1,4-dimethylpiperazine

Endpoint summary

Description of key information

Repeated dose toxiciy - oral:

A key, K1 repeated dose toxicity study in rats was performed according to OECD guideline 407 and according to GLP requirements (Malleshappa HN, 2020). The NOAEL for systemic toxicity was considered to be 600 mg/kg bw/day.

A key, K1 subchronic repeated dose toxicity study in rats was performed according to OECD guideline 408 and according to GLP requirements (Malleshappa HN, 2022). The NOAEL for systemic toxicity was considered to be 350 mg/kg bw/day. 

Repeated dose toxicity - inhalation: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure

Repeated dose toxicity - dermal: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-02-27 to 2022-07-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): 1,4-dimethylpiperazine
- Physical state: liquid
- Analytical purity: 99.91 A%. The substance is total titratable amine content (17.39 meq/g)
- Lot/batch No.: PFW210769
- Expiration date of the lot/batch: 2023-11-25

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (+15 to +22°C), protect from light/humidity. At temperature no higher than +22ºC under inert gas blanket.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability of the test item in the vehicle was established at 1 and 100 mg/mL under Eurofins Advinus Study No. G24817 in Milli-Q water. Based on the results, the test item was stable in the vehicle up to 48 hours when stored at room temperature.

OTHER
- pH 11.4
- density: 0.84 g/cm³
- specific gravity: 0.822 at 20°C

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology (India) Pvt. Ltd., 4B MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078 (licensed breeder of Charles River)
- Age at study initiation: 6 weeks old
- Weight at study initiation: 200.06 to 235.87 grams (males), 153.72 to 204.26 grams (females)
- Fasting period before study: no data
- Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in recovery group of each sex was housed individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week. During the experimental period, animals were housed in a single experimental room of barrier area (SC 22). Steam sterilized corn cob was used as bedding and changed along with the cage at least twice a week.
- Diet (e.g. ad libitum): ad libitum, Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany.
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: the rats were acclimatized for five days before start of the treatment. During acclimatization period, rats were observed once daily for any abnormalities
- Based on the latest analytical certificate/s available, there were no known contaminants in the food, water and bedding that are expected to interfere with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 - 24.2 °C
- Humidity (%): 62-67 %
- Air changes (per hr): 12.6-13.3 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2022-03-04 To: 2022-06-30

Route of administration:

oral: gavage

Details on route of administration:

Route of test item administration was through oral gavage. The oral route was chosen because it provides an exaggerated model of possible exposure in humans.

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Pre-calibration of the beaker to desired volume: Milli-Q® water was measured in a graduated cylinder to the final volume of 70 and 80 mL. The measured water was transferred into a clean beaker and upper and lower meniscus of water was marked on the beaker using a marker. The water was discarded, and the beaker was dried.
- dose formulation preparation: Required quantities of the test item was weighed in a beaker (previously calibrated to a desired volume*) for each dose levels separately and a small volume of vehicle (Milli-Q® water) was added and stirred using a glass rod till a uniform solution was obtained. The volume was made up to the mark using the vehicle to get the final desired concentration of 17.5, 35 and 75/85 mg/mL for the G2, G3 and G4/G4R groups, respectively.
- The test item was prepared at the appropriate concentrations as a solution in distilled water.
- The dose formulations were prepared once daily before start of each day dosing.
- From Day 43, the high dose formulation was prepared with changed concentration, the same procedure was followed
- The volume of dose formulation prepared was varied depending on the requirement and/or body weights of the rats recorded during experimental period.

VEHICLE
- Justification for selection of vehicle: The Formulation Analytical Method Validation and Stability was established in Milli-Q water under Eurofins Advinus study (G24817). Further, the same vehicle was used in 14-day repeated dose range finding toxicity study (Study No. N6321) Hence, Milli-Q water was used as vehicle for dose formulation preparation.
- Concentration in vehicle: 0, 17.5, 35, 75/85 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For test item concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during 2nd (Day 53) and 3rd (Day 71) month of the treatment period and analysed in-house.

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1, vehicle control

Dose / conc.:

175 mg/kg bw/day (nominal)

Remarks:

G2, low dose

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

G3, mid dose

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

G4, high dose during days 1 - 42

Dose / conc.:

850 mg/kg bw/day (nominal)

Remarks:

G4 high dose, from day 43 till sacrifice

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1R, vehicle control recovery

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

G4R, high dose recovery during days 1 - 42

Dose / conc.:

850 mg/kg bw/day (nominal)

Remarks:

G4R, high dose recovery from day 43 till sacrifice

No. of animals per sex per dose:

10 animales/sex/dose; 6 groups

Control animals:

yes, concurrent vehicle

Details on study design:

- The dose levels are selected for this study in consultation with the Sponsor as below based on the results of the 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No. N6321). In 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats, the dose levels of 500, 750, and 1000 mg/kg bw/day were tested. Based on the results, the dose levels of 0, 175, 350 and 750 mg/kg bw/day were selected in the subchronic toxicity study.
For selecting dose levels in viewing above results, 175 mg/kg bw/day was chosen as the low dose that would not cause any toxic effects. The highest dose of 750 mg/kg bw/day was selected expected to produce target organ or nonspecific toxicity. The intermediate dose of 350 mg/kg bw/day was selected to provide information on dose response relationship for toxicity. The limit dose 1000mg/kg bw/day caused mortality and clinical signs in the 14-
day study which makes it inappropriate for a 90-day study. During the course of the main study, treatment at 750 mg/kg bw/day dose did not induce any clinical, changes in body weight and food consumption. Hence, the high dose of 750 mg/kg bw/day was increased to 850 mg/kg bw/day from treatment day 43 to observe toxicity of the test item at the top dose. Finally the study was conducted with 175, 350 and 850 mg/kg bw/day as low (G2), mid (G3) and high dose (G4)/high dose recovery (G4R) groups, respectively. The high dose (G4/G4R) is represented as 750/850 mg/kg bw/day with a note that the high dose (G4/G4R) group rats were treated with 750 mg/kg bw/day during days 1 – 42 and then 850 mg/kg bw/day from day 43 till sacrifice

- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in recovery groups: 28 days following termination of treatment

Positive control:

no

Observations and examinations performed and frequency:

MORBIDITY AND MORTALITY:
- Time schedule: twice daily i.e., once in the morning and once in the afternoon except during holidays wherein the observation was done once daily as there were no clinical signs observed.

CLINICAL SIGNS:
- Time schedule: twice daily during treatment period. On the days of scheduled detailed clinical examination (once weekly), clinical signs were included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.

DETAILED CLINICAL EXAMINATION:
- Time schedule: prior to the test item administration on Day 1 and at weekly intervals thereafter (± 1 days) during treatment period.
- During detailed clinical examination, all rats were observed cage-inside/outside for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour (e.g. selfmutilation, walking backwards).

OPHTALMOLOGICAL EXAMINATION:
- Time schedule: prior to start of the treatment, at the end of the treatment period for main groups (Day 90) and at the end of recovery period (Day 117) for recovery groups.
- Before examination, mydriasis was induced using a 1 % solution of Tropicamide.

FUNCTIONAL OBSERVATION BATTERY TESTS (FOB):
- Time schedule: during the 13th week (Day 88) for main toxicity groups and towards the end of recovery period for recovery groups (Day 116).
* HOME CAGE OBSERVATIONS:
- Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.
* OBSERVATION DURING REMOVAL OF ANIMAL FROM HOME CAGE AND HANDLING:
The objective of this phase of neurological examination was to observe the subject’s response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following examinations:
- ease of removal from home cage
- handling reactivity
- palpebral closure
- eye examination
- piloerection
- lacrimation
- salivation
- skin/fur examination
- perineum wetness
- respiration
- muscle tone and
- extensor thrust response
The observations were recorded using scores/ranks.
* OPEN FIELD OBSERVATION:
Rat was placed (one at a time) in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each group. During this observation period, rat was evaluated as it moves about freely/unperturbed and the following observations were made and observations were recorded using score/ranks:
- gait
- posture
- tremors
- mobility score
- arousal level
- clonic or tonic movements
- stereotypic behaviour
- bizarre behaviour
- urination
- defecation
- rearing
- abnormal vocalizations
* FUNCTIONAL TESTS:
- Functional testing includes motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.
- motor activity:
- The motor activity of rats was measured using an automated animal activity measuring system equipped with a computer analyzer.
- Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes.
- During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large ambulatory movement) in seconds, distance travelled in centimeter and resting time in seconds were monitored. The Opto-Varimex 5 data was analyzed in 10 minutes intervals and the same was reported.
- sensory reactivity measurements:
After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/ranks.
- approach response
- touch response
- click response
- tail-pinch response
- pupil response
- aerial righting reflex
- landing hindlimbs footsplay:
- The landing hind limbs foot splay was performed by dropping the rat onto a horizontal surface of the tabletop from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat were gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and
then dropped from a height of approximately 30 cm on to a SOP format, which contains the details such as Study no., Animal no, Group and Sex. A clean recording SOP format was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values is presented in the report along with the individual footsplay values.
- grip performance:
- Hindlimbs and forelimbs grip performance was tested using computerized dual grip strength meter (Model: Columbus Instruments). Three trials were conducted for each rat i.e., three trials each for forelimb and hind limbs. Averages of three trials for both forelimb and hindlimbs are calculated and presented in the report along with the individual grip strength values.
* PHYSIOLOGICAL OBSERVATIONS:
- Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer.
- At the end of the functional test, body weight of each rat was measured.

BODY WEIGHT:
- Time schedule: prior to test item administration on Day 1 and weekly thereafter (± 1 day) for all groups of rats during treatment and recovery period except for week 13 wherein the animals were weighed on day 5 of that week.
- Fasting body weight was recorded prior to sacrifice for all surviving toxicity group animals.

FOOD CONSUMPTION:
- Time schedule: weekly intervals (± 1 day) during treatment and recovery period except on week 13, wherein the food consumption was measured on day 5 of that week.
- The cage wise average food intake (g/rat/day) was calculated.

OESTROUS CYCLE EVALUATION:
- Vaginal smear was examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy.

HAEMATOLOGY:
- Time schedule: at the end of the treatment and recovery periods, approximately 4.0 mL of blood was collected under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture.
- Anaesthetic used for blood collection: yes, isoflurane
- Animals fasted: yes, overnight (water allowed)
- How many animals: all
- Aliquots of blood (0.7mL) were collected into tubes containing K2EDTA (1.6 mg/mL blood)
- Haematological parameters were determined using ADVIA 2120i haematology system (Siemens Healthcare Diagnostics Inc., NY, USA): red blood corpuscles, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocytes count, white blood corpuscles, differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelets
- blood smears were prepared from the haematology (K2EDTA tube) samples and stained with Giemsa stain (solution-. Blood smear evaluation was not performed manually as the automated counts were adequate for the data interpretation. All the blood smear slides will be discarded at the time of final report preparation.

COAGULATION:
- Time schedule: at the end of the treatment and recovery periods, approximately 4.0 mL of blood was collected under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture.
- Anaesthetic used for blood collection: yes, isoflurane
- Animals fasted: yes, overnight (water allowed)
- How many animals: all
- Aliquots of blood (0.5 mL) were collected into tubes containing trisodium citrate (3.2 mg/mL blood)
- Blood samples collected for coagulation analysis were centrifuged at 2500 times gravity (xg) for 10 minutes at 15ºC for separation of plasma and analysed for the following parameters in plasma sample using Ceveron Alpha automated coagulation analyser: prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY:
- Time schedule: at the end of the treatment and recovery periods, approximately 4.0 mL of blood was collected under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture.
- Anaesthetic used for blood collection: yes, isoflurane
- Animals fasted: yes, overnight (water allowed)
- How many animals: all
- Aliquots of blood (1.8 mL) were collected into tubes containing lithium heparin (10 unit/mL blood)
- Plasma was separated after centrifugation of the whole blood samples at 4ºC, 5000 rpm for 5 minutes and analysed using Dimension RxL MaX Clinical Chemistry System
- parameters: alanine aminotransferase, alkaline phosphatase, albumin, albumin/globulin ratio (calculated value), aspartate aminotransferase, blood urea nitrogen, chloride, creatinine, calcium, calcium, glucose, globulin (calculated value), HDL cholesterol, inorganic phosphorous, LDL cholesterol, potassium, sodium, total cholesterol, total plasma protein, triglycerides, total bilirubin

URINANALYSIS:
- Time schedule: at the end of the treatment period and at the end of the recovery period in urine collection tube.
- how many animals: all
- Each rat was placed overnight in a specially fabricated cage (water allowed) and the next morning the collected urine was sent for analysis.
- parameters: specific gravity, nitrite, pH, proteins, glucose, ketone bodies, urobilinogen, bilirubin, erythrocytes, leukocytes, appearance (colour and clarity), volume (approximate)
- also, urine was subjected to microscopic examination: crystals, epithelial cells, casts

HORMONE ANALYSIS:
- At the end of the treatment and recovery periods, blood was collected from all rats along with blood collection for clinical pathology investigation for the determination of total T3, T4 and TSH hormones.
- Blood samples were collected in plain labelled tubes and kept on bench top for 90 min before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4° C. The serum samples were placed in labelled plastic tubes and was stored at ~ -70 °C until they were analysed. The left-over samples from hormone analysis were discarded following data review by analyst.

THYROID PROFILE HORMONES:
- thyroid hormones were estimated by Enzyme Linked Immuno Sorbent Assay (ELISA) method using BIO-RAD microplate washed and BIO-RAD model 680 reader
- parameters: rodent thyroid stimulating hormone (TSH), rodent thyroxine (T4), rodent triiodothyronine (T3)

Sacrifice and pathology:

NECROPSY:
- Rats were euthanized by exsanguination under isoflurane anesthesia.
- All rats were subjected to detailed necropsy (examination of external surfaces of the body, all orifices, cranial, thoracic and abdominal cavities and their contents) by a veterinary pathologist and findings were recorded. All rats were subjected to necropsy after overnight fasting (water allowed) at the end of treatment period for main group rats and at the end of recovery period for recovery group rats. Terminal body weights were recorded for all animals immediately prior to euthanasia.

TISSUE COLLECTION, WEIGHING AND PRESERVATION:
- The following tissues and organs were collected and weighed from all rats: artery, aorta; bone, femur/joint, femorotibial (decalcified prior to sectioning); bone marrow, femur; bone marrow, sternum; bone, sternum; brain (cerebrum, cerebellum, medulla oblongata and pons); cervix; epididymis; esophagus; eye (collected with optic nerve and preserved in Davidson's fluid); gland, adrenal; gland, coagulating (weighed after fixation); gland, mammary; gland, parathyroid (weighed after fixation); gland, pituitary (weighed after fixation); gland, prostate (prostate and seminal vesicles with coagulating glands were weighed as a whole; subsequently prostate was separated and weighed. The derived weight was presented for the seminal vesicles and coagulating glands); gland, salivary; gland, seminal vesicle (prostate and seminal vesicles with coagulating glands were weighed as a whole; subsequently prostate was separated and weighed. The derived weight was presented for the seminal vesicles and coagulating glands); gland, thyroid (weighed after fixation); gross lesion/mass/nodule; gut-associated lymphoid tissue; heart; kidney; large intestine, cecum; large intestine, colon; large intestine rectum; liver; lung (inflated with 10%NBF before immersion in the fixative); lymph node, mandibular; lymph node, mesenteric; muscle, skeletal; nerve, optic; nerve, sciatic; ovary; oviduct; pancreas; small intestine, duodenum; small intestine, ileum; small intestine, jejunum; skin; spinal cord; spleen; stomach; testis (collected in modified Davidson's fluid); tongue; trachea; thymus; urinary bladder (inflated with 10%NBF before immersion in the fixative); uterus (weighed with cervix); vagina
- The tissues/organs were preserved in 10 % Neutral Buffered Formalin (NBF) except for the testes and eyes.
- Bone marrow smears were prepared from femur marrow from all main group and recovery group animals on their respective termination days, air dried, fixed in methanol and stained with Giemsa stain. However, the bone marrow smears were not examined since the intact marrow of sternum and femur were normal histologically

ORGAN WEIGHT:
- the organ weight ratios as percentage of fasting body weight and brain weight were determined. The paired organs were weighed together and combined weight were presented.
- organs weighed: brain, epididymis, adrenal gland, pituitary gland, prostate gland, seminal vesicle gland, thyroid gland, heart, kidney, liver, ovary, spleen, testis, thymus, uterus

HISTOPATHOLOGY:
- Histopathological examination was carried out on the preserved organs of vehicle control (G1) and high dose group animals (G4) (full list above). Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis. In addition, all gross lesions from all the animals were examined microscopically. Stomach was examined in the lower dose groups (G2 and G3) and recovery groups (G1R and G4R) as test item related findings were noted in stomach at high dose (G4).
- The tissues were processed for routine paraffin embedding and 4-5 micron sections were stained with Haematoxylin and Eosin stain. Additional sections were taken for testes at 3-4 µm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis.

Statistics:

Data was analysed using the Provantis(TM) laboratory information management system (LIMS).
Parameters such as body weight, body weight change, body temperature, hindlimbs footsplay, grip performance, food consumption, organ weights, organ weight ratios (organ to body weight and organ to brain weight), laboratory Investigations – Haematology, Coagulation & Clinical Chemistry and transferred (motor activity, thyroid profile) data were evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be nonhomogeneous or of nonnormal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA found to be significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data captured outside of ProvantisTM: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0.
Descriptive statistics Mean, SD, Percentages & Numbers were presented by Treatment group and Day.
All hypothesis testing was carried out at the 5% (2-sided) significance level unless otherwise specified. Significant differences are designated throughout the report as below:
*: Statistically significant difference from the control group at p < 0.05

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 750/850 mg/kg bw/day, transient clinical sign of slight salivation was observed soon after the dose administration from treatment Day 11.
At 350 mg/kg bw/day, transient clinical sign of slight salivation was observed soon after the dose administration from treatment Day 23. In addition, isolated occurrence of sparse hair loss was observed in one female rat.
Nevertheless, the salivation subsided within a few minutes and the rats were found to be normal.
There were no clinical signs observed during the treatment at 175 mg/kg bw/day dose group in either sex.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities observed among all the tested doses.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly lower mean body weight was observed on days 8 and 15 at 750/850 mg/kg bw/day main toxicity group males, on day 22 at 750/850 mg/kg bw/day recovery females.
Significantly lower absolute body weight gain during days 1-8 in high dose males, during days 111-117 and in high dose recovery males during the recovery period, during days 22-29 at 175 mg/kg bw/day in females, during days 78-85 in high dose recovery females was observed. Significantly higher absolute weight gain during days 36-43 at 175 mg/kg bw/day in males, during days 15-22 at 750/850 mg/kg bw/day in main and recovery group females was observed. These sporadic incidences of significant differences were considered incidental as no consistency was observed. Further, total absolute gains were comparable to vehicle control.
The terminal fasting body weights were unaffected by treatment.
See data tables for detailed information

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males, the food consumption was significantly lower during Days 1-8, 8-15, 29-36, 36-43, 50-57, 57-64, 64-71, 71-78, 78-85 and 85-90 at high dose, during days 1-8 and 43-50 in high dose recovery group was observed. In females, the food consumption was significantly lower during days 1-8, 8-15, 57-64 and 71-78 at high dose, during days 29-36, 36-43, 50-57, 57-74, 64-71, 78-85, and 111-117 in high dose recovery group, during days 1-8 and 85-90 at low dose and during days 85-90 at mid dose was observed.
Significantly higher food consumption during days 1-8 in low dose males, during days 111-117 in high dose recovery group males andduring days 64-71 at mid dose in females was observed.
These significant differences were not considered toxicologically relevant as the body weights were not altered by the treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmological examination carried out with an ophthalmoscope prior to start of treatment, at the end of the treatment and recovery period did not reveal any abnormalities in the eyes of the experimental rats.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Increase in the neutrophil count (62%) was noted at 750/850 mg/kg/day in females, considered as the secondary changes associated with test item related inflammation in stomach.
All other statistically significant changes observed in haematology parameters were considered incidental in nature, as the changes were of minimal magnitude and/or not dose progressive.
No test-item related changes in coagulation parameters were observed in both sexes. All the differences noted in the coagulation data were sporadic, minimal with lack of dose progression and hence were not considered toxicologically relevant.
See data tables for detailed information

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test-item related changes in clinical chemistry parameters were observed. All statistically significant changes observed in clinical chemistry parameters were considered incidental in nature either in absence of associated histopathological findings or the changes were of minimal magnitude.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed in total T3, T4 and TSH hormones analyzed in all group animals.
Statistically significant decrease in thyroxine (T4) level in males at 750/850 mg/kg/day was not related to test item administration in the absence of correlating changes in T3/TSH and no associated microscopic findings in thyroid gland were observed. In addition, no effect on T4 levels was observed in the high dose recovery group.
See data tables for detailed information

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test-item related changes in urinalysis parameters were observed.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related abnormalities were observed in any of the tested dose groups in either sex during home cage and handling observations, open field observations, and sensory observations.
- Motor activity: There were no significant differences observed at all the tested doses in both sexes. Incidence of significantly lower distance travelled during interval 2 was observed at all treated groups in females. This significant difference was considered toxicologically not relevant as the total distance travelled was comparable the vehicle control.
The following statistically significant differences were observed in the motor activity in rats when compared to concurrent vehicle control groups:
- Higher:
Males: Resting time during interval 2 at 750/850 mg/kg bw/day recovery group.
Females: Stereotypic time during interval 2 and 3, total stereotypic time at 750/850 mg/kg bw/day in main toxicity group.
- Lower:
Males: Total distance and distance travelled during interval 2 and 3 at 750/850 mg/kg bw/day recovery group. Total ambulatory time and ambulatory time during interval 3, total stereotypic time and stereotypic time during intervals 2 and 3, total distance and distance travelled during interval 2 and 3 at 750/850 mg/kg bw/day, distance travelled during interval 2 at 350 mg/kg bw/day in main toxicity groups.
The above observed statistical variations in the motor activity measurements are considered to be incidental as there were no changes observed in the home cage or open field observations. Further, there were no clinical signs observed during daily clinical observation.

Neuromuscular observation:
- landing hind limb footsplay: Significantly lower hindlimb footsplay was observed at all tested doses in main toxicity groups males. This significant difference was considered to be toxicologically not significant as there were no changes observed in the home cage or open field observations. Further, there were no clinical signs observed during daily clinical observation.
- grip strength: Significantly higher hindlimb grip strength in males and forelimb grip strength in females at 750/850 mg/kg bw/day main toxicity group. These significant differences were considered toxicologically not relevant as the animals were normal in the home cage and open field observations.

Physiological observation:
- body temperature: No treatment-related abnormalities were observed at any of the groups in either sex.
- body weight: There were no significant differences observed in the body
weights, measured at the end of neurological observations at any of the doses
tested in both sexes.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The terminal fasting body weights were not affected by the test item administration.
Decrease in weights of seminal vesicles with coagulating glands and spleen at 750/850 mg/kg/day in males and increase in weights of liver at 750/850 mg/kg/day in females were noted. These were considered as test item related secondary changes and there were no microscopic correlates at 750/ 850 mg/kg/day.
There were no test item- related changes in the organ weight and their ratio in recovery group animals.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Grossly, test item related non-glandular/ glandular multifocal thickening, red focus and discoloration in both the sex at 750/ 850 mg/kg/day dose were correlated with either mucosal hyperplasia/ hyperkeratosis, inflammation or ulceration. The stomach lesions showed complete recovery after 28 Days of dosing free period.
- Incidences of unilateral enlarged testes in control male (Rab8147) was associate with dilatation of seminiferous tubules and decreased sperm, whereas in 750/ 850 mg/kg/day male (Rab8207) was associated with dilatation and degeneration of seminiferous tubules.
- Single incidence of unilateral enlarged epididymis in 750/ 850 mg/kg/day male (Rab8207) was associated with decreased sperm and cell debris. All these findings were considered as incidental/spontaneous change.
- Few incidences of dilatation of uterus with cervix observed across the groups were considered as normal spontaneous changes related to estrous cycle and not related to test item administration.
See data tables for detailed information

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- The qualitative assessment of spermatogenesis in testes did not reveal any changes at 750/ 850 mg/kg/day and in vehicle control rats.
- Histopathological examination of stomach revealed ulcer (at 750/ 850 mg/kg/day in both sexes), hyperplasia/hyperkeratosis (at ≥175 mg/kg/day in both sexes), submucosal and mucosal inflammation of non-glandular and/ or glandular region (at ≥350 mg/kg/day in males and 750/ 850 mg/kg/day in females).
- The ulceration (focal/ multifocal/ diffuse) of non-glandular and glandular stomach was characterized by loss of either partial or complete mucosa to muscularis layer with inflammatory cell infiltration in mucosa and submucosa. Hyperplasia/ hyperkeratosis (multifocal) in non-glandular stomach was characterized by epithelial proliferation with thickened keratin layer.
- The ulceration in stomach mucosa was considered as test item-related local adverse change whereas the other local effects were not considered as adverse.
- Single incidence of hyperplasia/hyperkeratosis of non-glandular mucosa was also observed at 750/ 850 mg/kg/day in recovery female. This finding was considered as test item-related non-adverse change in absence of ulceration.
All other histologic lesions were considered as background/spontaneous changes and not considered as test item related.
See data tables for detailed information

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Oestrus cycle evaluation: the vaginal smear was examined for all female rats prior to necropsy and details of various stages are observed. Most of the rats are showed diestrous, estrous, and metestrous at necropsy.

Details on results:

The administration of the test item for 90 consecutive days through oral gavage to Wistar rats at 175, 350 and 750/850 mg/kg/day did not show any test item-related changes in coagulation, clinical chemistry, thyroid profile, urinalysis parameters. The terminal fasting body weights were unaffected by treatment.
The following test item related changes were noted:
At 750/850 mg/kg/day:
- Increase in neutrophil count in females correlated with inflammation in stomach histologically.
- Grossly, test item related stomach findings were correlated with either mucosal hyperplasia/ hyperkeratosis, inflammation or ulceration microscopically. The stomach lesions showed complete recovery after 28 Days of treatment free period.
- Test item related microscopic findings of ulcer with submucosal and mucosal inflammation in glandular/ non-glandular stomach and hyperplasia/hyperkeratosis of non-glandular stomach were noted in both sexes. Grossly, these findings were noted as non-glandular/ glandular multifocal thickening, red focus and discoloration with increased neutrophil count in females.
- The ulcers were considered as adverse findings whereas the other changes were the secondary changes. All these findings reversed at the end of recovery period. The minimal degree non glandular epithelial hyperplasia in the recovery female showed the reversibility tendency.
At 175 and 350 mg/kg/day:
- Microscopically, test item related hyperplasia/ hyperkeratosis of non-glandular stomach was noted in both sexes. This was considered as a local adaptive non adverse finding.

Key result

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Dose Formulation Analysis of the Test Item

The dose formulations were considered acceptable as the mean results are within 80-120 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 20 %. The analyses results were within 90-116% of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 1-6 %. This indicates that the prepared dose formulation met the acceptance criteria for concentration and % RSD.

Conclusions:

The results of the study indicated that the oral administration of test item for 90 consecutive days in Wistar rats at dose levels of 175, 350 and 750/850 mg/kg/day did not cause any toxicological effect on general health, neurological findings, ophthalmological examination, body weights, food consumption, coagulation, clinical chemistry, thyroid profile, urinalysis parameters. Transient clinical sign of slight salivation was observed in all animals at 350 and 750/850 mg/kg bw/day soon after the dose administration. Nevertheless, the symptoms subsided within a few minutes and the rats were found to be normal. No mortality was observed at any dose tested.

There were no test item-related changes in terminal fasting body weights and organ weights at all dose levels tested.

Considering the changes observed in haematology and microscopic changes in the stomach at 750/850 mg/kg bw/day, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the test item is considered to be 350 mg/kg bw/day under the test conditions and doses employed.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 2022-01-19 to 2022-06-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this 14-day repeated dose toxicity study was to evaluate the toxicity potential of the test item in Wistar rats when administered orally through gavage for a period of 14 days. This study was also intended to define the dose levels for subsequent definitive toxicity studies. All procedures were in compliance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines of India. The protocol was approved by Institutional Animal Ethics Committee (IAEC) of Eurofins Advinus Limited (Proposal No.: 017/July - 2019).

GLP compliance:

no

Remarks:

This study is not intended to be conducted in compliance with Good Laboratory Practice (GLP) as it is a preliminary range-finding study, but it will follow the mutually agreed Study Plan and the test facility's SOPs (AAALAC approved).

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 1,4-dimethylpiperazine
- Source and lot/batch number of test material: PFW210769
- Expiration date of the lot/batch: 2023-11-25
- Purity: 99.91 A% (by GC)
- Physical appearance: liquid
- pH: 11.4
- density: 0.84 g/cm³
- specific gravity: 0.822 at 20°C

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (+15 to +22 °C), protect from light/humidity, at temperature no higher than +22 °C under inert gas blanket
- Stability under storage conditions: not indicated
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the vehicle: Stability of the test item in the vehicle (Milli-Q water) at 1 and 100 mg/mL was established under Eurofins Advinus Study No. G19071. The test item was found to be homogeneous and stable in the vehicle up to 48 hours when stored at room temperature.

OTHER:
- the substance is total titratable amine content (amine content 17.39 meq/g)

Species:

rat

Strain:

Wistar

Remarks:

Crl:Wistar IGS

Details on species / strain selection:

The rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology (India) Pvt. Ltd., 4B MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078, India (licensed breeder of Charles River)
- Females (if applicable) nulliparous and non-pregnant: not indicated
- Age at start of treatment: 6 weeks
- Weight at start of treatment: 224.35 to 276.06 g (males), 167.79 to 192.57 g (females)
- Fasting period before study: Not indicated
- Housing: The rats were housed in groups of two per sex per cage in solid floor standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for providing pelleted food and drinking water in polycarbonate bottle with stainless steel sipper tubes. Polycarbonate rat huts were provided to the animals from first day of acclimatization as environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall well-being. Steam sterilized clean corn cob was used as bedding and changed along with the cage twice a week.
- Diet (e.g. ad libitum): ad libitum, Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany
- Water (e.g. ad libitum): ad libitum, deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India was provided in polycarbonate bottles with stainless steel sipper tubes
- Acclimation period: 5 days (2022-01-19 to 2022-01-23). After detailed clinical examination for good health and the suitability for the study, the rats were acclimatized for five days before start of the treatment. During acclimatization period, rats were observed once daily for any abnormalities. Only nulliparous and non-pregnant females were used in the study

DETAILS OF FOOD AND WATER QUALITY:
The feed and water provided to the rats were tested for contaminants. Based on the latest analytical certificate(s) available, there were no known contaminants in the food, water and bedding that are expected to interfere with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 24.1 °C
- Humidity (%): 64 - 67 %
- Air changes (per hr): 13.9 -14.1 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2022-01-19 To: 2022-02-07

Route of administration:

oral: gavage

Details on route of administration:

Route of test item administration was through oral gavage. The oral route has been chosen because it provides an exaggerated model of the normal exposure in humans.

Vehicle:

water

Remarks:

Milli-Q water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Pre-calibration of the beaker to desired volume: Milli-Q® water was measured in a graduated cylinder to the final volume of 20 mL. The measured water was transferred into a clean beaker and upper and lower meniscus of water was marked on the beaker using a marker. The water was discarded, and the beaker was dried.
- The required quantity of test item was weighed into a pre-calibrated beaker and small volume of vehicle (Milli-Q® water) was added and mixed using glass rod. The final volume was made up with vehicle to the desired volume to get concentration was 50, 75 and 100 mg/mL.
- The dose formulations were prepared daily prior to dosing.
- The volume of dose formulation prepared, and the quantity of test item weighed varied depending on the requirement.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The Formulation Analytical Method Validation, Homogeneity and Stability, was established in Milli-Q water under Eurofins Advinus study (G19071). Further, OECD 407 (G19072) and OECD 421 (G19073) studies were conducted using Milli-Q water as vehicle for dose formulation preparation. Hence, Milli-Q water was used as vehicle for dose formulation preparation.
- Amount of vehicle (if gavage): 10 mL/kg
- Dose levels: 0, 500, 750 and 1000 mg/kg bw/day
- Concentration in vehicle: 0, 50, 75, 100 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For test item concentration analysis, prepared formulations from each dose level were sampled in duplicate sets on Day 1 of treatment period and analysed in-house. For each set, composite sample was drawn in six replicates from each preparation and in case of control, a duplicate composite sample was drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G24817. One set of samples were analyzed for concentration analysis and other set (second set) of samples were stored at ambient condition for reanalysis purpose as backup. The second set of samples were discarded, as the analysis results of first set of samples were within the limits.
The dose formulations were considered acceptable, as the mean percent recovery was within ± 15 % at each dose level and %RSD at each dose level was less than 10%.

Duration of treatment / exposure:

14 consecutive days

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 vehicle control group

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

G2

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

G3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4

No. of animals per sex per dose:

4 males and 4 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the Sponsor’s recommendation, the dose levels of 500, 750, and 1000 mg/kg/day were selected. In addition to the test doses, vehicle control group was included. Animals in the vehicle control was handled in a manner similar to the treatment groups except for test item administration.
- Fasting period before blood sampling for clinical biochemistry: At the end of the treatment period, all rats were fasted overnight (water allowed) and approximately 3.0 mL of blood was collected under isoflurane anaesthesia, with the help of a fine capillary tube, by retro-orbital sinus puncture.

Positive control:

No

Observations and examinations performed and frequency:

MORBIDITY and MORTALITY:
All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon except during holidays wherein the observation was done once daily as there were no clinical signs of concern which requires observation.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each rat was observed for clinical signs twice daily during treatment i.e. once prior to dosing and 1-2 hour after the dosing. On the days of scheduled detailed clinical examination (once weekly as described in 8.3), clinical signs (after dosing) were included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was carried out prior to the treatment and observations for general clinical signs was done after dosing the animals. On the day of euthanasia of rats, observations for clinical signs were recorded once in the morning prior to scheduled euthanasia.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examination was done prior to the test item administration on Day 1 and at weekly intervals thereafter (±1 day) during treatment period.
- Parameter examined: During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded prior to test item administration on Day 1 and on Days 4, 8, 11 and 14 of the treatment periods. At term, terminal body weight was measured on Day 15 following overnight fasting.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The food consumption was measured on Day 4 (representing the feed consumption between Days 1-4) and on Days 8, 11 and 14 (representing the feed consumption between Days 4-8, Days 8-11 and Days 11-14, respectively).
The cages were checked for any spillage during cage change and at each food leftover recording session.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, approximately 3.0 mL of blood was collected with the help of a fine capillary tube, by retro-orbital sinus puncture.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight (water allowed)
- How many animals: All rats
- Aliquots of blood were collected (0.7mL) with anticoagulant K2EDTA (1.6 mg/mL of blood)
- Parameters examined: Red Blood Corpuscles, Haemoglobin, Haematocrit, Mean Corpuscular Volume, Mean Corpuscular Haemoglobin, Mean Corpuscular Haemoglobin Concentration, Reticulocytes count, White Blood Corpuscles, Differential leukocyte count (Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils), Platelets
- Coagulation: Blood samples collected for coagulation analysis (0.5mL aliquot, trisodium citrate 3.2 mg/mL blood) were centrifuged at 2500 times gravity (xg) for 10 minutes at 15ºC for separation of plasma and analysed for the following parameters in plasma sample : Prothrombin Time, Activated Partial Thromboplastin Time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, approximately 3.0 mL of blood was collected with the help of a fine capillary tube, by retro-orbital sinus puncture.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight (water allowed)
- How many animals: All rats
- Aliquots of blood were collected (1.8 mL) with anticoagulant lithium heparin (10 unit/mL)
- Parameters examined: Alanine Aminotransferase, Alkaline Phosphatase, Albumin, Albumin/Globulin ratio (calculated value), Aspartate Aminotransferase, Blood Urea Nitrogen, Chloride, Creatinine, Calcium, Gamma Glutamyl Transpeptidase, Glucose, Globulin (calculated value), Inorganic Phosphorous, Potassium, Sodium, Total Cholesterol, Total Plasma Protein, Triglyceride, Total Bilirubin.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

- Necropsy: All rats including the found dead female at high dose were subjected to detailed necropsy and findings were recorded. The necropsy included examination of external surfaces of the body, all orifices; cranial, thoracic and abdominal cavities and their contents. Terminal fasting body weights were recorded for all surviving animals immediately prior to terminal sacrifice and used in calculation of relative organ weights. All terminally sacrificed rats were fasted overnight (water allowed), euthanized with isoflurane (as per the random numbers generated for the study), exsanguinated and subjected for gross examination.

- Tissue Collection: On completion of gross pathology examination, the tissues and organs listed below were collected and weighed from all terminally sacrificed rats. The organ weight ratios (organ to body weight and organ to brain weight) as percentage of fasting body weight and brain weight were determined. The paired organs were weighed together. The tissues were preserved in 10% Neutral Buffered Formalin (NBF) except for the testes and eyes: Artery, Aorta; Bone, femur/joint, femorotibial; Bone marrow, femur; Bone marrow, sternum; Bone, Sternum; Brain (cerebrum, cerebellum, medulla oblongata and pons) (incl. organ weight); Cervix; Epididymis (incl. organ weight); Esophagus; Eye; Gland, adrenal (incl. organ weight); Gland, coagulating; Gland, mammary; Gland, parathyroid; Gland, Pituitary (incl. organ weight); Gland, prostate (incl. organ weight); Gland, salivary; Gland, seminal vesicle (incl. organ weight); Gland, thyroid (incl. organ weight); Gross lesions/masses/nodules; Gut associated lymphoid tissue; Heart (incl. organ weight); Kidney (incl. organ weight); Large intestine, Cecum; Large intestine, Colon; Large Intestine, Rectum; Larynx; Liver (incl. organ weight); Lung; Lymph node, mandibular; Lymph node, mesenteric; Muscle, skeletal; Nerve, Optic; Nerve, sciatic; Ovary (incl. organ weight); Oviduct; Pancreas; Pharynx; Small intestine, Duodenum; Small intestine, Ileum; Small intestine, Jejunum; Skin; Spinal cord; Spleen (incl. organ weight); Stomach; Testis (incl. organ weight); Tongue; Trachea; Thymus (incl. organ weight); Ureter; Urinary bladder; Uterus (incl. organ weight); Vagina

- organ weight determined for: brain (cerebrum, cerebellum, medulla oblongata and pons), epididymis, adrenal gland, pituitary gland, prostate gland, seminal vesicle gland, thyroid gland, heart, kidney, liver, ovary, spleen, testes, thymus, uterus

HISTOPATHOLOGY: No
Histopathology examination of preserved organs/tissues was not carried out as there were no test item related gross findings. The preserved tissues were discarded before finalization of report.

Statistics:

Data was captured using the Provantis(TM) laboratory information management system (LIMS).
Parameters such as body weight, body weight change, food consumption, organ weights, organ weight ratios (organ to body weight and organ to brain weight), laboratory Investigations – Haematology, Coagulation & Clinical Chemistry data was evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data
found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be nonhomogeneous or of nonnormal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA found to be significant, pairwise comparisons of treated groups to the control group was made using a
parametric test, Dunnett, to identify statistical differences.
Descriptive statistics Mean, SD, Percentages & Numbers was presented by Treatment group and Day.
All hypothesis testing were carried out at the 5% (2-sided) significance level unless otherwise specified. Significant differences were designated throughout the report as below:
*: Statistically significant difference from the control group at p < 0.05.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs observed throughout the treatment period in either sex at 500 mg/kg/day.
At 750 mg/kg/day, transient clinical sign of slight salivation was observed soon after dose administration in all animals from treatment Day 8. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal.

At 1000 mg/kg/day, transient clinical sign of slight salivation was observed soon after the dose administration in all animals from treatment Day 8. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal. One female rat (Rab8031) exhibits a clinical sign of emaciation, dehydration, nasal discharge, abdominal respiration and weakness on Day 8 during morning observation. Subsequently, this female rat died during afternoon observation. Grossly, glandular stomach discoloration and non-prominent thymus were noted. In addition, one male rat (Rab8026) showed the clinical signs of piloerection and emaciation during days 11-14 of the treatment period.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female rat (Rab8031) at 1000 mg/kg/day was found dead on day 8. Grossly, glandular stomach discoloration and non-prominent thymus were noted.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day, treatment did not affect the mean body weights in males and females. The absolute body weight gains were lower (statistically not significant) during initial period of treatment. However, from day 4 onwards, the body weight gains were comparable to control group, suggesting adaptation to the treatment. The overall body weight gain and total percent weight gain during days 11-14 was significantly lower in males.
At 750 mg/kg/day in males, the mean body weights were apparently lower throughout the treatment, statistically significant on day 14 with the reduction of 12.80% on Day 14, compared to vehicle control group. The body weight gain was significantly lower during Days 1-4, 8-11 and overall weight gain during Days 1-14 as compared vehicle control. In females, a significantly lower mean body weights on Day 8, lower body weight gain during Days 4-8 and overall lower weight gain and lower total percent weight gain during Days 1-14 were observed as compared vehicle control.
In males at 1000 mg/kg/day, the mean body weights were apparently lower throughout the treatment, statistically significant on days 11 and 14, with the reduction of 19.01% and 18.2% on days 4 and 14 respectively, compared to vehicle control group. The body weight gain was significantly lower during Days 1-4, 4-8, 8-11 and overall weight gain during days 1-14 as compared vehicle control. In females, a significantly lower mean body weights on Day 8, lower body weight gain during Days 4-8 and overall lower weight gain and total percent weight gain during Days 1-14 were observed as compared vehicle control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day, the food consumption was significantly lower during Days 8-11 and 11-14 in females with the reduction of -5.87 to -14.53%. These significant difference in food consumption did not alter body weight and body weight gains.
At 750 mg/kg/day, the food consumption was significantly lower during Days 1-4, 8-11 and 11-14 and overall food consumption in males, during Days 4-8, 8-11 and 11-14 and overall food consumption with the reduction of -12.1% in males and -22.8 in females, compared to vehicle control group.
At 1000 mg/kg/day, the food consumption was significantly lower throughout the treatment in males, during 4-8, 8-11 and 11-14 in females and overall food consumption with the reduction of -27.5% in males and -30.6 in females, compared to vehicle control group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The neutrophil and monocyte counts were higher at ≥750 mg/kg/day in males and at all dose levels in females. However, these changes were not dose related. The lymphocyte count decrease was noted in males at ≥750 mg/kg/day and in females at 1000 mg/kg/day. These changes were considered to be associated with the stress due to test item administration.
The coagulation parameters were not affected by the test item administration.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, decreased glucose was noted at ≥750 mg/kg/day, a secondary observation to the decreased feed intake with decreased albumin level at 1000 mg/kg/day. The minimal increase in AST was observed at 1000 mg/kg/day males. The triglycerides were higher at ≥750 mg/kg/day in males and all dose levels in females.

Endocrine findings:

no effects observed

Description (incidence and severity):

Any effects seen in adrenal glands or thymus were seen as secondary.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The terminal fasting body weights were lower in both males and females at ≥750 mg/kg/day. Organ weight showed higher adrenal weights (absolute and relative) in males at ≥750 mg/kg/day and considered as a secondary stress associated finding. The decrease in seminal vesicles, coagulating gland weights at ≥750 mg/kg/day and thymus weights at all dose levels were considered as the secondary findings to the decreased body weights.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related gross lesions in the terminally sacrificed rats. The gross findings of glandular stomach discolouration and non-prominent thymus were noted in a found dead female at 1000 mg/kg/day on Day 8.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

Oral gavage administration of the test item at 500 and 750 mg/kg/day doses to Wistar rats for 14 consecutive days did not result in any mortality. Transient clinical sign of slight salivation was observed soon after the dose administration in all animals from treatment Day 8 at >=750 mg/kg/day. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal. One female rat (Rab8031) exhibited clinical signs of emaciation, dehydration, abdominal respiration, nasal discharge and weakness at 1000 mg/kg/day. Subsequently this female rat was died during afternoon observation. Grossly, glandular stomach discoloration and non-prominent thymus were noted. In addition, one male rat showed clinical sign of emaciation and piloerection during days 11 to 14 of the treatment period.
At 500 mg/kg/day, the mean body weights and food consumption were not altered by the treatment. Minimal increase in neutrophil/monocyte counts and higher triglyceride level in females were noted with a minimal decrease in thymus weights.
At 750 mg/kg/day, the mean body weights were apparently lower throughout the treatment with the reduction of 12.80% on day 14 in males associated with reduction in food consumption (-5.02 to -20.52%). The absolute body weight gain and overall weight gain during were lower as compared vehicle control. In females, the mean body weights during initial period of treatment and overall weight gain were significantly lower associated with the reduction in food consumption (-13.23 to -36.96%) as compared vehicle control. The increased neutrophil, monocyte counts were noted in both sexes and decreased lymphocyte counts were noted only in males. Glucose level was lower in males whereas the triglycerides were high in both sexes. Terminal fasting body weight was lower in both gender with higher adrenal weight and decreased seminal vesicle and coagulating gland and thymus weights.
At 1000 mg/kg/day, the mean body weights were apparently lower throughout the treatment with the reduction of 18.20 to 19.01% in males associated with reduction in food consumption (-6.06 to -34.53%). The absolute body weight gain and the overall weight gain were lower as compared vehicle control. In females, the mean body weights and overall weight gain were significantly lower associated with reduction in food consumption (-20.81 to -47.55%) as compared vehicle control. Increased neutrophil and monocyte counts were present in both sexes with decreased lymphocyte count. Lower glucose, albumin and increase in AST (males) and triglycerides were noted. The terminal fasting body weight was lower in males with secondary changes of decreased seminal vesicles, coagulating glands and thymus and the increased adrenal weight in males was considered as stress associated secondary finding. The test item related gross findings: glandular mucosa discoloration in stomach and non-prominent thymus were noted in the found dead female on Day 8. The mortality and the higher percentage of terminal body weight decrease at 1000 mg/kg/day were considered as adverse findings.
Due to the mortality observed in 1/4 females in 1000 mg/kg/day dose group, the clinical signs in female and males, the significantly lower body weight and food consumption in both sexes the dose 1000 mg/kg/day is not recommended for a sub-chronic repeated dose toxicity study.
At 750 mg/kg bw/day, the animals showed mild clinical signs, lower body weight and lower food consumption but no mortality. This dose level is recommended for the sub-chronic repeated dose toxicity study as it is not expected to cause severe pain or suffering or excessive death of the test animals.

Key result

Dose descriptor:

dose level:

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Key result

Critical effects observed:

no

Dose formulation analysis of the test item

Dose formulations were analyzed for concentration and homogeneity of test item prior to start of treatment on Day 1. Formulations were considered acceptable as the mean results are within ± 15 % of the claimed concentration and the relative standard deviation (% RSD) was less than 10%. No test item peak was detected in the vehicle samples.

Conclusions:

Due to the mortality observed in ¼ females in 1000mg/kg bw/day dose group, the clinical signs in female and males, the significantly lower body weight and food consumption in both sexes the dose 1000mg/kg bw/d is not recommended for a sub-chronic repeated dose toxicity study.

At 750 mg/kg bw/day, the animals showed mild clinical signs, lower body weight and lower food consumption but no mortality. This dose level is recommended for the sub-chronic repeated dose toxicity study as it is not expected to cause severe pain or suffering or excessive death of the test
animals.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

No data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Not enough information on material and methods

Qualifier:

no guideline followed

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): 1,4-dimethylpiperazine

Species:

rat

Sex:

male

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

0.125%, 0.25% and 0.5%

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

30 days

Frequency of treatment:

no data

Dose / conc.:

0.125 other: %

Remarks:

Basis: nominal in diet

Dose / conc.:

0.25 other: %

Remarks:

Basis: nominal in diet

Dose / conc.:

0.5 other: %

Remarks:

Basis: nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

No data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: at the start and at the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Yes

FOOD EFFICIENCY:
- Yes, mean food intake (g/rat/day) and mean weight gain (g/g food consumed) have been determined.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No

Clinical signs:

no effects observed

Description (incidence and severity):

All animals had a healthy appearance and normal behavior during the feeding period.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Animals fed 0.5% of the product had a marked, significant (P<0.05) reduction in mean body weight and mean food intake.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Animals fed 0.25% of the product had a slight, but significant decrease in mean food intake.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no apparent effect on the efficicency of food utilization at any of the feeding levels.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the end of the feeding period, all animals were killed and given a thorough gross autopsy. No relevant pathology was noted.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Critical effects observed:

not specified

Mean dosage: 0, 0.12, 0.24 and 0.48 g/kg/day

Conclusions:

The NOAEL observed in this supporting study was 120 mg/kg bw after an exposure period of 30 days.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2020-01-14 to 2020-02-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline available

Principles of method if other than guideline:

The purpose of this repeated dose toxicity study was to assess the systemic toxicological potential of the test item in Wistar rats when administered orally by gavage for a period of 14 consecutive days. The results of this study are used to select suitable doses for 28-day repeated dose toxicity study and reproduction/developmental toxicity screening test in the rats. All procedures were in compliance with the guidelines provided by the Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA) India. This Study Plan was approved by the Institutional Ethics Committee (IAEC) of the Test Facility (Approval No.: 017/July - 2019).

GLP compliance:

no

Remarks:

The mutually agreed study plan and the Standard Operating Procedures (SOPs) of Eurofins Advinus Limited. This study was performed in an AAALAC approved laboratory.

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: PFW190423
- Expiry Date: 2021-08-05
- Physical Appearance : Liquid
- Purity: 99.9 A%
- Purity test date: 2019-11-08

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to +25ºC). Temperature no higher than +25ºC.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability and homogeneity of the test item in the vehicle was established at 1 and 100 mg/mL under Eurofins Advinus Study No. G19071. Based on the results, the test item was stable and homogenous in the vehicle up to 24 hours when stored at room temperature.

OTHER SPECIFICS
- pH:11.4
- Density: 0.84 g/cm³
- Specific gravity: 0.84

Species:

rat

Strain:

Wistar

Remarks:

HanTac:WH

Details on species / strain selection:

The rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 20 male and 20 female rats from Vivo Bio Tech Ltd. Sy. # 349/A, Pregnapur Village, Gajwel mandal, Medak District, Telangana, India
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 121.40-144.03 grams, Females: 110.18-128.80 grams
- Fasting period before study: not indicated
- Housing: Rats were housed in a group of two animals of same sex per cage in sterilized suspended polysulfone cages with solid floor (Size: L 425 x B 266 x H 185 mm) with stainless steel top grill having facilities for providing pelletted food and drinking water in polycarbonate bottle with stainless steel sipper tubes. Polycarbonate rat hut was provided to the animals as environmental enrichment objects and changed along with cage once a week. Steam sterilized clean Corn cob was used as bedding and changed along with the cage at least twice a week.
- Diet: ad libitum, Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany
- Water: ad libitum, deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India was provided in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: After clinical examination for good health and the suitability for the study, the rats were acclimatized for five days before start of the treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24°C
- Humidity (%): 50 to 66 %.
- Air changes (per hr): 12.9 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2020-01-24 To: 2020-02-12

Route of administration:

oral: gavage

Details on route of administration:

Route of test item administration was through oral gavage. The oral route was chosen because it provides an exaggerated model of the normal exposure in humans.

Vehicle:

water

Remarks:

Milli-Q

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Required quantities of the test item were weighed in a beaker (previously calibrated to a desired volume*) for each dose levels separately and a small volume of vehicle (Milli-Q® water) was added and stirred using a glass rod till a obtaining uniform suspension. The volume was made up to the mark using the vehicle to get the final desired concentration of 5, 15, 30 and 60 mg/mL for the G2, G3, G4 and G5 groups, respectively.
- Pre-calibration of the beaker to desired volume: Milli-Q® water was measured in a graduated cylinder to the final volume of 30 mL. The measured water was transferred into a clean beaker and upper and lower meniscus of water was marked on the beaker using a marker. The water was discarded and the beaker was dried.
- From treatment day 8, the dose levels were increased to 100, 300, 600 and 1000 mg/kg/day. For this, required quantities of the test item were weighed for each dose levels separately and mixed with vehicle [Milli-Q water] to get the final desired concentration of 10, 30, 60 and 100 mg/mL for the G2, G3, G4 and G5 groups, respectively. The dose formulations were prepared once daily before start of each day dosing. Homogeneity of the test item solutions prior to sampling and during dosing was maintained by constant stirring using a magnetic stirrer. The volume of dose formulation to be prepared was varied depending on the requirement and/or body weights of the rats recorded during experimental period.

VEHICLE
- Justification for use and choice of vehicle: Based on solubility test, the test item forms homogeneous solution/suspension in Milli-Q water. The Formulation Analytical Method Validation, Homogeneity and Stability was established in Milli-Q water under Eurofins Advinus study (G19071). Hence, Milli-Q water was used as vehicle for dose formulation preparation.
- Concentration in vehicle: 10, 30, 60, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and 8 of the treatment period and analysed in-house. For each set, duplicate sample was drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer was drawn. The analysis was done as per the method validated under Eurofins Advinus Study No.: G19071. One set of samples were analyzed and other set (second set) of samples were stored at room temperature for possible reanalysis as a backup and these samples were discarded, as the analysis results of first set of samples were within the acceptable limits. Dose formulations were considered acceptable as the overall mean results were within ± 15 % of the theoretical concentration and the overall relative standard deviation (RSD) was less than 10%.

Duration of treatment / exposure:

14 consecutive days

Frequency of treatment:

once daily at approximately the same time (± 3 hours)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control (G1)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Low dose (G2) until Day 7

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose (G2) from day 8

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Mid dose (G3) until Day 7

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid dose (G3) from day 8

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid-Intermediate dose (G4) until Day 7

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

Mid-Intermediate dose (G4) from day 8

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

High dose (G5) until Day 7

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose (G5) from day 8

No. of animals per sex per dose:

5 groups, 4 males and 4 females per group

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dose levels of 100, 300, 600 and 1000 mg/kg bw/day were selected in consultation with the Sponsor. During the course of the experiment, treatment had not cause mortality and clinical signs of toxicity up to 600 mg/kg /day till treatment day 7. Hence, the dose levels of 50, 150, 300 and 600 mg/kg/day were increased to 100, 300, 600 and 1000 mg/kg/day, respectively from day 8 of the treatment period in consultation with the sponsor. Accordingly, the dose levels were represented as 50/100, 150/300, 300/600 and 600/1000 mg/kg/day with a note that the 50, 150, 300 and 600 mg/kg/day were treated during days 1 – 7 and then 100, 300, 600 and 1000 mg/kg/day from day 8 till termination. In addition to the test doses, vehicle control group was included. Animals in the vehicle control group was handled in a manner similar to the treatment group except for test item administration.
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups: Rats were randomly distributed to different groups by the body weight stratification method using ProvantisTM Software (Version 10.1.0.1, Instem LSS, Staffordshire ST15OSD, United Kingdom). Rats that are not selected were killed humanely without any further investigation. The grouping was done one day prior to initiation of treatment during acclimatization.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
*Acclimatization period: once daily for any abnormalities.
*Morbidity and mortality: twice daily, except during holidays wherein the observation was done once daily.
*Clinical signs: twice daily during the treatment i.e. once prior to dosing and 1-2 hour after the dosing. On the days of scheduled detailed clinical examination, clinical signs (after dosing) was included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to treatment on Day 1 and at weekly intervals thereafter during treatment period. On the days of detailed clinical examination, observation for general clinical signs was not performed except on Day 1.
- During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour (e.g. self-mutilation, walking backwards).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights (g) were recorded prior to test item administration for all animals on Day 1 and on Days 4, 8, 11 and 14 for all animals. Additionally, fasting body weight was measured on Day 15 following overnight fasting.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule for examinations: The food output was measured on Days 4, 8, 11 and 14 of the experiment.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: The blood samples were collected from all rats on Day 15.
- Anaesthetic used for blood collection: Yes, blood samples were collected under isoflurane anesthesia, with the help of fine capillary tube, by retro-orbital sinus puncture.
- Animals fasted: Yes, fasted overnight (water allowed).
- Approximate volume collected (mL): Haematology: 0.7 mL (K2EDTA at 1.6 mg/mL blood) and Coagulation: 0.5 mL (trisodium citrate 3.2 mg/mL blood)
- The following haematological parameters were determined using ADVIA 2120i haematology system (Siemens Healthcare Diagnostics Inc., NY, USA): Red Blood Corpuscles (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Haemoglobin Concentration (MCHC), Reticulocytes count (Retic), White Blood Corpuscles (WBC), Differential leukocyte count (DLC) (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Platelets (Plat).
- Blood samples collected for coagulation analysis were centrifuged at 2500 times gravity (xg) for 10 minutes at 20 ºC for separation of plasma and analysed for the following parameters in plasma sample using STart Max coagulation analyzer (Diagnostica stago, 92600 Asnieres, France): Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The blood samples were collected from all rats on Day 15.
- Anaesthetic used for blood collection: Yes, blood samples were collected under isoflurane anesthesia, with the help of fine capillary tube, by retro-orbital sinus puncture.
- Animals fasted: Yes, fasted overnight (water allowed).
- Approximate volume collected (mL): 1.8 mL (lithium heparine at 10 unit/mL blood)
- Plasma samples were separated following centrifugation of whole blood at 5000 rpm, 4 °C for 5 minutes and analyzed using Dimension RxL MaX Clinical Chemistry System (Dade Behring Inc. Newark, DE 19714, USA) for the following parameters: Alanine Aminotransferase (ALT), Alkaline Phosphatase (Alp), Albumin (ALB), Albumin/Globulin ratio (calculated value) (A/G), Aspartate Aminotransferase (AST), Blood Urea Nitrogen (BUN), Chloride (Cl), Creatinine (Creat), Calcium (Ca), Gamma Glutamyl Transpeptidase (GGT), Glucose (Glu), Globulin (calculated value) (GLOB), Inorganic Phosphorous (Pi), Potassium (K), Sodium (Na), Total Cholesterol T. (Chol), Total Plasma Protein (T. Pro), Triglycerides (Trig), Total Bilirubin (T. Bil), Direct Bilirubin (D. Bil) Indirect Bilirubin (calculated)(Ind. Bil).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- necropsy: All rats were fasted overnight (water allowed), euthanized with isoflurane, exsanguinated and subjected for detailed necropsy (examination of external surfaces of the body, all orifices, cranial, thoracic and abdominal cavities and their contents) and findings were recorded by a Veterinary Pathologist. Terminal fasting body weights were recorded for all animals immediately prior to terminal sacrifice and used in calculation of relative organ weights.
- tissue collection: On completion of the gross pathology examination, the tissues and organs noted below were collected and weighed from all rats. The organ weight ratios (organ to body weight and brain weight) were determined and presented in the report as percentage of fasting body weight and brain weight. The paired organs were weighed together and combined weight was presented. The tissues were preserved in 10 % Neutral Buffered Formalin (NBF) except for the testes and eyes: Brain including medulla/pons, cerebellum and cerebrum, Cecum, Cervix, Colon, Duodenum, Epididymides, Esophagus, Eyes, Femoral muscle (Skeletal Muscle), Femur Bone with joint, Glands, adrenal, Glands, salivary, Gross lesions/masses/nodules, Gut associated lymphoid tissue, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lungs (with bronchi and bronchioles), Lymph nodes, mandibular, Lymph nodes, mesenteric, Mammary gland, Nerves, sciatic, Ovaries, Oviducts, Pancreas, Pharynx, Pituitary, Prostate, Rectum, Seminal vesicles and coagulating glands, Skin, Spinal cord (cervical, thoracic and lumbar), Spleen, Sternum with marrow, Stomach, Testes, Thymus, Thyroid and Parathyroid, Tongue, Trachea, Ureters, Urinary bladder, Uterus and Vagina.
- list organ weights: brain including medulla/pons, cerebellum and cerebrum, epididymides, adrenal glands, heart, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles and coagulating glands, spleen, testes, thymus, thyroid and parathyroid, uterus

HISTOPATHOLOGY: Yes
- Histopathological examination was carried out on the preserved organs of all group rats.
- The tissues were processed for routine paraffin embedding and approximately 4 micron sections were stained with Haematoxylin and Eosin stain. Unused tissues were archived.

Statistics:

Data captured using Provantis™ for the parameters body weight and organ weights; laboratory Investigations – Haematology, Coagulation and Clinical Chemistry parameters were analyzed using built-in statistical tests.
Derived data like net body weight change, food consumption and organ weight ratios were also be analyzed using above mentioned methods.
The integrated decision tree of ProvantisTM:
(i) Test variance homogeneity by Levene’s method was tested. When variances are heterogeneous, suitable transformation was performed automatically by the software.
(ii) Further Two sample t-test was performed
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated by the following throughout the report:
*: Significantly different from vehicle control group.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- There were no clinical signs in the 100 and 300 mg/kg/day dose groups.
- At 300 mg/kg/day, no treatment-related clinical signs were observed up to treatment Day 7. Hence, the dose was increased to 600 mg/kg on treatment Day 8. From treatment Day 10, clinical signs of slight salivation was observed in both sexes.
- At 600 mg/kg/day, no treatment-related clinical signs were observed up to treatment Day 7. Hence, the dose was increased to 1000 mg/kg on treatment Day 8. From treatment Day 9, clinical signs like salivation (slight to moderate) and nasal discharge in males and slight salivation in females were observed in most of the animals.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities observed at any of the tested doses.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment did not affect the mean body weights at all the doses tested in either sex up to treatment Day 7. After increasing dose levels, significantly lower mean body weight on Days 11 and 14, absolute body weight gain during Days 8-11 and 11-14 and total body weight gains during Days 1-14 were observed in males at 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Treatment did not affect the mean food consumption at all the doses tested in either sex up to treatment Day 7. After increasing dose levels, significantly lower mean food consumption was observed during Days 8-11 and 11-14 in males and females at 1000 mg/kg/day. Thus, the treatment decreased the body weight and food consumption in males and food consumption in females at 1000 mg/kg/day.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 600/1000mg/kg/day, test item related decrease in total WBC count in males and lymphocyte count in both sexes was observed. Neutrophil count was lower in females. Decrease in terminal body weights was noted in males. Reduced thymus weight with lymphoid depletion was observed in both sexes. Increase in blood glucose noted in females. At 300/600mg/kg/day in females, lower neutrophil count, increase in blood glucose concentration were considered as test item related. Haematological parameters analysis revealed, decrease in the total leukocyte count in males and lymphocyte count in both sexes at 1000 mg/kg/day. Increased neutrophil count at ≥600mg/kg/day in females was observed. However, microscopic examination of hematopoietic organs did not reveal any associated morphological changes.
There were no test item related changes in the Prothrombin time (PT) and Activated partial thromboplastin time (APTT) at all the tested doses in either sex.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Administration of test item, by oral gavage to Wistar rats for 14 consecutive days at dose levels of 50/100, 150/300, 300/600 and 600/1000 mg/kg/day did not reveal any test item related change in clinical chemistry parameters. Clinical chemistry parameters analysis revealed, increase in blood glucose concentration at 1000 mg/kg/day in females.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The terminal fasting body weights were lower at 1000 mg/kg/day in males. Organ weights showed decrease in absolute and relative weights of thymus associated with microscopic correlate of decreased cellularity of thymus in both sexes at 1000 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Grossly, depressed foci in non-glandular of stomach was observed in males at 1000 mg/kg/day and was microscopically confirmed as non-glandular epithelial
hyperplasia.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 600/1000mg/kg/day, vacuolation of tubular epithelium in kidneys, duct epithelium of sub-mandibular and parotid salivary glands, acini of sublingual salivary glands, uterine glandular epithelium, glandular epithelium of stomach and ependymal cells of choroid plexus in brain were considered as test item related. The changes in glandular stomach, uterine glandular epithelium and ependymal cells of choroid plexus were considered as inconclusive effects as no evidence of general function alteration in organs /tissues affected. Hyperplasia of non-glandular epithelium and inflammation in lamina propria of stomach and tubular basophilia in kidneys at this dose were also considered test item related.
Grossly, depressed foci in non-glandular of stomach was observed in males at 1000 mg/kg/day and was microscopically confirmed as non-glandular epithelial hyperplasia. Hyperplasia of non-glandular epithelium and inflammation in lamina propria of stomach and tubular basophilia in kidneys at this dose were also considered test item related.

Histopathological findings: neoplastic:

not examined

Details on results:

Analysis of the test item
- identity of the test item: the identity of the test item was provided by the study sponsor by a Certificate of Analysis. The test item was not authenticated at the test facility.
- Dose Formulation Analysis of the Test Item: The dose formulations were analyzed for concentration and homogeneity of the test item on Days 1 and 8 of the treatment. The results were considered acceptable, as the percent agreement of the analyzed concentrations of all the layers and mean of each layer are in the range, 90% to 103% of the claimed concentrations and the relative standard deviation (RSD) was equal to or less than 10 %.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Conclusions:

Based on the findings in this oral 14 days dose range finding study, the NOAEL for systemic toxicity of the test item is considered to be 300 mg/kg/day following 14 day repeated oral administration to Wistar rats. Considering the body weight and microscopic changes, the Maximum Tolerated Dose is considered to be ≥600 mg/kg/day following 14 day repeated oral administration to Wistar rats.

Reference 5

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-03-13 to 2020-07-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008-10-03

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: PFW190423
- Expiration date of the lot/batch: 2021-08-05
- Purity:99.9%
- Physical appearance: liquid
- pH: 11.4
- density: 0.84 g/cm³
- specific gravity: 0.84

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (+15 to +25 °C), temperature no higher than +22 °C
- Stability under storage conditions: not indicated
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Stability of the test item in the vehicle was established at 1 and 100 mg/mL. The test item was stable in the vehicle up to 24 hours when stored at room temperature.
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): not indicated

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology Pvt. Ltd., Plot 4B, AKP, Turkapally Village, Shameerpet Mandal, RR Dist, Telangana 500078, India
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at start of treatment: 5 weeks
- Weight at start of treatment: 178.20 to 211.25 g (males), 144.47 to 174.78 g (females)
- Fasting period before study: No
- Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in each group and sex was housed individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage twice a week. During the experimental period, animals were housed in a single experimental room of barrier area (SC-33). Steam sterilized corn cob was used as bedding and changed along with the cage twice a week.
- Diet (e.g. ad libitum): ad libitum, Altromin Rat/Mice Maintenance diets
- Water (e.g. ad libitum): ad libitum, deep bore-well water passed through activated charcoal filter and exposed to UV rays in 'Aquaguard' on-line water filter-cum-purifier
- Acclimation period: 5 days (2020-03-13 to 2020-03-17). After clinical examination for good health and the suitability for the study, the rats were acclimatized for five days before start of the treatment. During acclimatization period, rats were observed once daily for any abnormalities.

DETAILS OF FOOD AND WATER QUALITY:
The feed and water provided to the rats were tested for contaminants. Based on the latest analytical certificate(s) available, there were no known contaminants in the food, water and bedding that are expected to interfere with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24 °C
- Humidity (%): 49 - 58 %
- Air changes (per hr): 12.4 -12.7 air change per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2020-03-18 To: 2020-04-15

Route of administration:

oral: gavage

Details on route of administration:

Route of test item administration was through oral gavage. The oral route was chosen because it provides an exaggerated model of the normal exposure in humans

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
* pre-calibration of the beaker to desired volume: Milli-Q water was measured in a graduated cylinder to the final volume of 40 and 50 mL. The measured water was transferred into a clean beaker and upper and lower meniscus of water was marked on the beaker using a marker. The water was discarded and the beaker was dried.
* Required quantities of the test item were weighed in a beaker (previously calibrated to a desired volume) for each dose level separately and a small volume of vehicle (Milli-Q water) was added and stirred using a glass rod until a uniform suspension was obtained. The volume was made up to the mark using the vehicle to get the final desired concentration of 15, 30 and 60 mg/mL. The suspensions were mixed well by stirring using a magnetic stirrer.
* The dose formulations were prepared once daily before start of each day dosing and administered within 2 hours after preparation. Homogeneity of the test item suspensions prior to sampling and during dosing was maintained by constant stirring using a magnetic stirrer.
* The volume of dose formulation to be prepared was varied depending on the requirement and/or body weights of the rats recorded during experimental period.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The Formulation Analytical Method Validation, Homogeneity and Stability was established in Milli-Q water under Eurofins Advinus study (G19071). Further, the same vehicle was used in the dose range finding toxicity study (study no. N4793). Hence, Milli-Q water was used as vehicle for dose formulation preparation.
- Amount of vehicle (if gavage): 10 mL/kg
- Dose levels: 0, 150, 300 and 600 mg/kg bw/day
- Concentration in vehicle: 0, 15, 30, 60 mg/mL

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

For homogeneity and test item concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during week 4 (Day 26) of the treatment period and was analysed in-house. For each set, composite sample was drawn in six replicates from each preparation and in case of control duplicate composite samples was drawn. The analysis was done as per the method validation under Eurofins Advinus Study G19071. One set of samples were analyzed and other set (second set) of samples were stored at room temperature for possible reanalysis as a backup and these samples were discarded, as the analysis results of first set of samples were within the acceptable limits. Dose formulations were considered acceptable as the overall mean results were within ± 15 % of the theoretical concentration and the overall relative standard deviation (RSD) was less than 10%.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 control (vehicle only)

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

G2 Low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3 Mid dose

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

G4 High dose

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1R vehicle control recovery

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

G4R high dose recovery

No. of animals per sex per dose:

5 males and 5 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of 150 (G2), 300 (G3) and 600 (G4/G4R) mg/kg/day were selected for this study based on the results of 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No.N4793) and in consultation with the Sponsor. The 14-day repeated dose oral toxicity study in Wistar rats was initiated with 50, 150, 300 and 600 mg/kg/day doses. During the course of treatment, there were no clinical/toxic signs observed till Day 7 up to 600 mg/kg/day. Hence, the dose levels of 50, 150, 300 and 600 mg/kg/day were increased to 100, 300, 600 and 1000 mg/kg/day, respectively from treatment day 8. The results were:
* clinical signs and mortality: at 1000 mg/kg/day, the clinical signs of toxicity included, salivation (slight to moderate) and reddish colour nasal discharge in males and slight salivation in females was observed in most of the animals from 9th day of treatment. At 600 mg/kg/day, clinical sign of slight salivation was observed from 10th day of treatment period.
* body weight, body weight gains and food consumption: at 1000 mg/kg/day, the body weight and body weight gains were lower, associated with decrease in the food consumption in males
* hematology: decrease in the total leukocyte count in males and lymphocyte count in both sexes at 1000 mg/kg/day. Increased neutrophil count at >=600 mg/kg/day in females was observed. However, microscopic examination of hematopoietic organs did not reveal any associated morphological changes
* coagulation and clinical chemistry: the treatment did not induce any test item-related changes in coagulation parameters at all the tested doses in either sex. Clinical chemistry parameters analysis revealed, increase in blood glucose concentration at 1000 mg/kg/day in females.
* terminal fasting body weights and organ weights: organ weights showed decrease in absolute and relative weights of thymus associated with microscopic correlate of decreased cellularity of thymus in both sexes at 1000 mg/kg/day.
* gross and histopathology: grossly, depressed foci in non-glandular of stomach was observed in males at 1000 mg/kg/day and was microscopically confirmed as non-glandular epithelial hyperplasia. Microscopically, vacuolation of tubular epithelium in kidneys, duct epithelium of sub-mandibular and parotid salivary glands, acini of sublingual salivary glands, uterine glandular epithelium, glandular epithelium of stomach and ependymal cells of choroid plexus in brain were observed at 1000 mg/kg/day.
The stomach lesions observed in 1000 mg/kg/day were considered due to a test item-related irritant effect of the test item.
- Rationale for animal assignment (if not random): randomly distributed to different groups by the body weight stratification method using Provantis(TM) Software. Rats that are not selected were killed humanely without any further investigation. The grouping was done one day prior to initiation of treatment during acclimatization.
- Fasting period before blood sampling for clinical biochemistry: Animals were fasted overnight (water allowed) and blood samples were collected on day 29 from main groups and on day 43 from recovery group rats.
- vehicle control group: in addition to the test doses, a vehicle control group was included. Animals in the vehicle control were handled in a manner similar to the treatment groups except for test item administration.
- treatment: the dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (+/-3 hours) each day for a period of 28 consecutive days. Similarly, the vehicle was administered to rats in vehicle control/vehicle control recovery group once orally for 28 consecutive days. The dose formulation and the vehicle were administered at an equivolume of 10 mL/kg/day. The dose volume was calculated for individual animals on the first day of the treatment period and was adjusted according to the most recent body weights recorded during the treatment period.

Positive control:

No

Observations and examinations performed and frequency:

MORBIDITY and MORTALITY:
Each rat was observed twice daily for morbidity and mortality except during holidays wherein the observation was done once daily as there were no clinical signs observed.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each rat was observed twice daily except during holidays wherein the observation was done once daily. Each rat was observed for clinical signs twice daily during the treatment period. On the days of scheduled detailed clinical examination, clinical signs (after dosing) was included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals. On the day of euthanasia of rats, observations for clinical signs was done once in the morning prior to scheduled euthanasia.
- Cage side observations checked: special attention was paid to posture and for presence or absence of abnormal vocalizations, tremors and convulsions.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examination was done prior to treatment on Day 1 and 1-2 hours after dosing and at weekly intervals thereafter during treatment period. On the days of detailed clinical examination, observations for general clinical signs were not performed except post-dose observations on treatment Day 1.
- Parameter examined: During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour (e.g. self-mutilation, walking backwards).

BODY WEIGHT: Yes
- Individual body weights (g) were recorded prior to test item administration on Day 1 and weekly thereafter (± 1 day) for all rats. Fasting body weight was recorded prior to euthanasia for all animals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, the food consumption was measured at weekly intervals (± 1 day) during treatment and recovery period. The cage wise average food consumption (g/rat/day) was calculated and presented in the report.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Ophthalmological examination of all animals was performed with an ophthalmoscope prior to start of the treatment, at the end of the treatment for main toxicity groups and at the end of recovery period for recovery groups. Before examination, mydriasis was induced using a 1 % solution of Tropicamide.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 29 from main groups and on day 43 from recovery group rats; approximately 3 mL of blood was collected from each rat with the help of a fine capillary tube, by retro-orbital sinus puncture
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight (water allowed)
- How many animals: All
- Aliquots of blood (0.7mL) were collected into tubes containing coagulant K2EDTA (1.6mg/mL blood)
- Parameters examined, determined using ADVIA 2120i hematology system: Red Blood Corpuscles, Haemoglobin, Haematocrit, Mean Corpuscular Volume, Mean Corpuscular Haemoglobin, Mean Corpuscular Haemoglobin Concentration, Reticulocytes count, White Blood Corpuscles, Differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Platelets
Additionally, blood smears were prepared from the hematology (K2EDTA tube) samples and stained with Giemsa stain (solution). As the study findings did not warrant any further evaluation, all the blood smear/slides will be discarded at the time of final report preparation.
- Coagulation: Aliquots of blood (0.5mL) were collected into tubes containing anticoagulant trisodium citrate (3.2 mg/mL blood). Blood samples collected for coagulation analysis were centrifuged at 2500 times gravity (xg) for 10 minutes at 15ºC for separation of plasma and analysed for the following parameters in plasma sample using Start Max coagulation analyzer: Prothrombin Time, Activated Partial Thromboplastin Time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 29 from main groups and on day 43 from recovery group rats; approximately 3mL of blood was collected from each rat with the help of a fine capillary tube, by retro-orbital sinus puncture
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight (water allowed)
- How many animals: all
- Aliquots of blood (1.8mL) were collected into tubes containing coagulant lithium heparin (10 unit/mL blood)
- Plasma was separated after centrifugation of the whole blood samples at 4°C, 5000 rpm for 5 minutes and analysed using Dimension RxL MaX Clinical Chemistry System
- Parameters examined: Alanine Aminotransferase, Alkaline Phosphatase, Albumin, Albumin/Globulin ratio (calculated value), Aspartate Aminotransferase, Total Bile acids, Blood Urea Nitrogen, Chloride, Creatinine, Calcium, Gamma Glutamyl Transpeptidase, Glucose, Globulin (calculated value), Inorganic Phosphorous, Potassium, Sodium, Total Cholesterol, Total Plasma Protein, Triglyceride, Total Bilirubin, Direct Bilirubin, Indirect Bilirubin (calculated).

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from all rats at the end of the treatment and recovery period in the urine collection tube.
- Metabolism cages used for collection of urine: Yes, for urine collection, each rat was placed overnight in a specially fabricated cage (water allowed) and the following morning the collected urine was sent for analysis.
- Animals fasted: Not specified
- Parameters examined: Specific gravity, Nitrite, pH, Proteins, Glucose, Ketone bodies, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes, Appearance (colour and clarity), Volume (approximate)
- Urine was also subjected to microscopic examination for sediments such as crystals, epithelial cells and casts.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during 4th week of treatment (day 24) period for main groups and towards the end of recovery period (Day 39) for recovery groups.
- Dose groups that were examined: all
- Home cage observations: each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions
- Observations during removal of animal from home cage and handling: The objective of this phase of neurological examination was to observe the subject's response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following parameters: ease of removal from home cage, handling reactivity, palpebral closure, eye examination, piloerection, lacrimation, salivation, skin/fur examination, perineum wetness, respiration, muscle tone and extensor thrust response. The observations were recorded using scores.
- Open field observation: Rats were placed (one at a time) in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each group. During this observation period, rat was evaluated as it moves about freely/unperturbed and the following observations were made and observations were recorded using score/ranks: gait, posture, tremors, mobility score, arousal level, clonic or tonic movements, stereotypic behaviour, bizarre behaviour, urination, defecation, rearing, abnormal vocalizations
- Functional tests: functional testing includes sensory evaluation, measurement of grip performance, rectal temperature and motor activity:
- Sensory Reactivity Measurements: After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but had positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for the following: approach response, touch response, click response, tail-pinch response, pupil response, aerial righting reflex. Observations were recorded using proper scores.
- Motor Activity: The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large ambulatory movement) in seconds, horizontal counts and ambulatory counts were monitored. The Opto-Varimex 4 motor activity measurement system provides the data at 1 minute intervals and the data was analyzed in blocks of 10 minutes intervals and the same was reported.
- Landing Hindlimbs Footsplay: The landing hind limbs foot splay was performed by dropping the rat on to a horizontal surface of the table top from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat was gently pressed on an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on to a SOP format, which contained the details such as Study no., Animal no, Group and Sex. A clean recording paper sheet was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values are presented in the report along with the individual footsplay values.
- Grip Performance: Hindlimbs and forelimbs grip performance was tested using computerized dual grip strength meter (Model: Columbus Instruments). Animal was allowed to grasp the mesh bar/triangular pull bar connected to the force gauge with its fore paws. Slowly the animal was pulled away from the pull bar until it releases forelimb pull bar. For measuring hindlimbs grip strength, the animal was allowed to grasp hindlimbs push bar with its hind paws and pulled until it releases hindlimbs push bar. Three trials were conducted for each rat i.e., three trials each for forelimb and hind limbs. Average of three trials for both forelimb and hindlimbs are calculated and presented in the report along with the individual grip strength values.
- Physiological Observations: Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Necropsy: At the end of the treatment (main groups) and recovery period (recovery groups), all rats in the study were subjected to detailed necropsy and findings were recorded. The rats euthanized at term were fasted overnight (water allowed) prior to necropsy, weighed, anaesthetized with isoflurane and exsanguinated.
- Tissue Collection: On completion of the gross pathology examination, the tissues and organs listed below were collected and/or weighed from all rats.
The tissues were fixed in 10 % Neutral Buffered Formalin (NBF): Aorta, Bone marrow smear collected from femur marrow and stained with Giemsa stain), Brain including medulla/pons, cerebellum and cerebrum, Cecum, Cervix, Colon, Duodenum, Epididymides, Esophagus, Eyes (collected with optic nerve and preserved in Davidson's fluid), Femoral muscle (Skeletal Muscle), Femur Bone with joint (decalcified prior to sectioning), Glands, adrenal Glands, salivary, Gross, lesions/masses/nodules, Gut associated lymphoid tissue, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lungs (with bronchi and bronchioles) (inflated with 10% NBF before immersion in the fixative), Lymph nodes, mandibular, Lymph nodes, mesenteric, Mammary gland, Nerves, sciatic, Ovaries, Oviducts, Pancreas, Pharynx, Pituitary (weighed after fixation), Prostate (prostate and seminal vesicles with coagulating glands were weighed as a whole; subsequently prostate was separated and weighed. The derived weight was presented for the seminal vesicles and coagulating glands), Rectum, Seminal vesicles and coagulating glands, Skin, Spinal cord (cervical, thoracic and lumbar), Spleen, Sternum with marrow, Stomach, Testes (collected in modified Davidson's fluid), Thymus, Thyroid and Parathyroid (weighed after fixation), Tongue, Trachea, Ureters, Urinary bladder (inflated with 10% NBF before immersion in the fixative), Uterus (weighed with cervix), Vagina.
- organ weight: The organ weight ratios as percentage of body and brain weight were determined. The paired organs were weighed together and combined weights were presented. The organs weighed are: brain including medulla/pons, cerebellum and cerebrum, epididymides, adrenal glands, heart, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles and coagulating glands, spleen, testes, thymus, thyroid and parathyroid, uterus

HISTOPATHOLOGY: Yes
- Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose (G4) group rats. In addition, all gross lesions from all the rats were examined microscopically. There were no test item-related histopathological changes observed in any organ/tissue in in high dose (G4) group; hence, histopathological evaluation was not carried out in the lower dose (G2 and G3) and recovery groups (G1R and G4R).
- The tissues were processed for routine paraffin embedding and 4-5 micron sections were stained with Mayer’s Haematoxylin and Eosin stain. Unused tissues will be archived.
- The tissues examined were: Aorta, Bone marrow smear collected from femur marrow and stained with Giemsa stain), Brain including medulla/pons, cerebellum and cerebrum, Cecum, Cervix, Colon, Duodenum, Epididymides, Esophagus, Eyes (collected with optic nerve and preserved in Davidson's fluid), Femoral muscle (Skeletal Muscle), Femur Bone with joint (decalcified prior to sectioning), Glands, adrenal Glands, salivary, Gross, lesions/masses/nodules, Gut associated lymphoid tissue, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lungs (with bronchi and bronchioles) (inflated with 10% NBF before immersion in the fixative), Lymph nodes, mandibular, Lymph nodes, mesenteric, Mammary gland, Nerves, sciatic, Ovaries, Oviducts, Pancreas, Pharynx, Pituitary (weighed after fixation), Prostate (prostate and seminal vesicles with coagulating glands were weighed as a whole; subsequently prostate was separated and weighed. The derived weight was presented for the seminal vesicles and coagulating glands), Rectum, Seminal vesicles and coagulating glands, Skin, Spinal cord (cervical, thoracic and lumbar), Spleen, Sternum with marrow, Stomach, Testes (collected in modified Davidson's fluid), Thymus, Thyroid and Parathyroid (weighed after fixation), Tongue, Trachea, Ureters, Urinary bladder (inflated with 10% NBF before immersion in the fixative), Uterus (weighed with cervix), Vagina.

Optional endpoint(s):

not applicable

Statistics:

Data captured using Provantis™ for the parameters body weight and organ weights; laboratory Investigations – Haematology, Coagulation, Clinical Chemistry and urine parameters were analyzed using built-in statistical tests.
Derived data like net body weight change, food consumption and organ weight ratios were also analyzed using above mentioned methods.
The integrated decision tree of ProvantisTM:
i. Test variance homogeneity by Levene’s method was tested. When variances were heterogeneous, suitable transformation was performed automatically by the software.
ii. Further one-way analysis of variance (ANOVA) was performed. When ANOVA was significant, Dunnett’s control versus treatment group mean comparisons was performed.
Data captured outside of Provantis™: The statistical analysis of the experimental data was carried out using the validated package in Excel and/or using licensed copies of SYSTAT Statistical package Ver.12.0. All quantitative variables, neurological observations (neuromuscular observation/body temperature/body weights) were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before performing ANOVA. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test was found to be significant.
In case of recovery groups, data was analysed by Student ‘t’ test. Comparison of means between treatment recovery group(s) and vehicle control recovery group was performed
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated by the following throughout the report:
*: Significantly different from vehicle control group

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 600 mg/kg/day, transient clinical sign of slight salivation was observed soon after the dose administration in all animals from treatment Day 03. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal. During the recovery period no clinical signs observed. There were no clinical signs observed during the treatment at 150 and 300 mg/kg bwt/day dose group in either sex.
See data tables for detailed information

Mortality:

no mortality observed

Description (incidence):

No mortalities observed during the treatment at all dose groups in either sex.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Treatment did not affect the mean body weights in all the tested doses tested doses in either sex during the treatment period. During the recovery period, significantly lower mean body weights on Days 35 and 42 were observed at 600 mg/kg/day in males. This significant differences were toxicologically not significant as the absolute body weight gains were comparable to vehicle control recovery group.
- Significantly lower absolute body weight gain during days 1-8 in main toxicity group males and during days 8-15 in recovery group males was observed at 600 mg/kg/day. The total absolute and percent body weight gains from day 1-28 were significantly lower in 600 mg/kg/day high dose recovery group males. These significant differences were toxicologically not significant as the same changes were not observed either in the main or recovery groups during the same intervals.
In females, the absolute and total percent weight gains were comparable to vehicle control during the treatment and recovery period.
- there were no significant differences observed in the body weights, measured at the end of neurological observations at any of the doses tested in both sexes.
See data tables for detailed information

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption was significantly lower during days 8-15 and 15-22 in main group males, during days 8-15, 15-22, 22-28, 28-35 and 35-42 during the treatment and recovery period in recovery group males at 600 mg/kg/day. These significant decrease in food consumption were toxicologically not significant due lack of consistency. Further, the body weights were not altered by the treatment.
In females, the food consumption was comparable to vehicle control during the treatment and recovery period.
Thus, the treatment did not affect the body weight and food consumption in males and females at all the tested doses in either sex.
See data tables for detailed information

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmological examination was carried out with an ophthalmoscope prior to start of treatment and at the end of the treatment period did not reveal any abnormalities in the eyes of the experimental rats.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in hematology parameters at all dose levels tested.
All the variations in hematology parameters were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.
A few variations in coagulation parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in clinical chemistry parameters at all dose levels tested.
A few variations in clinical chemistry parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no effects observed in glands, thymus, thyroid and parathyroid after histopathological examination.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes in urinalysis parameters at all dose levels tested.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Home cage and Handling observations: No treatment-related abnormalities were observed at all the tested doses in both sexes.
- Open field observations: No treatment-related abnormalities were observed at all the tested doses in both sexes.
- Sensory observations: No treatment-related abnormalities were observed in any of the groups in both sexes.
- Motor Activity: There were no significant differences observed at all the tested doses in both sexes.
- Landing hind limb footsplay: Significantly lower hind limb footsplay in males at 600 mg/kg/day and at all the treated groups in main toxicity group females and at 600 mg/kg/day high dose recovery group females was observed. The above observed statistical variations in the landing hind limb footsplay were considered to be incidental as there were no changes observed in the home cage or open field observations. Further, there were no clinical signs observed during daily clinical observation.
- Grip strength: No significant changes were observed at all the tested doses in both sexes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in terminal fasting body weights and organ weights at all dose levels tested.
A few variations in organ weights were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.
See data tables for detailed information

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross changes at all dose levels tested.
See data tables for detailed information

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic changes at all dose levels tested.
All other single or few incidences of microscopic findings observed were considered incidental and not related to test item as they were randomly distributed.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Body temperature: no treatment-related abnormalities were observed in any of the groups in either sex.

Details on results:

Analysis of the test item:
- identity of the test item: the identity of the test item was provided by the study Sponsor by a Certificate of Analysis. The test item was not authenticated at the test facility.
- dose formulation analysis of the test item: the results indicated the percent agreement of the analyzed concentrations are in the range, 85% to 115% of the claimed concentrations. Homogeneity of the dose formulation was considered acceptable as the %RSD from six replicates at each dose level were <10.0%.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

In conclusion, oral gavage administration of test substance at 150, 300 and 600 mg/kg/day doses to Wistar rats for 28 consecutive days did not result in any treatment-related clinical signs, mortality or changes in the body weights and food consumption. The transient clinical sign of slight salivation was observed soon after test item administration and subsided within few minutes in both sexes at 600 mg/kg/day. This finding may be attributed to local oral mucosa irritation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity. In the present study there were no gross or microscopic changes in the gastrointestinal tract. Therefore, it was considered that the transient salivation observed in this study was of no toxicological significance. There were no test item-related changes in body weights, body weight gains, food consumption and neurological findings. Administration of test item did not reveal any test item-related changes in clinical pathology parameters, terminal fasting body weights, organ weight, gross pathology and histopathology in both the sexes at all the dose levels tested.

As there were no adverse treatment-related adverse effects observed up to the highest dose, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the test item is considered to 600 mg/kg bw/day under the test conditions and doses employed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP-compliant study, according to OECD guideline

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Dose-Range finding study prior to OECD407 (Malleshappa, 2020)

A 14-day Repeated Dose toxicity study by oral gavage in Wistar rats (K1; Malleshappa, 2020; study N4793) is conducted as Dose-Range Finding study (50/100, 150/300, 300/600 and 600/1000 mg/kg/day). The study was initiated with 50, 150, 300 and 600 mg/kg/day doses. During the course of treatment, there were no clinical/toxic signs observed till day 7 up to 600 mg/kg/day. Hence, the dose levels of 50, 150, 300 and 600 mg/kg/day were increased to 100, 300, 600 and 1000 mg/kg/day, respectively from treatment day 8. The test item was dissolved in Milli-Q water and administered to rats at the graduated dose levels. The rats in the vehicle control group received vehicle alone. The dose volume administered was 10 mL/kg body weight. Each group consisted of 4 male and 4 female rats. In a separate study (G19071), the test item was found to be stable for up to 24 hours when stored at room temperature. The prepared dose formulations were analyzed for test item concentrations on day 1 and day 8 of the treatment period. The results indicated that dose formulation concentrations were found to be within the acceptance limit of +/-15% of target and % RSD was less than 10.0%.

At 1000 mg/kg/day, the clinical signs of toxicity included, salivation (slight to moderate) and reddish colour nasal discharge in males and slight salivation in females was observed in most of the animals from 9th day of treatment. At 600 mg/g/day, clinical sign of slight salivation was observed from 10th day of treatment period. At 1000 mg/kg/day, the body weight and body weight gains were lower, associated with decrease in the food consumption in males. Decrease in the total leukocyte count in males and lymphocyte count in both sexes at 1000 mg/kg/day. Increased neutrophil count at ≥ 600 mg/kg/day in females was observed. However, microscopic examination of hematopoietic organs did not reveal any associated morphological changes. The treatment did not induce any test item-related changes in coagulation parameters at all the tested doses in either sex. Clinical chemistry parameters analysis revealed, increase in blood glucose concentration at 1000 mg/kg/day in females. Organ weights showed decrease in absolute and relative weights of thymus associated with microscopic correlate of decreased cellularity of thymus in both sexes at 1000 mg/kg/day. Grossly, depressed foci in non-glandular tissue of stomach were observed in males at 1000 mg/kg/day and was microscopically confirmed as non-glandular epithelial hyperplasia. Microscopically, vacuolation of tubular epithelium in kidneys, duct epithelium of sub-mandibular and parotid salivary glands, acini of sublingual salivary glands, uterine glandular epithelium, glandular epithelium of stomach and ependymal cells of choroid plexus in brain were observed at 1000 mg/k/day. However, the stomach lesions observed in 1000 mg/kg/day were likely to be caused by the corrosivity/irritation of the test item. Based on these observations, the Maximum Tolerated Dose is considered to be 1000 mg/kg/day in the 28-day Repeated Dose toxicity study in rats. 

 

Repeated dose toxicity - oral; short-term 28d, (Malleshappa, 2020):

The systemic toxicity profile of the test item is determined in a repeated dose toxicity study in Wistar rats when administered orally by gavage for 28 consecutive days and by assessing the reversibility of any effects during a subsequent 14 days recovery period. This study was also intended to provide the information on major toxic effects, target organs and an estimation of a No Observed Adverse Effect Level (NOAEL).

The test item was dissolved in Milli-Q water and administered to rats at the graduated dose levels of 150, 300 and 600 mg/kg/day to low dose (G2), mid dose (G3) and high dose (G4) / high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1)/ vehicle control recovery (G1R) groups received vehicle Milli-Q water alone. The dose volume administered was 10 mL/kg body weight. Each group in the experiment was comprised of five male and five female rats.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not done at the test facility. The stability of test item in the vehicle was carried out separately under Eurofins Advinus Study No.G19071 at 1 and 100 mg/mL concentrations. Based on the results, the test item was found to be stable for up to 24 hours when stored at room temperature.

During the conduct of this study, the prepared dose formulations and vehicle were analyzed forthe test item concentration on Day 1 and during week 4 (Day 27) of treatment. The results indicated that the overall mean concentration of the test item in the tested formulation was found to be within ± 15% of the targeted concentration with the relative standard deviation (RSD) less than 10%. This indicates that the prepared dose formulation met the acceptance criteria for concentration and % RSD.

Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and at the end of recovery period for recovery groups. The body weights and food consumption were measured at weekly intervals during the course of the in-life phase. The clinical laboratory investigations such as haematology, coagulation, clinical chemistry and urine analysis were performed at termination.

All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratios were derived as percent fasting body weight and brain weight.

Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose (G4) group rats. None of the tissues were showing test item related histopathological changes in high dose group (G4), hence lower dose and recovery groups tissue were not examined. Transient clinical sign of slight salivation was observed in all animals at 600 mg/kg/day soon after the dose administration. Nevertheless, the symptoms subsided within a few minutes and the rats were found to be normal. No mortality were observed at any dose tested. The incidence of post-dosing salivation observed in 600 mg/kg/day, indicating that it was caused by the administration of test item. This finding may be attributed to oral mucosa irritation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity. In the present study there were no gross or microscopic changes in gastrointestinal tract. Therefore, it was considered that the transient salivation observed in this study was of no toxicological significance. Ophthalmological examination did not reveal any ocular abnormalities. No treatment-related neurological abnormalities/dysfunctions were observed at all the tested doses in either sex. Treatment did not affect body weight at all the tested doses in either sex. There were no test item-related changes in hematology coagulation, clinical chemistry and urine analysis parameters at all the tested doses in either sex. There were no test item-related changes in terminal fasting body weights and organ at all the tested doses in either sex. There were no gross pathology and histopathology findings at all the tested doses in either sex.

As there were no adverse treatment-related adverse effects observed up to the highest dose, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the test item is considered to 600 mg/kg bw/day under the test conditions and doses employed.

 

Dose-range finding study (prior to OECD408) (Malleshappa, 2022)

The systemic toxicological potential of the test item in Wistar rats when administered orally by gavage for a period of 14 consecutive days. 

The test item was mixed in Milli-Q® water and administered to rats at the dose levels of 500, 750 and 1000 mg/kg bw/day for low (G2), mid (G3) and high dose (G4) group rats, respectively. The rats in the vehicle control group (G1) received vehicle (Milli-Q® water) alone. The dose volume administered was 10 mL/kg body weight. Each group consisted of 4 male and 4 female rats.
The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA) and TIDS. The stability of test item in vehicle was established at 1 and 100 mg/mL under Eurofins Advinus Study No. G24817. Based on the results, the test item was found to be homogeneous and stable in the vehicle up to 48 hours when stored at room temperature.
Dose formulations were analyzed for active ingredient concentration prior to start of treatment on Day 1. Formulations were considered acceptable as the mean results are within ± 15 % of the claimed concentration and the relative standard deviation (% RSD) was less than 10%.
All animals were observed for clinical signs twice daily (pre dose & approximately 30-60 minutes post-dosing). Detailed clinical examination was done on Days 1, 8 and 15. Body weights were measured on Days 1, 4, 8, 11 and 14 of treatment period. Food consumption was measured during Days 1-4, 4-8, 8-11 and 11-14 of treatment period. The clinical laboratory investigations
such as hematology, coagulation and clinical chemistry analysis were performed at termination. Terminal fasting body weights were measured and necropsy was performed on all the rats. Study plan specified organs were collected, weighed and preserved.

Under the experimental conditions employed, the following results were obtained: 

* clinical signs and mortality: There were no clinical signs observed at 500 mg/kg/day. Transient clinical sign of slight salivation was observed soon after dose administration in males at 750 and 1000 mg/kg dose and became normal within 30 minutes post dose. In addition, one female rat (Rab8031) at 1000 mg/kg/day exhibits a clinical sign of emaciation, nasal discharge, abdominal respiration and weakness on day 8. Subsequently, this rat was found dead on the same day. Grossly, glandular stomach discoloration and non-prominent thymus were noted. One male rat (Rab8026) showed the clinical signs of piloerection and emaciation at 1000 mg/kg/day during days 12-14 of treatment.

* body weights, body weight gains and food consumption: At 500 mg/kg/day, the mean body weights and food consumption were not altered by the treatment. At 750 and 1000 mg/kg/day, the lower mean body weights, absolute body weight gain and overall weight gain were attributable to test item-related decrease in food consumption (-12.1% to -27.5% in males and -22.8% to 30.6% in females).

* hematology: The neutrophil and monocyte counts were higher at ≥750 mg/kg/day in males and at all dose levels in females. However, these changes were not dose related. The lymphocyte count decrease was noted in males at ≥750 mg/kg/day and in females at 1000 mg/kg/day. These changes were considered to be associated with the stress due to test item administration.

* coagulation:The coagulation parameters were not affected by the test item administration.

* clinical chemistry parameters: In males, decreased glucose was noted at ≥750 mg/kg/day, a secondary observation to the decreased feed intake with decreased albumin level at 1000 mg/kg/day. The minimal increase in AST was observed at 1000 mg/kg/day males. The triglycerides were higher at ≥750 mg/kg/day in males and all dose levels in females.

* terminal fasting body weights and organ weights: The terminal fasting body weights were lower in both males and females at ≥750 mg/kg/day. Organ weight showed higher adrenal weights (absolute and relative) in males at ≥750 mg/kg/day and considered as a secondary stress associated finding. The decrease in seminal vesicles, coagulating gland weights at ≥750 mg/kg/day and thymus weights at all dose levels were considered as the secondary findings to the decreased body weights.

* gross pathology: There were no test item related gross lesions in the terminally sacrificed rats. The gross findings of glandular stomach discolouration and non-prominent thymus were noted in a found dead female at 1000 mg/kg/day on Day 8.

To conclude, due to the mortality observed in ¼ females in 1000mg/kg bw/day dose group, the clinical signs in female and males, the significantly lower body weight and food consumption in both sexes the dose 1000mg/kg bw/d is not recommended for a sub-chronic repeated dose toxicity study. At 750 mg/kg bw/day, the animals showed mild clinical signs, lower body weight and lower food consumption but no mortality. This dose level is recommended for the sub-chronic repeated dose toxicity study as it is not expected to cause severe pain or suffering or excessive death of the test animals.

 

Repeated dose toxicity - oral, subchronic (Malleshappa, 2022)

The purpose of this repeated dose toxicity study was to evaluate the systemic toxicity profile of the test item in Wistar rats when administered orally by gavage for a period of 90 consecutive days. This study was also intended to provide the information on major toxic effects, target organs and an estimation of a NOAEL.
The test item was weighed and dissolved in Milli-Q® Water and administered to rats at the graduated dose levels of 175, 350 and 750 mg/kg bw/day to low dose (G2), mid dose (G3) and high dose (G4)/high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1) and recovery control group (G1R) received vehicle Milli-Q® Water alone. The dose volume administered was 10 mL/kg body weight. Each group in the experiment was comprised of ten male and ten female rats. The study was initiated with 175, 350 and 750 mg/kg bw/day doses. During the course of the experiment, treatment at 750 mg/kg bw/day dose did not induce any clinical, changes in body weight and food consumption. Hence, the high dose of 750 mg/kg bw/day was increased to 850 mg/kg bw/day from treatment day 43 to observe toxicity of the test item at the top dose. Finally, the study was conducted with 175, 350 and 850 mg/kg bw/day as low (G2), mid (G3) and high dose (G4)/high dose recovery (G4R) groups, respectively. The high dose (G4/G4R) is represented as 750/850 mg/kg bw/day with a note that the high dose (G4/G4R) group rats were treated with 750 mg/kg bw/day during days 1 – 42 and then 850 mg/kg bw/day from day 43 till sacrifice. The characterisation detail of the test item was provided by the study sponsor by a certificate of analysis and TIDS with the details of quality system adopted ISO. The test item was not authenticated at the Test Facility. The stability of the test item in the vehicle was established at 1 and 100 mg/mL
under Eurofins Advinus Study No. G24817. Based on the results, the test item was stable in the vehicle up to 48 hours when stored at room temperature. During the conduct of this study, the prepared dose formulations and vehicle [Milli-Q® Water] were analyzed for Test item concentration on test Day 1 and during 2nd (Day 53) and 3rd (Day 71) month of the treatment. The results indicated that the percent agreement of the analyzed concentrations was in the range, 80-120 % of the claimed concentrations and the relative standard deviation (% RSD) is equal to or less than 20 %. This indicates that the prepared dose formulation met the acceptance criteria for concentration and % RSD.

Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment and at the end of treatment for main groups, and at the end of recovery period for recovery groups. The body weights and food consumption were measured during in-life phase of the experiment. Neurological examinations were conducted towards the end of treatment (Day 88)
for main groups and end of recovery period (Day 116) for recovery groups. The clinical laboratory investigations such as haematology, coagulation, clinical chemistry, hormone analysis and urine analysis were performed at termination. Vaginal smear was examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy. All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratios were derived as percent fasting body weights and brain weight. Histopathological examination was carried out on the preserved organs of vehicle control (G1) and high dose group animals (G4). Histopathological examination of the testes also included a qualitative assessment of stages of
spermatogenesis. In addition, all gross lesions from all the animals were examined microscopically. Stomach was examined in the lower dose groups (G2 and G3) and recovery groups (G1R and G4R) as test item related findings were noted in stomach at high dose (G4).
Under the experimental conditions employed, the following results were obtained:

* clinical sings: Transient clinical sign of slight salivation was observed in all animals at 350 and 750/850 mg/kg bw/day soon after the dose administration. Nevertheless, the symptoms subsided within a few minutes and the rats were found to be normal. No mortality was observed at any dose tested. The incidence of post-dosing salivation observed in 350 and
750/850 mg/kg/day, indicating that it was caused by the administration of test item. This finding may be attributed to oral mucosa irritation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity. Therefore, it was considered that the transient salivation observed in this study was of no toxicological significance.

* ophthalmological examination: Ophthalmological examination did not reveal any ocular abnormalities.

* neurological findings: No treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested.

* body weights: Treatment did not affect body weight at all the tested doses in either sex.

* food consumption: Treatment did not affect food consumption at all the tested doses in either sex.

* haematology: Increase in the neutrophil count (62%) was noted at 750/850 mg/kg/day in females, considered as the secondary changes associated with test item related inflammation in stomach.

* clinical chemistry and coagulation: There were no test item related alterations observed at any of the tested dose levels in either sex.

* urine parameters: There were no test item related alterations observed at any of the tested dose levels in either sex.

* thyroid hormone profile: Thyroid hormone profile (TSH, T4 and T3) was not affected in both sexes across the treated groups when compared to the concurrent vehicle control group.

* terminal fasting body weights and organ weights: There were no test item-related changes in terminal fasting body weights and organ weights at all dose levels tested.

* gross and histopathology:

- at 750/850 mg/kg/day: Haematological parameters revealed increase in neutrophil count in females at high dose correlated with inflammation in stomach histologically.
Grossly, test item related non-glandular/ glandular multifocal thickening, red focus and discoloration in both the sex were correlated with either mucosal hyperplasia/ hyperkeratosis, inflammation or ulceration. The stomach lesions showed complete recovery after 28 Days of dosing free period. Microscopically, ulcer with submucosal and mucosal inflammation in glandular/ non-glandular stomach and hyperplasia/ hyperkeratosis of nonglandular stomach were noted in both sexes. Grossly, these findings were noted as non-glandular/ glandular multifocal thickening, red focus and discoloration with increased neutrophil count in females.

The ulcers were considered as adverse findings whereas the other changes were the secondary changes. All these findings reversed at the end of recovery period. The minimal degree non glandular epithelial hyperplasia in the recovery female showed the reversibility tendency. 

- at 175 and 350 mg/kg/day: Microscopically, test item related hyperplasia/ hyperkeratosis of nonglandular stomach was noted in both sexes. This was considered as a local adaptive non-adverse finding

Considering the changes observed in haematology and microscopic changes at 750/850 mg/kg bw/day, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the test item is considered to be 350 mg/kg bw/day under the test conditions and doses employed.

 

Repeated dose toxicity - inhalation:

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.

Repeated dose toxicity - dermal:

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation, the test substance should not be classified for STOT repeated exposure via the oral route.