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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

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
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4-dimethylpiperazine
EC Number:
203-412-0
EC Name:
1,4-dimethylpiperazine
Cas Number:
106-58-1
Molecular formula:
C6H14N2
IUPAC Name:
1,4-dimethylpiperazine
Details on test material:
- Name of test material (as cited in study report): Jeffcat DMP
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

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.