1,4-dimethylpiperazine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-03-13-to 2020-08-19

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:

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 39 days, up to and including the day before scheduled euthanasia. This included a two-week period prior to mating, during the mating period and post mating. Females were dosed throughout the treatment period. This included two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and 13 days after delivery, up to and including the day before scheduled euthanasia.

Test material

Test material information

Constituent 1

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:

SOURCE OF TEST MATERIAL
- Name of the test item: 1,4-dimethylpiperazine
- Appearance: liquid
- Source and lot/batch number of test material: PFW190423
- Expiration date of the lot/batch: 2021-08-05
- Purity: 99.9 A%
- physicochemical properties: pH 11.42, specific gravity 0.84, density 0.84 g/cm³

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 under study G19071. 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

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. The Wistar rat was selected due to the large amount of background data available for this strain.

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 study initiation: 12 weeks
- Weight at study initiation: 344.68 - 398.16 g (males); 222.47 - 272.37 g (females)
- Fasting period before study: No
- Housing:
* pre-mating: Two rats of the same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless-steel sipper tubes.
* Mating and post-mating: During mating, two rats (one male and one female) were housed in standard polysulfone cages with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plug (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term (Gestation Day 20).
* Enrichment: Polycarbonate rat huts were provided to the animals as environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall wellbeing. These objects were provided during the pre-mating and post-mating period for males and during the pre-mating period for females and changed along with cage twice a week.
* Bedding: Steam sterilized clean corn cob was used as bedding and changed along with the cage twice a week.
- Diet (e.g. ad libitum): Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum.
- Water (e.g. ad libitum): Deep bore-well water passed through an activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Limited., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: After detailed clinical examination for good health and the suitability for the study, the rats were acclimatized for five days before the pretreatment period. During the acclimatization period, animals were observed once daily for any abnormalities. Only the animals that were determined by the veterinarian to be suitable for use were assigned to this study. Female rats used in this study were nulliparous and non-pregnant.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24°C
- Humidity (%): 49-66%
- Air changes (per hr): 12-15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 2020-03-13 To: 2020-06-29

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q

Details on 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 obtaining a uniform solution. The volume was made up to the mark using the vehicle to get the final desired concentration of 12.5, 25 and 50 mg/mL for the G2, G3 and G4 groups, respectively. The solution was mixed well by stirring using a magnetic stirrer.
- Pre-calibration of the beaker to desired volume: Milli-Q water was measured in a graduated measuring cylinder to the final volume of 100 mL. The measured Milli-Q water was transferred into a clean beaker and the upper and lower meniscus of the Milli-Q water were marked on the beaker using a marker. The Milli-Q water was discarded and the beaker was dried. These lines were used to make up to the volume for the dose formulation suspension preparation.
- 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 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 (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.
- Concentration in vehicle: 12.5, 25 and 50 mg/mL for the G2, G3 and G4 groups respectively.
- Amount of vehicle (if gavage): 10 mL/kg

Details on mating procedure:

One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there was evidence of sperms in the vaginal smear and /or vaginal plug. All the females were successfully copulated within five days from the day of cohabitation except for three females each in control, low and mid dose groups. These females were placed with a second proven male of the same group and when the presence of sperm in the vaginal smear and/or vaginal plug was not confirmed within 7 days, the female was euthanized 24-26 days after last day of mating period. Subsequently, pregnant females were housed individually until LD 14. All the mated females were maintained till they littered. Not-littered females were euthanised after 25 days from the day they were found sperm positive (by vaginal smear examination). The day of confirmed mating was designated as Gestation Day ‘0’ (GD ‘0’). The pre-coital time (days) was calculated for each female.

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 of the treatment period and during the 2nd month (day 42) of treatment and was analysed inhouse. For each set, composite sample was drawn in six replicates from each preparation and in case of control duplicate composite samples were 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:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily for 43 days at approximately the same time each day (varying by ± 3 hours) which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled euthanasia.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours). This includes 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 42-69 days), after which, pups were euthanised on LD 13 and parental females (dams) were euthanised on LD 14 after overnight fasting (water allowed).
The animals in the vehicle control group were handled in an identical manner to the treatment groups and were administered vehicle only.
The dose volume administered to each rat was 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of the individual rat.

Frequency of treatment:

Daily

Details on study schedule:

Route of administration and justification: The 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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

GI (Vehicle control)

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

G2 (low dose)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

G3 (Mid dose)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

G4 (High dose)

No. of animals per sex per dose:

10 males and 10 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of 125 (G2), 250 (G3) and 500 (G4) mg/kg/day were selected for this study in consultation with sponsor based on the outcome of 14 Day repeated dose oral toxicity study in Wistar Rats (study N4793). In addition to the test doses, 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
- Rationale for animal assignment (if not random): Animals were randomly distributed to different groups by 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. Grouping was done one day prior to start of treatment during pre-treatment period.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs of toxicity, the observation for morbidity and mortality was carried out once in the morning during weekend and holidays. Each rat was observed for clinical signs twice daily during the treatment period 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: Detailed clinical examination was done prior to the treatment on Day 1 and 1 and 1-2 hour after dosing at weekly intervals (±1 days) thereafter during the treatment period.
- 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 like self-mutilation, walking backwards). On the days of detailed clinical examination, observations for general clinical signs were not performed except post-dose observations on treatment Day 1.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of males were recorded on Day 1 and at weekly (±1) intervals thereafter. Individual body weights of females were recorded on Day 1 (±1) and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
- All dams were weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The food consumption was measured at weekly intervals (± 1 day) during treatment period. The cage wise average food intake (g/rat/day) was calculated.
- Food consumption was not measured during the cohabitation period.
- Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Days 4 and 13 of the lactation period.

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

OTHER:
- Hormone analysis: Blood samples were collected and serum was separated as per the following schedule for the determination of total T4 and TSH:
- all adult males, prior to euthanasia and
- all dams, prior to euthanasia.
Blood samples were not collected from the non-littered females and the dams in which all pups were dead/cannibalized during lactation.
- Parturition: The duration of gestation was calculated from Day ‘0’ of pregnancy to the day of parturition (Gestation Length). Females were observed for signs of difficult or prolonged parturition.

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular 4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or precoital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal. Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle except for Rx6375 and Rx6380 in control. Rx6394 in low dose was killed moribund and one female rat in high dose (Rx 6440) was found dead. Hence, stage of oestrous was not recorded for these animals. A deviation was recorded for these animals.

Sperm parameters (parental animals):

Histopathology examination of the testes involved a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. Testes were sectioned at 3-4 µm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis.

Litter observations:

STANDARDISATION OF LITTERS
- At birth each day in the morning, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.
- The number of pups born (litter size), sex and individual pup body weight of male and female pups on LD 0 and 4 were recorded.
- On Day 4 after birth, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size drop below the culling target (8 pups/litter).


PARAMETERS EXAMINED
- Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.
- After standardization, the individual pup body weight was recorded on Day 13 of lactation.
- The ano-genital distance (AGD) of each pup was measured on PND 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from the cube root of body weight.
- The number of nipples/areolae in male pups was counted on PND 13.
- The litters were observed daily to note the number of alive, dead and cannibalized pups.
- In addition to daily clinical observations, any abnormal behaviour of the offspring was recorded.
- Fertility index for dams, sires as well as the pup survival index until lactation day 4 was calculated.

GROSS EXAMINATION OF DEAD PUPS: yes,
- all the dead and euthanised pups were examined for malformations and subjected to gross pathological examination

HORMONE ANALYSIS
Blood samples were collected and serum was separated as per the following schedule for the determination of total T4 and TSH:
- Two available surplus pups on Day 4 after birth.
- two available pups per litter on day 13
Pups were anesthetized with isoflurane and incised at the jugular vein in the neck region. The collected samples were pooled together for each litter. Blood samples were collected in plain labelled tubes and kept on the bench top for approximately 20 minutes 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 stored at ca. -70°C until they were analyzed.

Postmortem examinations (parental animals):

SACRIFICE AND GROSS NECROPSY
All adult animals were examined macroscopically for any structural abnormalities or pathology changes. The adult animals killed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia.
The rats were subjected to detailed necropsy by a veterinary pathologist and findings were recorded. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.
The number of implantation sites were recorded for all the dams.

HISTOPATHOLOGY / ORGAN WEIGHTS
On completion of the gross pathology examination the tissues and organs noted above were collected and weighed from all terminally sacrificed adult animals. The organ weight ratios (organ to body weight) as percentage of fasting body weight were determined. The paired organs were weighed together and combined weight was presented. The tissues were preserved in 10% Neutral Buffered Formalin (NBF) unless noted otherwise.
- Organ weight determined for: adrenals, all gross lesions, epididymides, ovaries with oviducts, stomach, thymus, thyroid with parathyroids (weighed after formalin fixation), uterus with cervix, vagina, prostate (prostate + 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), seminal vesicles and coagulating glands, testes, levator ani bulbocavernosus muscle complex, Cowper’s glands, Glans penis
- Tissues collected from all terminally sacrificed adult animals were collected: adrenals, all gross lesions, epididymides, ovaries with oviducts, stomach, thymus, thyroid with parathyroids, uterus with cervix, vagina, prostate, seminal vesicles and coagulating glands, testes, levator anu bulbocavernosus muscle complex, cowper's glands, glans penis
- Histopathology: Tissues collected from all animals in the control and high dose groups and the found dead animal (Rx6440) were examined microscopically for histopathological changes including qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure in testes. Gross lesions were examined in all the animals. The reproductive organs were examined from Rx6405 (male failed to mate) , Rx6380 (non-pregnant)and Rx6375 (pregnant not littered), Rx6394 (unscheduled sacrifice on LD 7) and Rx6440 (found dead on LD 3 and all pups dead).The remaining tissues from the lower dose groups were not examined as no test item related changes were noted at high dose.
The available thyroid gland from a male and a female pup per litter (randomly selected) were also evaluated from all the groups.
The tissues were processed for routine paraffin embedding and 4-5 micron sections were stained with Mayer’s Haematoxylin Eosin stain. In addition, testes were sectioned at 3-4 µm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived.
List of tissues: adrenals, all gross lesions, epididymides, ovaries with oviducts, stomach, thymus, thyroid with parathyroids, uterus with cervix, vagina, prostate, seminal vesicles and coagulating glands, testes

Postmortem examinations (offspring):

SACRIFICE AND GROSS NECROPSY
- All pups were examined macroscopically for any structural abnormalities or pathology changes. All the surviving pups were necropsied on lactation Day 13 and findings were recorded. Dead pups were examined for defects and/or cause of death
- on lactation day 13, thyroid gland from available one male and female pup from each litter was collected and preserved in 10% NBF for the histopathological examination. The thyroid weight of pups were determined after fixation.

Statistics:

Data captured using ProvantisTM: Parameters of body weight, oestrous cycle, ano-genital distance, organ weights and terminal fasting body weight were analyzed using built-in statistical tests.
Derived data like net body weight change, food consumption, ano-genital ratio, oestrous cycle length, organ weight ratios, post implantation loss (%), number of nipples/areolae in male pups, no. of implantations, pre-coital interval, mean litter size, sex ratio and gestation length (days), mating, fertility and survival indices were also analyzed using above mentioned methods.
Hormone data was analyzed using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables 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 ANOVA was performed. 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. For two groups, the comparisons of mean between treatment and control group was done using Student’s ‘t’ test.
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 throughout the report as stated below:
*: Significantly different from the vehicle control group.

Reproductive indices:

Male mating index (%) = (Number of males with evidence of mating / Number of males cohabited) x 100
Male fertility index (%) = (Number of males siring a litter or impregnated a female / Number of males cohabited) x 100
Female mating index (%) = (Number of females mated / Number of females cohabited) x 100
Female fertility index (%) = (Number of pregnant females / Number of females used for mating ) x 100
Mean number of implantations or group = (Total number of implantations / Total number of pregnant animals) x 100
Post implantation loss (%) = ( (Number of implantations - Number of live pups) / Number of implantations ) x 100

Offspring viability indices:

Mean litter size per group = Total Number of pups born / Total Number of littered animals
Mean viable litter size = No. of viable pups / No. of females littered
Live birth index (%) = (No. of viable pups born (at first observation) / Total no. of pups born (at first observation) ) x 100
Day 4 survival index (%) = (Number of viable pups on lactation Day 4 / Number of viable pups born ) x 100
Sex Ratio (%) = (No. of male pups born / Total no. of pups born ) x 100
Ano-genital Distance Ratio (mm/g1/3 ) = Ano-genital distance / Cube root of body weight

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 125 mg/kg/day, clinical sign of hypoactivity, piloerection, weakness and nasal discharge was observed in one female rat (Rx6394) Day 46 (LD7). The microscopic findings of congestion in adrenals, lymphoid depletion in thymus were noted in this animal. Lymphoid depletion in thymus was a secondary finding associated with stress. However, the cause of moribundity could not be determined based on the gross or microscopic findings.

At 500 mg/kg/day, transient clinical sign of slight salivation was observed soon after the dose administration in all animals (males and females) from treatment Day 4. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal. In addition hypoactivity, piloerection (slight) and vaginal discharge was observed in one female rat (Rx6440) on treatment Day 44 (LD 3). Subsequently, this female rat was found dead on Day 45 (LD4). Grossly, this animal showed nodules on the durface of heart, lung and thymus.
There were no clinical signs or mortalities observed during the treatment at 250 mg/kg bwt/day dose group in either sex.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female rat (Rx6394) dosed at 125 mg/kg was sacrified on moribund on lactation Day 46 (LD7). The cause of moribundity could not be determined based on the gross or microscopic findings.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights and body weight gains were unaffected throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group.
Treatment had no effects on the maternal body weights measured during different intervals of the gestation period at all the tested doses when compared to vehicle control.
Treatment had no effects on the maternal body weights during measured different intervals of the lactation period at all the doses when compared to the vehicle control. Incidence of significantly lower body weight gain during days 4-13 was observed at 250 mg/kg/day. This statistically significant difference observed in absolute weight gain was toxicologically not significant as the total absolute weight gain from LD 0-13 was comparable to vehicle control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption was unaffected throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group. Incidence of significantly lower food consumption during days 1-8 in females at 500 mg/kg/day was observed. This statistically significant difference observed in food consumption was toxicologically not significant as the mean body weights were not altered by the treatment.

Treatment had no effects on the maternal food consumption measured during different intervals of the gestation period at all the tested doses when compared to vehicle control.

Treatment had no effects on the maternal food consumption during measured different intervals of the lactation period at all the doses when compared to the vehicle control.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no test item related changes in the T4 and TSH levels in Dams as well as in pups on Lactation Day 4 and Lactation Day 13.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item related microscopic changes. The observed findings were considered incidental/spontaneous and not related to test item administration.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks during the treatment period, prior to cohabitation with males.
The calculated mean oestrous cycle length was 4.7, 4.8, 4.5 and 4.6 days in vehicle control, low, mid and high dose groups, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on the mean pre-coital time, gestation length (average days to litter), number of pregnancies and number of dams littered. No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested.
No test item-related changes were observed in the number of implantations and percentage of post implantation loww at all the tested doses when compared to vehicle control

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Test item had no treatment-related effects on the number of dead pups at first observation. There were no external abnormalities in live or dead pups in any of the groups. The mean total number of pups born and number of live pups on Day 0, 1, 4 and 13 were comparable to vehicle control at all the tested doses.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No treatment-related changes were observed in the survival data of pups up to LD 4 at all the tested doses.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The litter mean pup body weight of male, female and combined sex pups per litter were not affected by the treatment at all the tested doses.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No changes attributable to the test item were detected in the Ano-genital distance and Ano-genital ratio in either sex.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The male pups did not exhibit areola/nipple retention on PND 13.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no effects on the thyroid hormone profile, terminal body weights/organ weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item related gross findings in pups.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no test item related histopathology findings in pups.

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Analytical verification

The results were considered acceptable, as the percent agreement of the analyzed concentrations were in the range, 85% to 115% of the claimed concentrations and the overall %RSD from six replicates at each dose level were <=10.0%.

Applicant's summary and conclusion

Conclusions:

Parameters up to and including 500 mg/kg bw/day, the No Observed Effect Level (NOEL) for Reproduction/Developmental Toxicity Screening Test for the test substance is determined to be 500 mg/kg bw/day under the test conditions and doses employed.

Executive summary:

The daily oral (gavage) administration of the test item to Wistar rats at the dose levels of 125, 250 and 500 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, food consumption, pre-coital time, gestation length, mating and fertility parameters. Male and female reproductive organs did not reveal any changes. There were no treatment-related effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

At 500 mg/kg/day, transient clinical sign of slight salivation was observed soon after the dose administration in all animals from treatment Day 2-5. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal. The incidence of post-dosing salivation observed in 500 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. One female rat (Rx6440) was found dead on LD 3. All the pups for this dam were found dead during LD 0-3. Grossly, this animal showed nodules on the surface of heart, lung and thymus. Microscopically, suppurative inflammation involving all these organs and uterus was noted. The systemic inflammation was related to the cause of death. One female rat (Rx6394) was sacrificed moribund on LD7 at 125 mg/kg/day. The microscopic findings of congestion in adrenals, lymphoid depletion in thymus were noted in this animal. Lymphoid depletion in thymus. was a secondary finding associated with stress. However, the cause of moribundity could not be determined based on the gross or microscopic findings

Treatment did not induce any test item-related adverse changes with respect to terminal fasting body weights, organ weights/ratios (including reproductive organs) and thyroid hormone profile. There were no test item related gross and microscopic changes findings at all the tested doses in either sex. 

 

Parameters up to and including 500 mg/kg bw/day, the NOEL for Reproduction/DEvelopmental Toxicity Screening Test for the test substance is determined to be 500 mg/kg bw/day under the test conditions and doses employed.