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Description of key information

Daily dosages of 0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of 12 male rats for 54 days (Zeljenkova, D, 2013). Satellite animals in a control and the highest dose group (1600mg/kg bw) with 5 individual each were also included. The test-article was formulated in drinking water and administered in 10 ml/kg bw.


At dosages of 100 and 400 mg/kg bw. the animals showed no differences to the control animals (NOAEL).


1600 mg ASC plus/kg bw., considered as the LOEL may have induced an influence on the body weight gain of the males (and the survival of pups until day 4 after birth).


The NOAEL of 400 mg/kg bw/day based on influence on the body weight gain of the males (and the survival of pups until day 4 after birth).


OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents):


Daily dosages of 0, 100, 400, and 1600 mg ASC plus/kg bw. in 10 ml/kg bw. destilled water were administered by stomach tube to groups of 10 male and 10 female Sprague-Dawley rats for 90 days. Additional groups of 6 male and 6 female animals were given dosages of 0 and 1600 mg/kg bw. for 90 days followed by a 21 day recovery period. (Oksana N. Khokhiova 2021)


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;


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;


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.


At dosages of 100 mg/kg bw. the animals showed no differences to the control animals (NOAEL).


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.


 


NOEL: >=100 mg/kg bw/day (nominal) (male) based on: (test mat.) clinical biochemistry


NOEL: >=400 mg/kg bw/day (nominal) (male) based on: (test mat.) organ weights and organ / body weight ratios


NOEL: >=1600 mg/kg bw/day (nominal) (female) based on: (test mat.) clinical biochemistry


NOEL: >=1600 mg/kg bw/day (nominal) (male) based on: (test mat.) haematology


NOEL: >=100 mg/kg bw/day (nominal) (female) based on: (test mat.) haematology ; organ weights and organ / body weight ratios


NOEL: >=1600 mg/kg bw/day (nominal) (male/female) based on: (test mat.) behaviour (functional findings) ; body weight and weight gain ; clinical signs ; dermal irritation ; gross pathology ; histopathology: neoplastic ; immunology ; mortality ; neuropathology ; ophthalmological examination ; serum/plasma hormone analyses (not measured/tested - food consumption, food efficiency, sperm measures, immunological findings, neuropathology findings)


NOEL: >=400 mg/kg bw/day (nominal) (male/female) based on: (test mat.) urinalysis


Target system / organ toxicity


Lowest effective dose /concentration: hepatobiliary : kidney ; liver ; testes ; thyroid gland (lowest effective dose/conc.: 400mg/kg bw (total dose) ;


treatment-related ; dose-response: yes )

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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:
28.04.2020 - 17.08.2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Full name of test item: 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid
Name in the study: ASCplus®
Lot Number: 1812004
CAS No.: 78521-39-8
EC No.: 278-934-5
Composition: 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid – 75.95%; Water – 23.7%
Molecular formula: C13H19NO4S
Molecular weight: 285.36 g/mol
Purity: > 99.5 % (6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid 75.95 %; water 23.7 %)
Appearance: White powder
Solubility: H2O: 0.1 - 1 g/L; EtOH: unknown; acetone: unknown; CH3CN: > 1 g/L; DMSO: > 1 g/L
Stability: H2O: 96 h h; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Manufactured date: 15 Dec. 2018
Expiry Date: 30 Nov. 2021
Amount of the Test Item: 3.0 kg (6 jars / 0.5 kg)
Provider: Metall-Chemie GmbH & Co.KG
Date of receipt: 06.04.2020
Storage conditions: room temperature (15-25°C)
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Species: Rattus spp.
Strain: Sprague-Dawley (SD), outbreed
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: Approximately 6-7 weeks old at the initiation of dose administration
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.
Body weight at first day of dosing, g (MEAN ± SD): Males: 147 ± 12, N = 52 Females: 129 ± 10, N = 52
Number of animals used (a): Males: 52, females: 52
(a) The number of animals received was 60 males and 60 females. After the experimental groups forming, the remaining animals were transferred to the stock colony of the BTL BIBC RAS.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals were kept in the two corridors barrier-type facility (barrier zone 2 of BTL BIBC RAS) with the automatic change of day and night time (08:00-20:00 - "Day", 20:00-08:00 - "Night") and the renewal of the room air at least 12 times hourly. 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
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
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.

Environmental Conditions
Temperature and humidity are constantly monitored in each room automatically by Eksis Visual Lab system (Practice-NC Ltd, Russia). Actual mean temperature ranged from 20 °C to 24 °C and 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 (see Appendix 2 Deviations from the Study Plan).

Adaptation/Acclimatization
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.

Assignment to Groups
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.

Animal Identification
An individual number indicated by ear punch identified each male and female. The cage card indicated the study number, study director, Bioethics Committee protocol number, animal number, and group.
Route of administration:
oral: gavage
Details on route of administration:
Assignment to Groups:
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.

Animal Identification:
An individual number indicated by ear punch identified each male and female. The cage card indicated the study number, study director, Bioethics Committee protocol number, animal number, and group.
Dosing Procedure:
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:
Dosing Procedure:
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).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 concentrati
Duration of treatment / exposure:
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).
Frequency of treatment:
daily
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)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study 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®) 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, 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid (name in the study - ASCplus®), in the vehicle (distilled water), was administered by gavage once daily to three groups of SD rats at the doses 100, 400 and 1600 mg/kg bw. A concurrent vehicle control group received the vehicle (distilled water) 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 91st day. 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.
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 at the end of the dosing period.

Clinical pathology evaluations (hematology, coagulation, serum chemistry, and thyroid hormones assay) 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.

Animals of the main subgroup were euthanized following completion of the 90 days dosing period, and animals of the satellite subgroup were euthanized after a 3-week recovery period. Complete necropsies were conducted on all animals, and selected organs were weighed. Selected 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.
Observations and examinations performed and frequency:
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 physical examinations were recorded for all animals before first dosing and regularly on a weekly basis throughout the study.
Body Weights:
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:
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.
Ophthalmological Examination:
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.
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
Diuresis and Collection of Urine:
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 (Section 8.9.1) 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.
Food Deprivation:
Animals were fasted overnight (approximately 16-18 hours) prior to euthanasia and blood collection being in metabolic cages.
Clinical Pathology:
Urinalysis:
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
Blood Sample Collection for Clinical Pathology Assay:
Blood samples for clinical pathology evaluations (hematology, coagulation, serum chemistry, and hormones assay) 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).
The blood was collected terminally following anesthesia (Telazol® / Xyla®, im) from the caudal vena cava after laparotomy using a syringe with 23G needle. For assessing the level of glucose, a drop of blood was obtained by tail-tip amputation prior to anesthesia.
For coagulation analysis, the blood samples (~0.9 ml) was placed in tubes with 3.2% sodium citrate. The standard whole-blood-to-anticoagulant ratio (9:1 vol/vol whole blood/3.2% citrate) was used in all cases. Plasma for coagulation analysis was obtained after centrifugation of citrate samples for 15 min at 1500 g and 22 °C. Plasma samples were analyzed immediately after being taken.
For hematology analysis (including blood smear and reticulocyte count), the blood samples (~0.5 ml) were placed in Microvette® tubes containing K3EDTA. For reticulocyte counting, ~50 μL of blood was placed in a tube with 1% cresyl blue dye for supra-vital staining followed by blood smear. For differential leukocyte count, the blood smear was prepared followed by Papendheim staining.
For clinical chemistry analysis, the blood samples (all remaining blood) was placed in a tube without anticoagulant. The blood was allowed to clot for 50 min at the room temperature and centrifuged (1600 x g, 4 °C, 15 min) for serum separation. Serum from each animal was divided into minimum 5 aliquots (for serum biochemistry, ion-selective, and three thyroid hormones analysis) and immediately frozen until assayed (at –20 °C for biochemistry and ion-selective analysis and at -70 °C for hormones analysis).
If possible, the remaining of serum from all animals was frozen at –70 °C and used as backup samples for clinical pathology assay if necessary.
Coagulation:
The fresh plasma was analyzed for the following parameters with a 4-channel semi-automatic coagulometer CL 4 (BehnkElektronik, Germany) using the reagents for human clinical trials (Renam, Russia).
Activated partial thromboplastin time (APTT), Prothrombine time (PT), Fibrinogen (Fg)
Hematology:
The blood samples in 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.
Serum Chemistry:
The serum was analyzed with a SAPPHIRE 400 (Prestige 24i) automatic biochemical analyzer (Tokyo Boeki, Japan) using reagents from Randox Laboratories Ltd. for the following parameters:
Total protein, 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 were measured by ion-selective electrodes using analyzer EX-D (JOKOH Co, Ltd) (SOP Chem/27).
Glucose level was estimated in the drop of blood before animal anesthesia by electrochemical method using Sattelite Express® glucometer (ELTA, Russia) routinely controlled with control strip.
Thyroid Hormones Assay
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. Specification information from manufacturer is the following:
Rat Thyroxin (T4) ELISA Kit, Manufacturer: Cusabio (Wuhan Huamei Biotech Co., Ltd.), Code of product: CSB-E05082r, Species: Rat specific, Analytical sensitivity: < 20 ng/mL (minimum detectable concentration), Detection range: 20 ng/mL – 320 ng/mL, Lot: C0663070689, Expiration Date: 28 Nov 2020.
Rat Tri-iodothyronine (T3) ELISA Kit: Manufacturer: Cusabio (Wuhan Huamei Biotech Co., Ltd.), Code of product: CSB-E05085r, Species: Rat specific, Analytical sensitivity: < 0.5 ng/mL (minimum detectable concentration), Detection range: 0.5 ng/mL – 8 ng/mL, Lot: C00663080690, Expiration Date: 29 Nov 2020.
Rat Thyroid Stimulating Hormon (TSH) ELISA Kit: Manufacturer: Cusabio (Wuhan Huamei Biotech Co., Ltd.), Code of product: CSB-E05115r, Species: Rat specific, Analytical sensitivity: < 0.3 IU/mL (minimum detectable concentration), Detection range: 0.6 IU/mL – 24 IU/mL, Lot: C0663090691, Expiration Date: 30 Nov 2020.
Sacrifice and pathology:
Scheduled Euthanasia
All animals were schedule sacrificed after the 90-day treatment (day 91) or 3-weeks recovery (day 112). Animals were euthanized by anesthesia (Telazol® / Xyla®, intramuscular) followed by terminal blood sampling from vena cava for clinical pathology, subjected to a gross necropsy, organ weight collection, and tissue preservation.
Necropsy:
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.
Organ Weights:
The following organs were weighed from all animals (sex corresponding). Paired organs excluding testes and epididymides were weighed together. Organ-to-body weight and organ-to-brain weight ratios in percentages were calculated.
Adrenals, Prostate, Brain, Seminal vesicles & coagulating glands, Epididymides (Left / Right), Spleen, Heart, Testes (Left / Right), Kidneys, Thymus, Liver, Thyroid, (after fixation), Ovaries, Uterus (including cervix), Pituitary (after fixation),
Tissue Collection
At the time of necropsy, the following tissues and organs from all sacrificed animal, were collected and placed in 10% neutral-buffered formalin (except as noted):
Adrenals, Nerve: Sciatic, Aorta (thoracic), Pancreas, Bone marrow, aspirated (smear), Pituitary, Bone: Sternum, Salivary glands (mandibular), Brain (cerebrum, cerebellum and pons), Skeletal muscle (biceps femoris), Esophagus+Trachea+ Thyroids (with parathyroids), Spinal cord (cervical, thoracic, lumbar), Eyes (including optic nerve) (a), Spleen,
Gross lesions:
Stomach (glandular and nonglandular), Heart, Thymus, Intestine (incl. Peyer's patches): Tongue, Duodenum, Ureter, Jejunum, Urinary bladder, Ileum, Males:
Cecum, Testes (c), Colon, Epididymides (c), Rectum, Prostate, Kidneys, Seminal vesicles with coagulating glands, Liver.
Females: Lungs with bronchus(b), Ovaries with oviducts, Lymph nodes, Uterus with cervix, Mesenteric, Vagina, Submandibular-, Mammary glands (male and female) with Skin,
(a) Have been preserved in Davidson’s fixative
(b) Have Preserved by inflation with fixative and then immersion
(c) To be placed in modified Davidson’s fixative
Bone Marrow Smear:
Bone marrow was collected from all animals immediately after euthanasia by aspiration from the right femur into a centrifuge tube for a smear. Two smear slides per animal were prepared by Pappenheim’s staining for following cell counting :
All erythroid cells (E) (%), Monocytes (%), Myeloid-granulocytic cell line (M) different. calc (%), Lymphoid cells (%), M:E ratio.
Bone marrow smears were examined for animals in the control and high-dose groups euthanized on day 91.
Microscopic Examination
Tissues listed in Section 8.10.4. 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.
Clinical signs:
no effects observed
Description (incidence and severity):
There was no morbidity and mortality of animals caused by the test item administration. No test item-related clinical external observations, ophthalmologic findings, and functional findings of neurotoxicity evaluated during FOB testing were revealed in any dose group.
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:
no effects observed
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.
Food consumption and compound intake (if feeding study):
not examined
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.
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.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematology:
Males:
100 mg/kg bw.: no testarticle related changes
400 mg/kg bw.: no testarticle related findings,
1600 mg/kg bw.: no testarticle related findings.
Females:
100 mg/kg bw.: no testarticle related changes
400 mg/kg bw.: decresed 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 biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
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 effects observed
Description (incidence and severity):
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. dose group than in the control animals. This seems to be a merely statistical effect as the mean T4 levels of the contol animals of the recovery group are a little bit higher than of the control animals of the dose group while the variance is accidentally lower.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw.: no treatment related findings
400 mg/kg bw.: no treatment related findings
1600 mg/kg bw. : males increased urine urobilinogen and decreased pH,
females:
100 mg/kg bw.: no treatment related findings
400 mg/kg bw.: no treatment related findings
1600 mg/kg bw. : increased urine urobilinogen
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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
The changes werd mild and only with females the increase in liver weights has a dose-reponse relationship.
No changes were seen at the end of the 21 days recovery period.
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.
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.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
Key result
Dose descriptor:
NOEL
Effect level:
>= 400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOEL
Effect level:
>= 1 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
Key result
Dose descriptor:
NOEL
Effect level:
>= 1 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
haematology
Key result
Dose descriptor:
NOEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
haematology
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOEL
Effect level:
>= 1 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical signs
dermal irritation
gross pathology
histopathology: neoplastic
immunology
mortality
neuropathology
ophthalmological examination
serum/plasma hormone analyses
Remarks on result:
not measured/tested
Remarks:
food consumption, food efficiency, sperm measures, immunological findings, neuropathology findings
Key result
Dose descriptor:
NOEL
Effect level:
>= 400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
urinalysis
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw (total dose)
System:
hepatobiliary
Organ:
kidney
liver
testes
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
Dosages of 0, 100, 400 and 1600 mg/kg bw of ASC+ in 10 ml/kg bw. destilled water were administered by gavage to groups of 10 male and female Sprague-Dawley rats for 90 days. Additional groups of 6 male and female animals were given dosages of 0 and 1600 mg/kg bw. For 90 days followed by a 21 day recovery period. The administration volume was 10 ml/kg bw.
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.

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

Hematology:
Males:
100 mg/kg bw.: no testarticle related changes
400 mg/kg bw.: no testarticle related findings,
1600 mg/kg bw.: no testarticle related findings
Females:
100 mg/kg bw.: no testarticle related changes
400 mg/kg bw.: decresed 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 testarticle related changes
400 mg/kg bw.: increased: Urea,
1600 mg/kg bw.: increased: urea, decreased: triglyceride,
females: no testarticle related findings ,
After 21 days recovery period no significant changes between the control group and the 1600 mg/kg bw. dose group were seen.
Endocrine findings:
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. 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.males and females: 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
The changes werd mild and only with females the increase in liver weights has a dose-reponse relationship.
No changes were seen at the end of the 21 days recovery period.

Gross pathological findings:
Not treatment related findings were observed

Non-neoplastic hiostopathology findings:
Liver, male, fatty change:
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 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.

Kidneys, 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,
Testes, hyperplasia. Leydig cells:
0 mg/kg bw.: 0/10,
100 mg/kg bw.: 2/10,
400 mg/kg bw. : 4/10,
1600 mg/kg bw.: 4/10,
Testes, degeneration/atrophy, tubule:
0 mg/kg bw.: 0/10,
100 mg/kg bw.: 0/10,
400 mg/kg bw. : 1/10,
1600 mg/kg bw.: 1/10,

Thyroid glands, c-cell hyperplasia, male:
0 mg/kg bw.: 0/10,
100 mg/kg bw.: 0/10,
400 mg/kg bw. : 1/10,
1600 mg/kg bw.: 3/10,
Thyroid glands, c-cell hyperplasia, females:
0 mg/kg bw.: 2/10,
100 mg/kg bw.: 2/10,
400 mg/kg bw. : 6/10,
1600 mg/kg bw.: 6/10,

With the animals of the recovery groups no histopathological changes between the control animals and those of the 1600 mg/kg bw. dose-group were detected.

All in all the treatment with ASC+ caused an increase in liver weight starting with females of the 400 mg/kg bw dosage group. With these animals also a fatty change of the liver tissue with increased liver weights, hepatocellular hypertrophy, kidney lipofuscinosis, calculi in pelvis, c-cell hyperplasia in the thyroid gland and a lowered APTT was seen.
With the males of the 400 mg/kg bw dose group a hyperplasia of Leydig cells in the testes, an increased serum urea were seen.
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.
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;

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.
Dosages of 100 mg/kg bw caused no treatment-related changes.
Executive summary:

Dosages of 0, 100, 400 and 1600 mg/kg bw/day of ASCplus® in 10 ml/kg bw destilled water were administered by gavage to groups of 10 male and female Sprague-Dawley rats for 90 days. Additional groups of 6 male and 6 female animals were given dosages of 0 and 1600 mg/kg bw for 90 days followed by a 21 day recovery period. The administration volume was 10 ml/kg bw.


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.

Endpoint:
repeated dose toxicity: oral, other
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study with no deviations
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
daily administration by stomach tube for 54 days
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany, D-97633 Sulzfeld
- Age at purchase: 11 weeks
- weight rage at time of grouping: males, 175-200 g
- Fasting period before study: no
- Housing: 2 per cage,
- Cages: TECHNIPLAST filter top cages type 2145 F with an G-Temp (PSU) durable filter cover, 480x265x210 mm³, floor area 940 cm²,
- Source: Techniplast Company, Italy
- Diet: ad libitum, M3, BONAGRO Ltd., reg. CZ 10174, Czech Republic
- Water: ad libitum, tap water
- Acclimation period: 9 days
Remark: Although female rats were investigated in this 54 d combined repeated dose and reproduction / developmental screening study, female rats are disregarded in this endpoint study record (7.5.1) because the condition "non-pregnant" is not applicable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE: Suspension in water

- Amount of vehicle: 10 ml/kg
- single daily adminisration at similar times each day
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
54 administrations, (2 weeks before mating, 2 weeks during mating and 26 days after mating)
dissection ca. 24 hours after the last administration
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 100, 400, 1600 mg/kg body weight (mg/kg bw)
Basis:
actual ingested
No. of animals per sex per dose:
12 males, satelite groups (control and highest dosage): 5 males each
Control animals:
yes
Details on study design:
- Control groups: drinking water
- Dose selection rationale: Results of an acute toxicity study with oral administration to male and female rats
- Result: no effects up to and including 2000 mg/kg bw.
- Rationale for animal assignment: randomly grouped
- Section schedule rationale: all animals were sacrificed
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on administration days 1, 8, 15, 22, 29, 36, 42, 50 and on day of autopsy

FOOD CONSUMPTION : 2 weeks before mating, after 2 week mating period and weekly until the end of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 14 (before mating) and prior to autopsy from the satelite groups and from the control and highest dose group.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:day 14 (before mating) and prior to autopsy from the satelite groups and from the control and high dose group.

URINALYSIS: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Organ weights: brain, heart, thymus, spleen, liver, testis, epididymis, kidney, adrenal gland

HISTOPATHOLOGY: Yes: high dose and control animals
- Organ: medulla oblongata, brain, heart, pancreas + lymphnode, spleen, liver, lung, small intestine, stomach, kidneys, adrenal gland, testes, prostrate, urinary bladder, bone + bone marrow, thymus, trachea, white + brown fat, muscle, pituitary gland
Statistics:
Statistical evaluation was operated using the software SPPS version 16.0. Group data were represented by mean, standard deviation and median. Statistical analysis in case of data measured once during the study (organ weight, haematology, clinical chemistry) : Mann-Whitney U test for pairwise comparison between control and individual experimental groups on significance level alpha = 0.05.
Statistical analysis in case of repeated data measurement (body weight, food intake): Repeated measures ANOVA (procedure General Linear Model (GLM) for Repeated Measures in SPSS statistical software).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
statistically significant decrease after dosages of 1600 mg/kg bw./day
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS:
- 100, 400 and 1600 mg/kg bw/day:
no differences to the control animals observable

MORTALITY: no

BODY WEIGHT GAIN: 1600 mg/kg bw: reduced

FOOD CONSUMPTION: no statistical differences

HAEMATOLOGY/ CLINICAL CHEMISTRY:
Haematology and clinical chemistry reveales some statistically significant differences, but these were neither related to dosage not confirmed by the findings in other groups, for example by the results of the satelite groups, or the effects are of biological low relevance i.e..

URINALYSIS: not examined

NEUROBEHAVIOUR: not examined

ORGAN WEIGHTS: no statistical differences

GROSS PATHOLOGY: no dosage related effects

HISTOPATHOLOGY: NON-NEOPLASTIC: no statistical differences

HISTOPATHOLOGY: NEOPLASTIC: no statistical differences

HISTORICAL CONTROL DATA: not given

Dose descriptor:
NOEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
clinical signs
gross pathology
haematology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Dose descriptor:
LOEL
Effect level:
1 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decrease in body weight (not exeeding 10%)
Critical effects observed:
not specified
Conclusions:
Daily dosages of 0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of 12 male rats for 54 days. Satellite animals in a control and the highest dose group (1600mg/kg bw) with 5 individual each were also included.The test-article was formulated in drinking water and administered in 10 ml/kg bw..
At dosages of 100 and 400 mg/kg bw. the animals showed no differences to the control animals (NOAEL).
1600 mg ASC plus/kg bw. , considered as the LOEC, may have induced an influence on the body weight gain of the males (and the survival of pups until day 4 after birth).
NOAEL: 400 mg/kg bw.
Executive summary:

Daily dosages of 0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of 12 male rats for 54 days. The test-article was formulated in drinking water and administered in 10 ml/kg bw..

At dosages of 100 and 400 mg/kg bw. the animals showed no differences to the control animals (NOEL).

All examined organs and tissues showed a normal histological structure.

1600 mg ASC plus/kg bw. may have induced an influence on the body weight gain of the males (and the survival of pups until day 4 after birth).

In regard to the results of the present study 400 mg ASC plus/kg bw. per day can be defined as "No Observed Adverse Effect Level" (NOAEL) for the systemic toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
reliable, according to OECD Guidelines under GLP

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

According to the present data the toxicity and the toxicokinetics of ASC plus (6-[[(4-methylphenyl) sulphonyl]amino]hexanoic acid) can be described as follows:


 



  1. 100 mg/kg bw.ASC+ administered orally for 90 days did not cause any toxicity.

  2. Repeated oral dosages of 400 mg/kg b. w. caused


with males increased serum urea and in the testes a hyperplasia of Leydig cells hyperplasia;


with females decreased APTT (Activated Partial Thromboplastin Time), increased liver weights;  fatty change in the liver tissue; lipofuscinosis in the kidneys and calculi in the pelvis of the kidneys and in the thyroid glands a C-cell hyperplasia.


These changes do not show clear tissue damage but seem to be a metabolic and or adaptive  reaction to the high load of foreign matter in the body. Only lipofuscinosis in the kidneys and calculi in the pelvis of the kidneys may indicate a toxic damage.



  1. ASC plus is nearly completely eliminated from serum within 24 hours after administration. There is no potential for accumulation in the body.

  2. ASC plus seems to be excreted mainly as unchanged substance via the urine.

  3. This is in good accordance with the data from ecotoxicity studies showing a slow but steady degradation together with a low toxicity.


 


In summary the present studies seem to be sufficient for the toxicological evaluation of ASC plus.

Justification for classification or non-classification

There was no treatment related effect that would fulfill any criteria for classification and labelling according to the criteria laid out in Regulation (EC) No.: 1272/2008 (especially 3.7 "Reproductive toxicity" and "3.9. Specific target organ toxicity — repeated exposure"). Regarding the specific target organ toxicity, the LOEL lays far above the Guidance Value of 100 mg/kg body weight/day in Table 3.9.3 in Annex I of the CLP regulation. Also, according to the criteria specified by Directive 67/548/EEC the substance is not classified.