. Animals not assigned to study were transferred to the BTL BIBC stock colony.
- Fasting period before blood sampling for clinical biochemistry: Animals were fasted overnight (approximately 16-18 hours) prior to euthanasia and blood collection being in metabolic cages.
- Rationale for selecting satellite groups: An additional satellite group of six animals per sex was included in the control and the top dose group for observation after the treatment period for the potential reversibility or persistence of any toxic effects.
- Post-exposure recovery period in satellite groups: 3-weeks recovery

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon at the same time, for morbidity and mortality. Each animal was also observed for signs of toxicity approximately 30 minutes following dose administration. In addition, the presence of findings at the time of dose administration was recorded for individual animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were recorded for all animals before first dosing and regularly on a weekly basis throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded during groups assignment, on the first day of dose administration, weekly thereafter throughout the study (at the same day as evaluation of food consumption: days 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 90, 98, 105, and 111), and prior to the scheduled euthanasia (day 91 or day 112). Mean body weights and body weight changes (g, %) from day 1 are presented.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was assessed for each cage quantitatively by weighing of feeder (cage lid) for the periods beginning of the day and 24 hours after. Food consumption was recorded prior to the initiation of dose administration (day 0-1), and weekly thereafter throughout the study (days 6-7, 13-14, 20-21, 27-28, 34-35, 41-42, 48-49, 55-56, 62-63, 69-70, 76-77, 83-84, 89-90, 97-98, 104-105, and 110-111). Food consumption are reported as g/kg of bw/day.

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmic examinations were done in each animal once in the pre-treatment period and at the end of the in-life period by direct ophthalmoscopy using Ri-mini® Riester ophthalmoscope. The external observation (conjunctiva, tears and exudation, pupillary reflex) of both eyes was done followed by an examination eye structure after applying mydriatic agent (0.5 % Cyclopentolat, A.S.S.S Novartis Farmaceutica, S.A., Spain). The cornea, anterior chamber, iris, lens, vitreous body, and fundus (posterior vitreous, retina, optic nerve head) were examined using a modified Hackett-McDonald scoring system.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked: The blood samples in K3EDTA 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/Monocytes (MON #)
Granulocytes (GRA #),
White Cell Differential by manually counting in blood smear was no done, because there were no changes in WBC in any treated group.
Reticulocytes were counted manually in 1000 red blood cells stained with 1% cresyl blue dye.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in all males and females at the end of an in-life phase. Blood samples for clinical pathology evaluations were collected from all surviving animals at the scheduled necropsies (as a part of euthanasia): at the next day after 90 days treatment period or following three weeks post-treatment (day 112)
- Animals fasted: Yes
- How many animals: all
- Parameters checked:
Coagulation: Activated partial thromboplastin time (APTT), Prothrombine time (PT), Fibrinogen (Fg)
Serum Chemistry: Total protein, Urea, Albumin, Uric acid, Globulin (by calculation), C-reactive protein, Albumin/Globulin ratio (by calculation), Total bilirubin, Alkaline phosphatase (ALP), Total Cholesterol, Aspartate aminotransferase (AST), Triglycerides, Alanine aminotransferase (ALT), Low-density lipoproteins (LDL), Total creatine kinase (Total CK), High-density lipoproteins (HDL), Glutamate Dehydrogenase (GLDG), Calcium, Gamma-Glutamyl Transferase (GGT), Inorganic phosphates, Creatinine, Chloride, sodium and potassium ions (measured by ion-selective electrodes using analyzer EX-D (JOKOH Co, Ltd) (SOP Chem/27)), Glucose level (estimated in the drop of blood before animal anesthesia by electrochemical method using Sattelite Express® glucometer (ELTA, Russia) routinely controlled with control strip)

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: in all males and females at the end of an in-life phase
- Animals fasted: Yes
- How many animals: all
Thyroid hormones (thyroxin (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH)) were assayed in serum from all males and females by competitive inhibition enzyme immunoassay technique using relevant ELISA kits (see below) and Multiskan™GO Microplate Spectrophotometer (Termo Scientific) and according to standard procedure of manufacturer and SOP of BTL BIBC RAS.

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis determination was performed in all animals before scheduled euthanasia
Before scheduled euthanasia (day 90-91 or day 111-112 for recovery subgroup), all animals were placed in metabolic cages (Tecniplast s.p.a) overnight to measure diuresis and urinalysis and for a sampling of urine for future analytical assay, if requested. Water was supplied to the metabolic cage in a drinking bottle, food was not provided.
The urine volume was determined the day after the animal had been in the metabolic cage for about 16 hours. Diuresis was calculated in mL/kg bw/24 hrs.
Samples of urine were collected after diuresis and urinalysis measurement individually for each animal into the appropriate tubes and frozen (-70 °C) until the Sponsor’s decision for assay or sample destruction. If the analytical procedures requested, they will be described in a separate study plan.
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: Glucose, Specific gravity, Protein, Blood cells, Bilirubin, Ketones, Urobilinogen, Nitrites, pH, Leukocytes

OTHER:
Functional Observational Battery (FOB) and locomotor activity data were recorded for half males and females at the end of the dosing period.
Functional Observation Battery Assessment (FOB):
FOB assessments and locomotor activity were recorded for half males and females from each dose group at the end of the dosing period (day 88). Animals were tested at the same time on each day following approximately 30 minutes of dosing in a randomized order. Recovery satellite subgroup animals were tested approximately at the same time after 3 weeks post-treatment (day 109).
All animals were observed for the following FOB parameters using score system as described in SOP Neu/7 of BTL BIBC RAS:
Locomotor Activity Count:
Locomotor activity was measured after FOB handling observation automatically using a personal computer-controlled system Multiple Activity Cage 47420 with CUB 2005 v.3.0.15 software, Ugo Basile utilizes a series of infrared photobeams surrounding a clear plastic, rectangular cage to quantify each animal’s motor activity. Each animal was tested separately for a 6-minute period to measure the following parameters (relative values based on the number of interruptions in the signal of infrared photobeams):
Horizontal activity, count
Vertical activity, count
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were schedule sacrificed after the 90-day treatment (day 91) or 3-weeks recovery (day 112).
A complete necropsy was conducted on all animals 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.
(Details are given under 'Any other information')

HISTOPATHOLOGY: Yes
Tissues (see respective table under ‘Any other information’) from all animals in the control and high-dose groups (in the main subgroup euthanized on day 91) were be trimmed, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically.
In other dose groups and recovery subgroups, the liver, kidney, thyroid glands, adrenals, testes, mammary glands (male), and salivary glands (female) were examined as potential target organ based on histopathological examination of tissues from the high-dose group and other parameters (organ weights, clinical pathology, etc.).
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.
Statistics:
Statistical Methods
All statistical tests were performed separately for each sex using Microsoft Excel (descriptive statistics) and statistical software Statistica for Window v.7.1 to compare the treated groups to the control group. Descriptive statistics (mean, standard deviation (SD), and N) was presented for all measurement data and shown in the summary tables. Urinalysis parameters were presented in quartiles (Median, 25th and 75th percentile).
Continuous data variables (mean body weights, body weight gain and food consumption data) were analyzed by multi-factor analysis of variance ANOVA-2, followed by the Duncan test, to determine inter-group differences.
Clinical pathology values and Functional Observational Battery data values were analyzed by a parametric one-way analysis of variance (ANOVA) with a post-hoc Dunnett's test to compare the treated groups to the control group. The t-test was used additionally to compare each dose group with control value. Functional Observational Battery parameters which yield scalar or descriptive data were analyzed by Fisher's Exact Test. Gamma glutamyltransferase data and urinalysis data were subjected to the Kruskal-Wallis nonparametric ANOVA test with Dunn’s test.
Organ weights (absolute and relative to body weights and relative to brain weights) were subjected to a parametric ANOVA test and Dunnett's test as described above. The t-test was used additionally to compare the organ weights of each dose group with the control value. Histopathological findings of each treated group were compared to those of the control group by the Fisher's Exact test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No test item related clinical findings were revealed in any dose group.
(Details on the results see 'Any other information'; Tabular summary of the results see attachment)
Mortality:
no mortality observed
Description (incidence):
There was no morbidity and mortality of animals caused by the test item administration.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The male body weight gain was slightly non-significant decreased in 400 and 1600 mg/kg bw/day dose groups. On the contrary, in females treated with the high dose, there was a slight increase in body weight by the end of the administration period.

The slight changes in the body weight were statistically insignificant and considered toxicologically unimportant.

(Details on the results see 'Any other information'; Tabular summary of the results see attachment)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption in the 100, 400, and 1600 mg/kg bw/day dose treated groups was approximately similar to that in the vehicle control group during all study days.
(Tabular summary of the results see attachment)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item related abnormalities in the eyes in all doses treated groups. In one female (No. 82) in the 400 mg/kg bw/day dose group, a punctured penetrating wound of the cornea with a pin trace on the lens was found in the right eye, accompanied by chromodacryorrhea in this eye observed beginning day 27. These findings in one eye are supposed to be caused by accidental mechanical damage to the eye and are not treatment-related.
(Tabular summary of the results see attachment)
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No test item related hematological changes were revealed in any dose groups. In males, hematological parameters were approximately similar in all groups. In females, the slight dose-dependent increase in hemoglobin level (HGB) can be noted. In the 1600 mg/kg bw/day dose group, this increase by 3.8 % (136 ± 6 g/L versus 131 ± 5 g/L) was associated to the same increase in hematocrit (HCT) value (0.409 ± 0.020 L/L versus 0.394 ± 0.014 L/L). These findings were slight, non-adverse, and presumably can be associated with increased diuresis in females and mild dehydration.
After the three-week high dose post-treatment, the hematological parameters were similar to the values in the control vehicle group in both males and females.

In females treated with the test item, the shortening of activated partial thromboplastin time (APTT) was observed. This effect was dose-dependent with statistical significance in the 400 mg/kg bw/day dose group (14.7 ± 1.7 s, p < 0.05) and 1600 mg/kg bw/day dose group (14.6 ± 1.3 s, p < 0.01) compared the control value (16.2 ± 1.0 s). Prothrombin time (PT) did not significantly change in females; however, the mean value in the 1600 mg/kg bw/day dose group was slightly decreased (17.4 ± 1.7 s versus 18.4 ± 1.8 s in the control group). In the recovery female high dose subgroup, the coagulation parameters were similar to the control group.
In males, there were no significant changes in the APTT, PT, and fibrinogen.

(Tabular summary of the results see attachment)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males of the 1600 mg/kg bw/day group, the decrease in cholesterol (by 14.2 %, p < 0.05) and triglycerides (by 31.0 %, p < 0.01) was observed. These changes were dose-dependent and recoverable. In addition, males treated with 400 and 1600 mg/kg bw/day doses had decreased glucose (4.5 ± 0.4 and 4.5 ± 0.3 mmol/L, versus 5.2 ± 0.7 mmol/L in the control group, p < 0.05), elevated urea, respectively, by 16.9 % and 12.4 % (10.4 ± 0.9 and 10.0 ± 1.4 mmol/L versus 8.9 ± 0.6 mmol/L), and significant increase in urea:creatinine ratio (162 ± 26 and 174 ± 21 versus 142 ± 13) with the decrease in creatinine in the high dose group (by 9.5 %, p < 0.05). The elevated urea associated with an increase in urea:creatinine ratio, hypolipidemic, and hypoglycemic effect is considered to reflect a non-adverse catabolic state rather than kidney damage. In females, there were no statistically significant changes in serum chemistry parameters.

(Details on the results see 'Any other information'; Tabular summary of the results see attachment)
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Neither the male animals nor the female animals of the dose groups showed any treatment-related changes. Only in the male recovery groups, there seems to be a lower T4 serum level in the 1600 mg/kg bw/day dose group than in the control animals. A comparison with the historical data shows that this is a merely statistical effect caused by accidentally low variances of the control and the dose group T4 levels.

(Details on the results see 'Any other information'; Tabular summary of the results see attachment)
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In the 1600 mg/kg bw/day dose group, the statistically significant increase in the urobilinogen level was observed in both males and females (p < 0.01, p < 0.05), that considered as test item related. Moreover, in males of this group, the pH was decreased (p < 0.01) compared to the control value. The excretion of urobilinogen is increased during liver damage or hemolysis. In animals treated with the test item, there were no hemoglobinemia and serum bilirubin changes. On the other hand, the excretion of urobilinogen is a pH-dependent process [Levy M. et al,1968] and, at least in males, an increase in urinary urobilinogen in the high dose group correlated with a decrease in urine pH.
In the hight dose recovery subgroup, the median value of urobilinogen still remained increased, but not significantly. Moreover, the increased specific gravity was noted in post-treated males with unclear relation to the test item.

Diuresis measured as mL/kg body weight/24 hours in the test item-treated males did not significantly differ from the control group after the 90-day administration period.
In females of 1600 mg/kg bw/day dose group, the calculated diuresis was slightly increased (57.8 ± 18.6 mL/kg/24 hrs, p < 0.05) compared to the control group 38.3 ± 19.7 mL/kg/24 hrs). However, diuresis has high variance values, and the mean parameter value in the vehicle control group was lower than the historical control value, making it difficult to interpret the obtained change as test item-related.

(Tabular summary of the results see attachment)
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
(Details on the results see 'Any other information'; Tabular summary of the results see attachment)
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw.: no treatment related changes,
400 mg/kg bw.: males: no treatment related changes, females: increased liver weights,
1600 mg/kg bw.: males and females: increased: liver weights and kidney weights.
No changes were seen at the end of the 21 days recovery period.

(Details on the results see 'Any other information'; Tabular summary of the results see attachment)
Gross pathological findings:
no effects observed
Description (incidence and severity):
The gross finding related to the test item was revealed in one female (No.85) from the 400 mg/kg bw/day dose group. This female had dark kidneys associated with a tubular brown pigmentation in the cortex of moderate grade (presumably lipofuscin accumulation).
Macroscopic findings in the thymus, lymph nodes, spleen, and thyroids were noted as well in the test item-treated groups as in the control group.
One female (No. 82) in the 400 mg/kg bw/day dose group had unilateral corneal injury discussed above, focal alteration of texture of urinary bladder, and unilaterally enlarged ovary. These findings were considered incidental and not related to the test item administration.

(Details on the results see 'Any other information'; Tabular summary of the results see attachment)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver, fatty change, male:
0 mg/kg bw.: 2/10,
100 mg/kg bw.: 0/10,
400 mg/kg bw. : 1/10,
1600 mg/kg bw.: 4/10,
Liver: Fatty change, female:
0 mg/kg bw.: 3/10,
100 mg/kg bw.: 3/10,
400 mg/kg bw. : 6/10,
1600 mg/kg bw.: 9/10,
Hepatocellular hypertrophy, female:
0 mg/kg bw.: 0/10,
100 mg/kg bw.: 1/10,
400 mg/kg bw. : 0/10,
1600 mg/kg bw.: 6/10,

A special view has to be taken on the histopathology of the kidneys. The kidneys of the control animals of both sexes show a high incidence of chronic
Kidneys, progressive nephropathy. With the control animals, we have an incidence of 7/10 (=70%) males and 6/10 (=60%) females, which is still higher than the historical controls (40% males, 25% females) and has most obviously no relation to the treatment. In comparison to the frequency of these changes, all the other more or less dose-related changes had a much lesser and inconsistent incidence, and glomerulosclerosis and tubular focal necrosis can be seen as directly connected to the chronic progressive nephropathy and are probably not a treatment-related change.
In the kidneys, lipofuscinosis and calculi were seen in a dose-related incidence. Therefore a treatment-related change could not be excluded.

Lipofuscinosis, male:
0 mg/kg bw.: 0/10,
100 mg/kg bw.: 1/10,
400 mg/kg bw. : 1/10,
1600 mg/kg bw.: 4/10,
Kidney, Lipofuscinosis, female:
0 mg/kg bw.: 2/10,
100 mg/kg bw.: 1/10,
400 mg/kg bw. : 4/10,
1600 mg/kg bw.: 0/10,
Kidney, Calculus in pelvis, female:
0 mg/kg bw.: 0/10,
100 mg/kg bw.: 0/10,
400 mg/kg bw. : 3/10,
1600 mg/kg bw.: 0/10,

Thyroid glands, C-cell hyperplasia, male:
0 mg/kg bw.: 0/10,
1600 mg/kg bw.: 3/10.
Thyroid glands, C-cell hyperplasia, females:
0 mg/kg bw.: 2/10,
400 mg/kg bw. : 6/10
1600 mg/kg bw.: 6/10

Testes, Hyperplasia, Leydig cells:
0 mg/kg bw.: 0/10,
400 mg/kg bw. : 4/10,
1600 mg/kg bw.: 4/10.

Despite the kidneys histopathology revealed, no tissue damage but adaptive reactions to the treatment with ASCplus in dosages of 400 and 1600 mg/kg bw for 90 days. In the kidneys, lipofuscinosis and calculi were seen in a dose-related incidence. Therefore a treatment-related change could not be excluded.

After a recovery period of 21 days, no treatment-related changes were observable.

(Details on the results see 'Any other information'; Tabular summary of the results see attachment)
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
(Details on the results see 'Any other information'; Tabular summary of the results see attachment)

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
urinalysis

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Result Test Item Stability in Formulation (tabular summary of results see attachment)


The stability of the test item ASCplus in the vehicle (water) prepared at concentrations of 10 and 160 mg/mL was checked following 7 days of storage at room temperature (20 - 25 °C) during method validation study. The actual concentration of the storage formulation sample was compared to the value on Day 0, and the concentration of freshly prepared sample used as Time 0 data for additional comparison of stability in case of variations or preparation errors.


The mean concentration of 10 mg/mL formulation was within the acceptable range of the target concentration (85 – 115 % with RSD < 10 %). In some analysis date, the percentage of concentration level was lower of acceptable range if compared to the Day 0 and/or Time 0 level with the maximum deviation on Day 5 (84.3 % and 79.7 %, respectively). However, on Day 7 of storage, the mean concentration of 10 mg/mL formulation was within the acceptable range of both point zero values. So, some deviations in actual concentration are considered to be due to errors in the assayed samples' preparation and not decrease instability.


The mean concentration of the high-level formulation of 160 mg/mL was within the acceptable range of the target concentration (85 – 115 % with RSD < 10 %). It should be noted a tendency to increase target concentration from Day 0 through shelf life. This finding may be due to the poor solubility of the test substance in water and the difficulty of homogenization in water. It also led to relatively high values of the percentage difference between the mean concentration of the storage sample and the values on Day 0 and Time 0. On storage Day 7, these differences were 120.0 % and 137.5 %, which is higher than acceptable at 115 %. However, the preparation of the formulations was every 4 days, and on Day 4 of storage, the percentage difference from fresh samples was in the acceptability range (88.9 - 113.7%), which met the acceptance criteria for suspension stability (>85% of the pre-storage value with <10% RSD).



It was concluded that the formulations of the test item ASCplus in a vehicle (suspension in water) are stable for at least seven days of storage at room temperature (20 - 25 °C). Some deviations in actual concentration are assumed to be caused by the test item homogenization difficulties in the water.


 


Dose Concentration and Homogeneity (tabular summary of results see attachment)


The re-mixing homogeneity assessments of the 10 and 160 mg/mL formulations prepared 22.04.2020 and analyzed after 4-day storage and re-mixing met the BTL BIBC SOP requirement, i.e., the RSD for the mean concentration was ≤15% at a concentration within the acceptable limits (within 85% to 115% of target concentration).



During the dosing period, the homogeneity assessment of the ASCplus® dose formulations of 10, 40, and 160 mg/mL met the BTL BIBC SOP requirement, except for the following points. The mean actual concentration of 40 mg/mL formulation was less of the target range value on date 05.06.2020 (80.2 %). The mean actual concentration of 160 mg/mL formulation was less of the minimally acceptable target concentration on 04.05.2020 (81.9 %) and 05.06.2020 (78.8 %).


For dose formulations 10 mg/mL, the analyzed concentrations were within the acceptable range of 85-115% of the target concentration at all analysis dates. For dose formulations of 40 and 160 mg/mL, deviations from target concentration were revealed for some dates. For dose formulations 40 mg/mL, the analyzed concentration was less of the acceptable target level on 05.06.2020 (84.4 %). The actual concentration of the test item in the 160 mg/mL formulation was less of the acceptable target value, approximately by 5 % on date 04.05.2020 and by 4 % on date 05.06.2020. No test item was detected in the analyzed vehicle administered to the control group (Group 1).



The changes in actual dose concentration were slight and supposed to be not critical. Deviations in 160 mg/mL concentration are assumed due to the difficulty of homogenizing a high dose formulation of the test item insoluble in water and the small increase in the volume of the finished formulation (approximately by 4 %). Based on the analysis data, the mean actual concentration of the high dose formulation was approximately 138 mg/mL for the all administration period, which lies within the acceptable range, closer to the lower bound.


 


Clinical Observations


No test item related clinical findings were revealed in any dose group.
In one female from the 100 mg/kg bw/day dose group (No.77), the body weight loss with a hunched posture and wheezing were recorded beginning day 16. The formulation aspiration is suggested as a likely cause of the observed clinical findings. The dose was failed to gavage for 5 days (at the period from day 16 to day 20, and the administration continued further without deviations with a positive body weight gain and the absence of wheezing. No associated gross observations were revealed in this female during scheduled necropsy.
In one female treated with 400 mg/kg bw/day dose (No.82), the unilateral chromodacryorrhea was recorded beginning from day 27 thought the all administration period. The ophthalmoscopy has revealed the corneal injury, which is not related to the test item.
In two males from the control group and low dose group, the non-treatment related focal alopecia was noted starting from the approximately eighth week of dosing.


 


Body Weights and Body Weight Gain


The absolute body weight of males was slightly dose-dependently decreased after approximately one-month dosing (Figure 1). To the end of the administration period, this statistically non-significant decrease was 3.5 % in the 100 mg/kg bw/day dose group (362 ± 26 g), 4.8 % in the 400 mg/kg bw/day dose group (357 ± 30 g), and 4.3 % in the 1600 mg/kg bw/day dose group (359 ± 26 g), respectively, compared to the control value (375 ± 33 g). The body weight gain in gram was statistically reduced in the high dose group to the day 56 of dosing, and the total percentage gain was non-significantly decreased compared the value in the control group (155.6 ± 25.0 %) in all dose groups: 148.9 ± 26.6 %, 143.2 ± 15.4 %, and 143.4 ± 20.5 %. After three weeks of recovery in the 1600 mg/kg bw/day dose group, the decrease in body weight remained noticeable (371 ± 27 g versus 390 ± 43 g), and the total percentage body weight gain differed by 15.8% the value in the control group (177.1 ± 8.0 % versus 192.9 ± 20.8 %).
In females treated with the test item, body weight did not decrease, and even on the 1600 mg/kg bw/day dose, there was a slight increase in body weight: by 3.7 % (227 ± 24 g) compared to the control group (219 ± 18 g) by the end of the administration period. The increase in female body weight in the high dose group was non-significant, also noted after the recovery period, and is supposed to be non-adverse.


 


Functional Observation Battery


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 88 (end of the treatment period). After a three-week recovery period, the FOB parameters in 1600 mg/kg bw/day dose group were also comparable with the control group.


 


Locomotor Activity


Locomotor activity patterns (horizontal movements and vertical activity) registered for 6 minutes were unaffected by test item administration at all doses when evaluated on day 88 (end of the treatment period) in males and females. After a three-week post-treatment period, the parameters of locomotor activity in the 1600 mg/kg bw/day dose group were comparable with the control group in both males and females.


 


Serum Chemistry


The following alterations in serum chemistry parameters were considered to be related to test item administration:
Males from the 400 and 1600 mg/kg bw/day dose groups had elevated urea level, respectively, by 16.9 % (p < 0.01) and 12.4 % (p < 0.05 using paired t-test) (10.4 ± 0.9 and 10.0 ± 1.4 versus 8.9 ± 0.6 mmol/L in the control group).


In addition, in the 1600 mg/kg bw/day treated males, the statistically significant decrease in creatinine (by 9.5 %, p < 0.05) and increase in urea:creatinine ratio (174 ± 21 versus 142 ± 13, p < 0.01) were observed


In contrast to the urea level, the increase in the urea:creatinine ratio was dose-dependent, and the change in these parameters is considered to be related to the test item. Urea and serum creatinine are routinely used to measure kidney damage; however, creatinine is a poor indicator of renal function in the situation of its reduction. Elevated urea:creatinine ratio may be recorded in the context of the catabolic state due to starvation or corticosteroids. The relative kidney weight was increased in the high dose male group; however, the histological alterations associated with the altered renal function were observed only in single males.


Moreover, males in the 1600 mg/kg bw/day dose had a slightly non-significant increase in total creatine kinase (by 21.3 % versus control group) and decrease in glucose level (4.5 ± 0.3 mmol/L versus 5.2 ± 0.7 mmol/L in the control group, p < 0.05) associated with a slight decrease in body weight and unchanged food intake. A decrease in serum creatinine and glucose levels on the ASCplus was not excluded due to the hypolipidemic effect of ASCplus and the catabolic status of treated animals. However, reduced serum creatinine and glucose were slight and remained within the historical control range, so these changes are not considered as a toxicological effect of the test item.



In addition, in males of the 1600 mg/kg bw/day dose group, the decrease in cholesterol (by 14.2 %, p < 0.05), triglycerides (by 31.0 %, p < 0.01) was observed with visible tendency to the decrease in Low-Density Lipoproteins (LDL) (by 20.0 %, not significant due large variability). The change in triglycerides was dose-dependent, significant, and considered toxicologically relevant. The decrease in the total cholesterol level was slight compared to the vehicle-treated group, remained at the same time historical control level, and considered implicit. Notably, after a withdrawal period, lipid levels in the high dose group recovered and were even slightly higher than control levels (not significant).



Interestingly, in females of all test item treated groups, there were no significant changes in serum chemistry parameters. However, it is noticeable that the mean cholesterol and High-Density Lipoproteins (HDL) level was slightly increased (not significantly) in the high dose group (2.58 ± 0.49 mmol/L and 0.86 ± 0.13 mmol/L versus 2.32 ± 0.41 mmol/L and 0.79 ± 0.10 mmol/L in the control group) following the tendency towards a decrease in cholesterol and HDL in the 400 mg/kg bw/day dose group (respectively, 2.08 ± 0.63 mmol/L and 0.71 ± 0.20 mmol/L) as well as a slight decrease in glucose (4.7 ± 0.6 mmol/L versus 5.0 ± 0.4 mmol/L). Such an inverse dynamic of lipid levels in females was non-significant; however, it correlated with the dynamics of body weight and microscopic findings in the kidneys and liver in the test item treated females. Females in the 1600 mg/kg bw/day dose group had a slightly increased body weight (by 3.7 %) to the end of the administration period, hepatocellular hypertrophy, and kidneys without pigmentation in tubules. Whereas 400 mg/kg bw/day dose female group showed the most incidence and severity of renal tubular pigmentation (presumable lipofuscinosis), there was no noticeable hypertrophy of hepatocytes.
So, the main clinical chemistry findings in male and female rats treated with ASCplus are related to serum lipids change. These observations are supposed to be due to the test item (or its primary metabolites) influence on cholesterol exchange via peroxisome proliferator-activated receptors, PPARs (discussed below); they were reversible and non-adverse. The revealed sex difference in lipid change is considered to be associated with a faster and more pronounced activation of the liver's metabolic enzyme system in females. No clinical chemistry findings were found to be biomarkers of any adverse changes in the liver and kidney.


 


Thyroid Hormones Assay Data


After the 90 day of ASCplus administration, the triiodothyronine (T3) level was decreased in the 1600 mg/kg bw/day dose (males: by 15.6 %, 1.417 ± 0.191 ng/mL versus 1.679 ± 0.389 ng/mL in the control group; females: by 8.4 %, 2.063 ± 0.461 ng/mL versus 2.251 ± 0.342 ng/mL). This change was not significant, however, with notable dose-dependence in males. Moreover, the statistically significant decrease in thyroxine (T4) level was observed in the high dose post-treatment males (by 13.1 %, 33.26 ± 2.38 ng/mL versus 38.28 ± 1.79 ng/mL), which was accompanied by a remaining low level of triiodothyronine 1.338 ± 0.168 ng/mL versus 1.515 ± 0.201 ng/mL). A decrease in the mean thyroxine level was also noted in 1600 mg/kg bw/day dose post-treatment females (37.88 ± 6.10 ng/mL versus 43.17 ± 3.89 ng/mL, non-significant).
A decrease in serum level of thyroid hormones was associated with increased liver relative weight in the high dose treated males and females and hepatocellular hypertrophy in females of this group. We did not assay microsomal enzymes in the study. However, there is evidence that an increase in liver weight correlates with the induction of microsomal enzymes, although the degree of induction does not necessarily correlate with the severity of an increase in liver mass or the degree of hepatocellular hypertrophy in rats [Hall A. et al, 2012]. Xenobiotic-dependent induction of liver enzymes can result in lower serum thyroid hormones [Li A. et al, 2019]. Enhanced thyroid hormones clearance from the serum due to chemically-induced hepatic enzyme up-regulation is recognized as a relevant endocrine mode-of-action [OECD 2018]. Thyroid hormones regulate the circulating level of TSH by negative feedback, the sustained increase of which influence of thyroid morphology as a standard end-point for thyroid toxicity evaluation.
There was no significant increase in the TSH level; however, in the 1600 mg/kg bw/day dose male group, the mean level of TSH exceeded by 17.3 % the level in the control group (0.774 ± 0.331 uIU/mL versus 0.660 ± 0.377 uIU/mL). In females of 1600 mg/kg bw/day dose recovery subgroup, the mean value of TSH was increased by 31.1 % (1.164 ± 0.377 uIU/mL versus 0.888 ± 0.245 uIU/mL). The change in the TSH level may be associated with the Hypothalamo-Pituitary-Thyroid feedback action, leading to pathomorphology changes in the thyroid gland. However, the increase in TSH in the high dose group was slight, and no indications of TSH-mediated thyroid gland activation were found, such as an increase in thyroids weight, thyroid hypertrophy, or follicular hyperplasia.
Thus, the test item ASCplus lowered the thyroid hormones in rat male and female in the dose of 1600 mg/kg bw/day, presumably via the activation of the hepatic enzymes. This change in thyroid hormones and accompanying rise in TSH was slight without apparent alterations in thyroid glands, so it was considered non-adverse.


 


Organ Weights


The absolute kidneys weight was non-significantly but notably increased in males and females in the 1600 mg/kg bw/day group (males, 2.2722 ± 0.2454 g versus 2.1581 ± 0.2184 g; females, 1.3453 ± 0.1141 g versus 1.2620 ± 0.1119 g) while the change in relative to body weight value was significant in males (by 9.3 %, 0.6645 ± 0.0348 versus 0.6078 ± 0.0370 g/100 g, p < 0.01), and females (by 9.5 %, 0.6114 ± 0.0191 versus 0.5887 ± 0.0242 g/100 g, p < 0.05). In the recovery high dose subgroup, the mean relative weight of kidneys in males remained slightly increased, but without statistical difference from the control group. The change in weight of kidneys in the high dose group was correlated to the clinical pathology and pathomorphology observations and considered to be test item-related.
The weight of the liver was increased by 9.5 % in the 1600 mg/kg bw/day treated males (relative to body weight, 3.0916 ± 0.1513 versus 2.8232 ± 0.1766 g/100 g, p < 0.001). In females, the change in liver weight was more pronounced. The increase in absolute weight was dose-dependent with a significant change relative control group by 25.8 % in the high dose group (7.2479 ± 0.6628 g versus 5.7599 ± 0.4665 g, p < 0.0001). Females in the 400 and 1600 mg/kg bw/day dose group had the significantly increased relative liver weight by 8.0 % and 22.2 %, respectively, (2.9091 ± 0.2219 g/100 g, p < 0.05, and 3.2927 ± 0.1317 g/100 g, p < 0.001) compared to the control group (2.6940 ± 0.2228 g/100 g). The relative to brain weight value of the liver weight was also significantly increased in the 1600 mg/kg bw/day female group. Change in the liver weight correlated to the clinical pathology and histological findings; however, it is supposed to be caused by adaptive activation of metabolic enzymes and considered as non-adverse. After the recovery period, the mean relative liver weight remained slightly increased, but without statistically difference from the control group.
In the 400 mg/kg bw/day dose female group, the relative weight of thymus was statistically increased by 23.6 % compared to the control group (p < 0.05). The relative weight of thymus in the high dose group was approximately similar to the control value. Non-dose-dependent change in the thymus weight was not correlated to the hematological observations and considered of unclear relationship to the test item. There was no test item related histological findings in the thymus of the high dose treated animals.
In 1600 mg/kg bw/day dose recovery males, the slight decrease in the brain weight (p < 0.05 for absolute value) and heart weight (p < 0.05 for relative to body weight value) was registered. No correlations in the histology of these organs in the high dose main subgroup were revealed. These changes can be associated with the decrease in body weight of high dose treated males and considered non-adverse. The slight increase in the relative to brain testes weight (g/100 g of brain weight, right testis: 101.07 ± 2.48 versus 92.00 ± 5.56 in the control group) and adrenals weight (g/100 g of brain weight: 2.748 ± 0.292 versus 2.370 ± 0.194) in the high dose recovery males can be caused partially by a decrease in the brain weight value and regarded to be of uncler relationship to the test item.


 


Macroscopic Observations


The gross finding related to the test item was revealed in one female (No.85) from the 400 mg/kg bw/day dose group. This female had dark kidneys associated with a tubular brown pigmentation in the cortex of moderate grade (presumably lipofuscin accumulation). Accumulation of pigment in cortex tubular was observed in a dose-dependent manner in males and was most pronounced in 400 mg/kg bw/day treated females.
Other gross findings revealed during necropsy are considered not related to the test item.
Macroscopic findings in the thymus, lymph nodes, spleen, and thyroids were noted in the test item-treated groups and the control group; they did not correlate to organ weight changes, clinical pathology, or any microscopic observations.
One female (No. 82) in the 400 mg/kg bw/day dose group had unilateral corneal injury discussed above, focal alteration of texture of urinary bladder, and unilaterally enlarged ovary. These findings were considered incidental and not related to the test item administration.


 


Microscopic Findings


The gross finding related to the test item was revealed in one female (No.85) from the 400 mg/kg bw/day dose group. This female had dark kidneys associated with a tubular brown pigmentation in the cortex of moderate grade (presumably lipofuscin accumulation). Accumulation of pigment in cortex tubular was observed in a dose-dependent manner in males and was most pronounced in 400 mg/kg bw/day treated females.


Other gross findings revealed during necropsy are considered not related to the test item.


Macroscopic findings in the thymus, lymph nodes, spleen, and thyroids were noted in the test item-treated groups and the control group; they did not correlate to organ weight changes, clinical pathology, or any microscopic observations.


One female (No. 82) in the 400 mg/kg bw/day dose group had unilateral corneal injury discussed above, focal alteration of texture of urinary bladder, and unilaterally enlarged ovary. These findings were considered incidental and not related to the test item administration.


Test item-related histological alterations were observed in liver, kidneys, thyroid glands, and testes.


 


The findings of unclear relation to the test item were observed in adrenals, pituitary gland, female salivary glands, lymph nodes, spleen, and glandular stomach.


 


Liver


Microvesicular vacuolation in hepatocyte cytoplasm was increased in 1600 mg/kg bw/day treated males as well in 400 and 1600 mg/kg bw/day treated females with a significantly higher incidence in 1600 mg/kg bw/day female group compared to the control females (p < 0.05). The specific morphological features of the cytoplasmic vacuolation are sufficiently consistent with lipid accumulation to warrant a presumptive diagnosis of fatty change.


Fatty change may be due to perturbations in lipid metabolism and disposition, correlated to the clinical serum chemistry, and considered as toxicological effect of the test item.


In the 1600 mg/kg bw/day female group, the hepatocyte hypertrophy of slight to moderate grade was observed with significantly increased incidence (p < 0.05). It is noteworthy that hepatocyte hypertrophy was revealed only in high-dosed females, which correlated to the significant increase in absolute and relative liver weight in this group. The increase in relative liver weight in males of the 1600 mg/kg bw/day group and females of the 400 mg/kg bw/day group was less pronounced and correlated to the increased incidence of fatty change. There were no associated changes in the serum enzymes considered to be biomarkers of hepatotoxicity (ALP, ALT, AST, GGT, GLDG) in both female and male treated rats.


Interestingly, 1600 mg/kg bw/day treated males had decreased body weight correlated to lipids metabolism perturbations in this group. Females from the high dose group developing hepatocyte hypertrophy had the reversible serum lipids level and increased body weight. The hepatocyte hypertrophy was not observed after a 3-weeks post-treatment period, whereas the liver weight remained slightly increased. Hypertrophy of hepatocytes on xenobiotics develops due to metabolic enzyme induction, increase in mitochondria and peroxisomes [Hall A. et al, 2012; Thoolen B. et al, 2010].


Hepatic hypertrophy in females can be responsible for the observed sex-related differences in lipid levels and the lack of dose-response in microscopic findings in the kidney in females. It was considered to be a test item related and toxicologically relevant.


In one female (No.91) from the 1600 mg/kg bw/day dose group, the focal hyperplasia of oval cells was observed. Oval cell proliferation is considered to arise from terminal ductule epithelial cells (canal of Hering cells). It can be a rare spontaneous lesion in rats but can be observed following xenobiotic-induced hepatic injury. However, the uniqueness of this finding among test item treated group does not allow concluding its test item relation. A similar finding was observed in the control male of the recovery subgroup.


Kidney


The tubular brown pigmentation in the cortex (presumably lipofuscin accumulation) was found in 4 males from the 1600 mg/kg bw/day dose group and in single males from 100 and 400 mg/kg bw/day dose groups. In females, pigment accumulation of minimal grade was found in two control animals; however, four females had the lipofuscinosis in more severe grade in the 400 mg/kg bw/day dose group. In one female (no.85), the moderate grade of pigment correlated to the dark kidney noted during necropsy.


Lipofuscin is found in the kidneys of most laboratory rodents, especially rats, is most often localized to the proximal tubules, and considered of limited or no functional significance


[Frazier K. et al, 2012]. However, there is a clear dependence of lipofuscinosis on the test item in our study. This finding shows the sex-dependence presumably related to differences in metabolic enzyme activation (see Discussions).


The mineralization in the renal pelvis was noted only in 400 mg/kg bw/day treated females, which correlated to more pronounced lipofuscinosis in this group. Mineralization in the kidney occurs spontaneously in laboratory animals with a dietary imbalance of calcium/phosphorus ratio, particularly in female rats and severity increase with age. There can be a much higher prevalence of spontaneous mineralization in the outer stripe of the outer medulla (as we can see in this study, see Table S19-3). The mineralization in 400 mg/kg bw/day treated females was more severe with calculus in the renal pelvis, which was considered test item-related.


Interestingly, that no test item related findings were revealed in the kidney of the high dose treated females with hepatocyte hypertrophy. There is an assumption that the early metabolic deactivation of the substance by the hypertrophied liver neutralizes the main test-related effects in 1600 mg/kg bw/day female group.


The signs of chronic progressive nephropathy (CPN) of minimal to slight grade (tubule basophilia, tubule dilation with hyaline casts in the cortex, OSOM, and ISOM) were observed in approximately similar frequency, including the control group. CPN is one of the most common spontaneous lesions in rats, and male rats are more severely affected than female rats. In females, the incidence and severity of CPN have not been exacerbated by the test item administration. In one male from the 1600 mg/kg bw/day dose group, CPN findings were more severe with glomerulosclerosis and tubular hypertrophy. Renal tubule hypertrophy is often observed in more severe CNP cases and is believed to be a compensatory mechanism related to a decline in renal function. One another male from the high dose group had focal tubular necrosis of slight grade.


Microscopic findings in kidneys of 1600 mg/kg bw/day administered males correlated to the clinical pathology changes and increase in kidney weight in males of this group. However, it should be noted that the kidneys of the control animals show a high incidence of chronic progressive nephropathy (CPN). Kidney glomerulosclerosis, glomerular hypertrophy, and tubular focal necrosis are a consequence of CPN, and single findings of an apparent exacerbation of chronic nephropathy in the high dose male group do not allow concluding of its test relation taking into account the high background of CPN.


 


Thyroids


In males and females of the 1600 mg/kg bw/day dose group, the slight decrease in the T3 was observed after 90 days of test item administration and a more pronounced decrease in T4 level in high dose animals. The increase in TSH in the high dose group due to the hypothalamo-pituitary-thyroid feedback was slight, and no indications of TSH-mediated thyroid gland activation were found in its histology. There were no findings of thyroid hypertrophy, or follicular hyperplasia, and changes in thyroids weight.


In the 400 and 1600 mg/kg bw/day dose groups, the increase in incidence and severity of multifocal C-cell hyperplasia was observed. Diffuse C-cell hyperplasia represents a physiological or pathophysiological response to the stimulation of calcitonin-producing cells, such as chronic hypercalcemia.


It can be assumed that C-cell hyperplasia reflects a disturbance in calcium balance associated with renal dysfunction and indirect parathyroid hormone stimulation on the high dose of the test item acting on kidney PPAR. However, there are no enough data to make this conclusion. The calcium level in serum of test item treated males and females was not changed. Despite the absence of explainable reasons for C-cell hyperplasia, its relationship with the test item cannot be excluded. But this change was slight and is not considered adverse.


In one female from the 1600 mg/kg bw/day group, follicular atrophy was revealed. This finding can reflect the decrease in thyroid hormones; however, it was of minimal grade and considered non-adverse without toxicological importance.


 


Testes


Hyperplasia of Leydig cells was found in two males from 100 mg/kg bw/day dose group, and four males from each of 400 and 1600 mg/kg bw/day dose group. These findings were multifocal, minimal to slight grade, and considered to be a physiologic response to hormonal imbalance. It is known that interstitial cell hyperplasia can be induced in rats by any chemical that increases circulating levels of luteinizing hormone (LH) via disruption of the hypothalamic-pituitary axis followed by stimulation of steroidogenic Leydig cell function.


It is assumed that the test item and/or its metabolites (4-(tosylamino)butyric acid and 2-[(4-methylphenyl)sulfonylamino]acetic acid) acts on peroxisome proliferator-activated receptor α (PPARα), which is highly expressed in liver and renal proximal tubules, as well as in Leydig cells [Gazouli M. et al, 2002; Guan Y., 2004]. The “classic” PPARα-agonists aryloxyisobutyric acid derivates (clofibrate and its analogs) are clinically proven lipid-lowering drugs. Some peroxisome proliferators, including clofibric acid, are known to lower serum testosterone levels causing testicular atrophy and impaired spermatogenesis [Gazouli et al., 2002; Harada Y. et al, 2016]. It was shown a critical role for PPARα in testicular dysfunction due to disrupted cholesterol/testosterone homeostasis in Leydig cells [Harada Y. et al, 2016]. To maintain normal reproductive function, a feedback mechanism is present, where decreased circulating testosterone increases circulating LH that stimulates testosterone synthesis and hyperplasia of Leydig cells. The physiological stimulation of steroidogenic Leydig cell function can be adverse, considering Leydig cell tumor outcome [Kotula-Balak M. et al, 2020].


The tubular degeneration/atrophy was revealed in one male (No.44) from the 1600 mg/kg bw/day dose group and one male (No. 36) in the 400 mg/kg bw/day dose group. The male from the high dose group had a slight change with partial germ cell depletion in some tubules of one testis. Male from 400 mg/kg bw/day dose group had a more severe bilateral observation with atrophy of some tubules correlated to the decrease in relative testes individual weight. Tubular degeneration is a common manifestation of toxicologic injury to the testis. However, it also can be seen as a low incidence background finding in rats and mice. This finding was revealed in the medium and high dose group that correlated to the increased incidence of Leydig cells hyperplasia on these doses and, presumably, can be caused by impaired testosterone as noted above. Therefore, the relationship of tubular degeneration/atrophy in testes of males in the 400 and 1600 mg/kg bw/day dose group with the test item is not excluded.


The mean testes weight (absolute and related) was not changed after 90 days administration, but in 1600 mg/kg bw/day dose recovery males, the testes weight was slightly increased relative to brain weight, which can be partially explained by the slightly decreased value of brain weight in this group. Simultaneously, the increase in the relative weight of the testes after the post-treatment period may reflect the rebound of testosterone level.


There was no test item related changes in the epididymides, seminal vesicles with coagulating glands, and prostate. In the prostate of males from the 1600 mg/kg bw/day dose group, two mononuclear infiltration incidents of the ventral lobe were observed. Focal extravasation of inflammatory cells into the interstitial tissue or the acinar lumen is common in the prostate of rodents of unknown etiology. This finding was unfrequent, slight, and considered as not treatment-related.


 


Mammary glands (male)


In two males (No. 38 and No. 39) from the 1600 mg/kg bw/day group and two males (No. 18 and No.22) from the 100 mg/kg bw/day group, the lobule atrophy of moderate grade was observed with a noticeable smaller of alveolar profiles and slight tubulo-alveolar appearance.


 


The change of mammary glands by the type of feminization was revealed in two males from each of 100 and 1600 mg/kg bw dose groups. These findings cannot be unambiguously attributed to the toxicological effects of the test item, considering the absence of dose-dependence (there were no such findings on medium dose) and their incidence in the historical control population.


 


Salivary glands (females)


The incidence of alteration in granular secretory ducts was slightly increased in all doses treated females and especially in the 400 mg/kg bw/day dose group. The submandibular salivary gland is sexually dimorphic and shows increased granularity of the convoluted (granular) ducts in males compared to females. When females have increased androgen levels, their granular ducts of the submandibular glands acquire male morphology. Both male and female salivary glands have androgen receptors AR, estrogen receptors ER‐β, and progesterone receptors PR [Konttinen et al., 2010], are sensitive to hormonal disbalances during stress; apparent cyclic fluctuations are observed in tissue morphology depending on the phases of the estrous cycle [Rybakova M.G., 1979]. As speculation, increased androgenic background in the test item treated females can be due to androgen/estrogen hormonal disturbance. However, the change from the control group was weak, which does not allow conclusions about its test item relation.


 


Adrenals and Pituitary


In the 1600 mg/kg bw/day male group, the slight increase in the incidence of vacuolation in zona fasciculata was observed; one male (No. 40) had the maximal of noted severity of this finding. Cortical vacuolation is increased during fatty change due to degeneration or lipid retention of the cortical cells. A variety of xenobiotics can increase lipid deposition and vacuolation in the adrenal cortex, usually by inhibiting steroid synthesis; adaptive hypertrophy can also be observed [Harvey P. et al, 2007]. The cortical hypertrophy was noted in single males from each group included control.


The complex effects of the test item in this study may be attributed to a sex-dependent alteration in cholesterol and steroid synthesis that discussed below. In male No.40, the moderate grade of cortical vacuolation was correlated to the incidence of microfocal hyperplasia of endocrine cells of the pars intermedia of the pituitary gland, which can be a consequence of hypothalamic-pituitary feedback ACTH activation. The relative weight of adrenals (relative to brain) was statistically increased in the high dose recovery males, but it can due to a decrease in body weight and brain weight and not test item related. An increase in cortical vacuolation was not dose-dependent, not correlated to the cortical hypertrophy, and was observed in the approximately same frequency in recovery control males.


Thus, the slight increase in adrenal cortical vacuolation in the high dose treated males, and one incidence of hyperplasia in the pars intermedia of the pituitary gland are slight, non-adverse and without clear relation to the test item.


In pituitary pars distalis, no changes of gonadotropin-producing and thyrotropin-producing cells were observed in the test item treated animals; there were no significant changes in the pituitary weight.


 


Lymph Nodes, Spleen, and Glandular Stomach


A slightly higher incidence of lymphoid hyperplasia was observed in mandibular lymph nodes in the 1600 mg/kg bw/day dose-treated males and females. In one male from this group, hyperplasia in spleen white pulp of moderate grade was observed. These findings reflect a change in lymphocyte kinetics due to antigen stimulation [Willard-Mack C. et al, 2019] .


In one male in 1600 mg/kg bw/day dose group, the macrophage accumulation of slight grade was noted in the mesenteric lymph node. Macrophages accumulate and form aggregates when they cannot completely degrade ingested macromolecules.


In the stomach, the increased incidence of infiltration of deep mucosa with inflammation cells was observed in the 1600 mg/kg bw/day dose group (male and female). A minimal/mild infiltration of eosinophilic leucocytes is common in the submucosa of the glandular stomach. The slightly high infiltration observed mainly in a deep mucosa can be potentially due to the local pro-inflammation/immunogen effect of the test item.


Findings in lymph nodes, spleen, and glandular stomach were of slight grade or observed as an individual case, did not correlate to the clinical pathology or gross observations and changes in organ weight (spleen), was not associated to the inflammation of the tissues, considered to be non-adverse and of unclear relation to the treatment.


The 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.

Applicant's summary and conclusion

Conclusions:
No mortality was observed. No treatment related changes were found in the clinical observation, the body weights, the food consumption, the ophthalmological examinations and in the Functional Observation Battery.

The following treatment-related changes were observed:
Males, 400 mg/kg bw/day: increased serum urea, testes, Leydig cells hyperplasia;
Males, 1600 mg/kg bw/day: increased urobilinogen, decreased urine pH, decreased serum triglyceride, increased serum urea, increased liver weights and kidney weights; liver, fatty change; kidney, lipofuscinosis; thyroid glands, C-cell hyperplasia; testes, Leydig cells hyperplasia;
Females, 400 mg/kg bw/day: decreased APTT, increased liver weights; liver, fatty change; kidney, lipofuscinosis, calculi in pelvis; thyroid glands, C-cell hyperplasia;
Females, 1600 mg/kg bw/day: increased urobilinogen, decreased: APTT, increased liver weights and kidney weights; liver, fatty change, hepatocellular hypertrophy; thyroid glands, C-cell hyperplasia.
Therefore, under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) is considered 100 mg/kg bw/day for males and 100 mg/kg bw/day for females.
Executive summary:

Design:


This 90-day study was designed to evaluate the possible health hazards likely to arise from repeated exposure of the test item 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid (ASCplus®, CAS 78521-39-8) over a prolonged period of time covering post-weaning maturation and growth into adulthood of the test animals. The study's goal was to get information on the significant toxic effects, indicate target organs, and estimate a no-observed-adverse-effect level (NOAEL) of exposure for establishing safety criteria for human exposure.


The test item as a suspension in the vehicle (distilled water), was administered by gavage once daily to three groups of Sprague-Dawley rats starting from the age of 6-7 weeks at the doses 100, 400 and 1600 mg/kg body weight (kg/bw/day). A concurrent vehicle control group received the vehicle on a comparable regiment and in the same volume of 10 mL/kg bw. Each group consisted of 10 males and 10 females used for 90-day dosing and euthanized on the day 91. An additional satellite group of six animals per sex was included in the control and the top dose group for observation after the treatment period for the potential reversibility or persistence of any toxic effects and was euthanized at day 112. All animals were observed twice daily for mortality and morbidity. Detailed clinical observations, body weights, and food consumption were recorded weekly. Ophthalmic examinations were done once in the pre-treatment period and at the end of the in-life period. Functional Observational Battery (FOB) and locomotor activity data were recorded for half males and females from each group at the end of the dosing period and in recovery subgroup. Clinical pathology evaluations (hematology, coagulation, serum chemistry, and thyroid hormones T3, T4, and TSH) were performed in all males and females at the end of an in-life phase. Urinalysis determination was performed in all animals before scheduled euthanasia. Complete necropsies were conducted on all animals, and selected organs were weighed. Tissues were examined microscopically from all males and females in the vehicle control and high-dose groups from the main subgroup; lower doses level groups and satellite subgroup were evaluated for presumptive target organs.


Results:


There was no morbidity and mortality of animals caused by the test item administration. No treatment related changes were found in the clinical observations, the food consumption, the ophthalmological examinations and in the Functional Observation Battery. A slight decrease in the body weight of males at doses of 400 and 1600 mg/kg bw/day was statistically insignificant and considered toxicologically unimportant.
The summary of significant findings related to the ASCplus treatment is presented in table below.


Urinalysis


males:
100 mg/kg bw.: no test article related change,
400 mg/kg bw.: no test article related changes,
1600 mg/kg bw.: increased: urobilinogen, decreased: pH
females:
100 mg/kg bw.: no test article related changes,
400 mg/kg bw.: no test article related changes,
1600 mg/kg bw.: increased urobilinogen.


Hematology:


males: no test article related findings
females:
100 mg/kg bw.: no test article related changes,
400 mg/kg bw.: decreased APTT,
1600 mg/kg bw.: decreased APTT.
After the three-week high dose post-treatment, the hematological parameters were similar to the values in the control vehicle group in both males and females.



Clinical chemistry:


males:
100 mg/kg bw.: no test article related changes,
400 mg/kg bw.: increased: urea,
1600 mg/kg bw.: increased: urea, decreased: triglyceride,
females: no test article related findings.



Endocrine findings: No test article related changes
A special situation is seen with the thyroid hormones serum levels.
Neither the male animals nor the female animals of the dose groups showed any treatment-related changes. Only in the male recovery groups, there seems to be a lower T4 serum level in the 1600 mg/kg bw/day dose group than in the control animals. A comparison with the historical data shows that this is a merely statistical effect caused by accidentally low variances of the control and the dose group T4 levels.



Organ weights:


100 mg/kg bw.: no treatment related changes,
400 mg/kg bw.: males: no treatment related changes, females: increased liver weights,
1600 mg/kg bw.: males and females: increased: liver weights and kidney weights.
No changes were seen at the end of the 21 days recovery period.


 


Gross pathological findings:
Not treatment related findings were observed


 


Histopathology:


Liver, Fatty change, male:
0 mg/kg bw.: 2/10,
1600 mg/kg bw.: 4/10.
Liver: Fatty change, female:
0 mg/kg bw.: 3/10,
400 mg/kg bw. : 6/10,
1600 mg/kg bw.: 9/10.


Liver: Hepatocellular hypertrophy, female:


0 mg/kg bw.: 0/10,
1600 mg/kg bw.: 6/10.
A special view has to be taken on the histopathology of the kidneys. The kidneys of the control animals of both sexes show a high incidence of chronic progressive nephropathy. With the control animals, we have an incidence of 7/10 (=70%) males and 6/10 (=60%) females, which is still higher than the historical controls (40% males, 25% females) and has most obviously no relation to the treatment. In comparison to the frequency of these changes, all the other more or less dose-related changes had a much lesser and inconsistent incidence, and glomerulosclerosis and tubular focal necrosis can be seen as directly connected to the chronic progressive nephropathy and are probably not a treatment-related change.


Kidney: Lipofuscinosis, male:
0 mg/kg bw.: 0/10,
1600 mg/kg bw.: 4/10.


Kidney: Lipofuscinosis, female:
0 mg/kg bw.: 2/10,
400 mg/kg bw.: 4/10.


Kidney: Calculi in pelvis, female:
0 mg/kg bw.: 0/10,
400 mg/kg bw.: 3/10.


Thyroid glands, C-cell hyperplasia, male:
0 mg/kg bw.: 0/10,
1600 mg/kg bw.: 3/10.


Thyroid glands, C-cell hyperplasia, females:
0 mg/kg bw.: 2/10,
400 mg/kg bw. : 6/10
1600 mg/kg bw.: 6/10


Testes, Hyperplasia, Leydig cells:
0 mg/kg bw.: 0/10,
400 mg/kg bw. : 4/10,
1600 mg/kg bw.: 4/10.


Despite the kidneys histopathology revealed, no tissue damage but adaptive reactions to the treatment with ASCplus in dosages of 400 and 1600 mg/kg bw for 90 days. In the kidneys, lipofuscinosis and calculi were seen in a dose-related incidence. Therefore a treatment-related change could not be excluded.
After a recovery period of 21 days, no treatment-related changes were observable.
Dosages of 100 mg/kg bw caused no treatment-related changes.


 


Conclusion:


No mortality was observed. No treatment related changes were found in the clinical observation, the body weights, the food consumption, the ophthalmological examinations and in the Functional Observation Battery.
The following treatment-related changes were observed:
Males, 400 mg/kg bw/day: increased serum urea, testes, Leydig cells hyperplasia;
Males, 1600 mg/kg bw/day: increased urobilinogen, decreased urine pH, decreased serum triglyceride, increased serum urea, increased liver weights and kidney weights; liver, fatty change; kidney, lipofuscinosis; thyroid glands, C-cell hyperplasia; testes, Leydig cells hyperplasia;
Females, 400 mg/kg bw/day: decreased APTT, increased liver weights; liver, fatty change; kidney, lipofuscinosis, calculi in pelvis; thyroid glands, C-cell hyperplasia;
Females, 1600 mg/kg bw/day: increased urobilinogen, decreased: APTT, increased liver weights and kidney weights; liver, fatty change, hepatocellular hypertrophy; thyroid glands, C-cell hyperplasia.
Therefore, under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) is considered 100 mg/kg bw/day for males and 100 mg/kg bw/day for females.