Alkanoic acid and polyoxomolybdate borate alkanoate clusters

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-07-19 to 2019-12-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-07-19 to 2019-12-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dated 06-05-2019

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected for this study because it is a preferred species for toxicity testing as recommended by test guidelines. The Wistar strain was chosen because it is used for non-clinical studies and recommended by regulatory agencies..

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Hylasco Biotechnology (India) Pvt. Ltd., Telangana, India (Under License for Charles River).
- No. of groups: 4 (Vehicle control + three treatment groups)
- No. of rats / group: 10 males and 15 females - Total: 40 males and 60 females
- Age at start of study: 11 to 15 weeks (Healthy virgin animals)
- Body Weight range at start of the treatment:
Male Rats: 366 - 454 g
Female Rats: 231 - 315 g
- Veterinary examination: Prior to final assignment to the study, the animals were subjected to a veterinary examination to ensure that the selected rats were in a good state of health.
- Housing:
Animals were housed in Room No. AR-46 of the experimental animal facility of INTOX.
Animals were housed maximum three per sex / cage in solid [Sized: 42 cm (L) x 29 cm (W) x 19 cm (H)] bottom polypropylene cages with stainless steel grill tops, facilities for food and water bottle, and with bedding of clean and sterilized corn cob. The cages were suspended on stainless steel racks.
Cages were suspended on movable stainless steel racks arranged in such a way that every cage would receive almost same amount of light. For this purpose, cages arranged on the first row were shifted to the last row, those on the second row were shifted to first row. Likewise all the cages were rotated. This kind of cage rotation was carried out every week throughout the treatment and observation period.
Animals were housed in groups or singly, as below:
Pre-mating period: males and females were housed in groups of maximum two or three per cage per
sex.
During mating (co-habitation) : one male : one female, per cage.
Post-mating: females were housed individually once they were successfully mated. Males were housed in groups of maximum three per cage. During the post-natal (lactation) period the dams were housed individually along with its litter.
- Acclimatisation: The animals were acclimatised for a period of seven days to the experimental room in the animal house of INTOX.

DETAILS OF FOOD AND WATER QUALITY:
- 'Altromin' brand pelleted rat feed manufactured by M/s Altromin Spezialfutter GmbH & Co. KG, Germany, was provided ad libitum. The diet has been tested and certified to be free from undesired levels of environmental contaminants.
- Drinking water, passed through ‘Aquaguard’ water filter, and subjected to ultra violet irradiation, was provided ad libitum in sterilized glass bottles. Water is analysed quarterly for potability and once in a year for various environmental contaminants.

ENVIRONMENTAL CONDITIONS:
- Air conditioned rooms with 10 to 15 air changes per hour,
- Temperature between 19 to 25 °C,
- Relative humidity 30 to 70%,
- Illumination cycle set to 12 hours light and 12 hours dark.

Route of administration:

oral: gavage

Details on route of administration:

The test substance was administered orally (gavage) as it is the route recommended by test guideline s. The oral route is also the most appropriate route of administration, having regard to the likely route of human exposure.

Vehicle:

corn oil

Details on oral exposure:

VEHICLE:
Test item was miscible in corn oil at the doses levels used in this study, hence corn oil was used as vehicle for dosing formulation.

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations were prepared at appropriate concentrations (i.e. 15, 30 and 60 mg/mL) to meet dosage level requirements. The dosing formulations were prepared freshly each day before dosing. Cyclomixer was used during formulation in order to obtain a homogeneous suspension. Concentrations were adjusted to allow a constant dosage of 5 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity of the test item in the vehicle for highest and lowest concentrations were determined at Analytical Chemistry Section of test facility, in a separate study (Study No. RA2932/AMV-SHA).
Test item formulations (for all concentrations) were subjected for verification of concentration twice (i.e. in first week after initiation of treatment and in last week at termination of treatment) during the study. Formulation analysis was performed at Analytical Chemistry Section of Test Facility. Molybdenum and Boron contents were checked.

Duration of treatment / exposure:

Males: minimum 28 days
Non-pregnant females/pregnant females: approximately 63 days

Frequency of treatment:

once daily

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

males and females

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Remarks:

males and females

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

males and females

No. of animals per sex per dose:

10 males and 15 females

Control animals:

yes, concurrent vehicle

Details on study design:

Repeated Dose 14 Days Oral Toxicity Study of test item in Wistar Rat (Dose Range Finding Study) was performed in compliance with the Study Plan (No. P/18697/SOR-14-DRF/19). Groups of five male and five female Wistar rats were administered with test item by oral gavage daily at the doses of 75, 150, 300 and 600 mg/kg body weight for 14 days and were sacrificed on day 15 to evaluate its toxicity. A concurrent vehicle control group received corn oil at the dose of 5 mL/kg.
The rats were examined daily for signs of toxicity, morbidity and mortality. Body weight and food consumption was recorded weekly. All animals sacrificed terminally were subjected to a detailed necropsy and weights of kidneys, liver, adrenals, testes, spleen, brain, ovaries and heart were recorded.
The findings of this study were as follows:
- no mortality in rats treated with test item at and upto the dose of 600 mg/kg body weight;
- no incidence of treatment related clinical abnormalities, at and up to 600 mg/kg body weight;
- no effect on the body weight gain by male and female rats treated at and upto the dose of 300 mg/kg body weight. Reduction in body weight gain on day 14 was observed in male rats at the dose of 600 mg/kg body weight;
- no effect on average daily food consumption by the male and female rats treated at and upto the dose of 300 mg/kg body weight. Reduction in food consumption was observed during last week of treatment in both male and female rats at the dose of 600 mg/kg body weight;
- dose dependent statistically significant changes (increased adrenal weights) observed in absolute and relative weights of adrenal in male rats;
- no treatment related gross pathological alterations in the tissues of male and female rats at and upto 600 mg/kg body weight.
Based on these findings, the doses selected for the "Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test of test item in Wistar Rat" are 75, 150 and 300 mg/kg body weight/day.

Positive control:

none

Observations and examinations performed and frequency:

CLINICAL SIGNS AND MORTALITY
- General Clinical Signs and Mortality:
All signs of illness, together with any behavioural changes or reaction to treatment were observed (cageside observation) once a day for individual animals. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets.
Throughout the study, all animals were checked early on each working day and again in the afternoon to look for dead or moribund animals to allow necropsy examination to be carried out during the working hours of that day.
- Detailed Clinical Examinations:
Once before the first exposure (to allow for within-subject comparisons), and once a week thereafter, detailed clinical observations were made in all parental animals. The rats were subjected to detailed clinical examinations before initiation of the treatment (to allow for within-subject comparisons) and weekly thereafter during the treatment period. These observations were made outside the home cage in a standard arena and preferably at the same time. Signs noted included changes in skin, fur, eyes and mucous membranes, occurrence of secretions, excretions and autonomic activity such as lacrimation, piloerection, pupil size and unusual respiratory pattern. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies, difficult or prolonged parturition or bizarre behavior were also recorded.

FUNCTIONAL OBSERVATION BATTERY
Five males and five females, randomly selected from each group were examined for assessment of sensory reactivity, assessment of grip strength and motor activity. These included the functional observational battery suggested by Moser V. C. (1989). [Animal Behavioral Methods in Neurotoxicity Assessment: SGOMSEC Joint Report; Beverly Kulig et. al, Environ Health Perspect 104 (Suppl 2):193-204 (1996)]
In males, these functional observations were made towards the end of their dosing period, shortly before scheduled sacrifice but before blood sampling for haematology or clinical chemistry.
In females these functional tests were made during lactation (e.g., LD 6-13), shortly before scheduled sacrifice.
This neurological examination included :
1. Examinations in home-cage and open field: Posture / Movement, Respiration, Palpebral closure, Lacrimation, Salivation, Skin and hair coat, Urination, Defecation, Locomotor activity, Rearing, Gait
2. Manipulative examination / Responses to stimuli: Tactile (touch) response, Response to non receptive stimuli (tail pinch), Pupil response to light, Proprioception – Righting reflex, Auditory response, Head shaking, Landing foot splay, Grip strength (fore limb and hind limb) - (By ‘Grip Strength Meter, Cat. No. 47200 by UGO Basile Biological Research Apparatus’).

BODY WEIGHT
Males and females were weighed on the first day of dosing, weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and then within 24 hours of parturition (day 1 post-partum), and at day 4 and day 13 post-partum. These observations were reported individually for each adult animal.

FOOD CONSUMPTION
During pre-mating, food consumption was measured weekly (on days 0, 7 and 14). During pregnancy it was measured on gestation days 0, 7, 14 and 20 and during lactation on days 0, 4 and 13. Food consumption during mating period was not measured.
Food consumption was computed as the amount of food consumed in grams per animal per day.The quantity of food consumed by rats in each cage was recorded weekly during premating period. Food consumption during mating period was not be measured.
During pregnancy period the quantity of food consumed by the female in each cage was calculated for the days 0 to 7, 7 to 14 and 14 to 20, whereas during lactation period it was calculated for the days 0 to 4 and 4 to 13.
Food consumption was computed as the amount of food consumed in grams per animal per day.

Sacrifice and pathology:

PATHOLOGY

HAEMATOLOGY AND CLINICAL CHEMISTRY
Animals were fasted overnight prior to blood sampling but had access to water ad libitum. Blood sampling was performed, under light CO2 anaesthesia, through the orbital sinus of all males and females sacrificed at termination (day 29 for males and day 14 of lactation for females). Samples were collected separately in tubes containing EDTA (dipotassium salt), for haematology, and Heparin, for clinical chemistry, as anticoagulants.
For hormone analysis (serum total T4), blood samples were collected separately in plain tubes to yield serum. Blood samples from the pups were collected by cardiac puncture. Pups were anesthetized by barbiturate anaesthetic (Sodium Thiopentone) agent prior to blood collection.
Baseline Sampling (Haematology and Clinical chemistry)
The base-line values for haematological and clinical chemistry parameters including the hormone estimation (Total T4) were obtained by randomly selected five males and five females. Sampling was performed before start of treatment.
- Haematology (at the end of premating period)
After completion of the premating period (day 14 from start of treatment), haematological examination was made in five males and five females randomly selected from each group. The following haematological parameters were estimated on blood samples listed below, together with an abbreviated title (used in Appendices and Tables).
The following estimations were performed using 'ADVIA 2120i Haematology System (Siemens Healthcare Diagnostic Ltd. Frimley, Camberley, UK GU16 80D):
Haemoglobin (HGB); Haematocrit (PCV); Erythrocyte Count; Total (Total RBC); Reticulocytes; Total Leukocyte Count (Total WBC); Platelet Count - Total (Platelet); Absolute RBC indices : Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH) and Mean corpuscular haemoglobin concentration (MCHC); Prothrombin time as a measure of blood clotting (seconds); Leucocytes were differentiated as Neutrophils (N), Lymphocytes (L), Eosinophils (E), Monocytes (M) and Basophils (B); General blood picture: Blood smear was assessed for abnormal and immature cells during microscopy.
- Clinical Chemistry (at termination)
At termination, clinical Chemistry examinations were made in five males and five females randomly selected from each group. The clinical chemistry parameterthat were measured are Sodium, Potassium, Glucose, Total cholesterol, Urea, Creatinine, Total protein, Albumin, Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Bile acids, Thyroid Hormone, total and Blood Urea Nitrogen.
Plasma samples were analysed individually for determination of clinical chemistry parameters using Dimension XPand Plus Clinical Chemistry System (Siemens Healthcare Diagnostics Inc. Newark, U.S.A.).
- Hormone Analysis
Blood (Serum) samples were taken based on the following schedule:
1. two of the surplus pups, pooled, and used for determination of serum total T4 levels on day 4 after birth - (as per 3.4.3: Litter Size)
2. at least two pups per litter at termination on Lactation day 13 - serum total T4
3. from all adult males at termination (day 29) - serum total T4
4. from all dams at termination on lactation day 14 - serum total T4 (dams were fasted over night on lactation day 13)
Blood (serum) samples were collected from all dams at termination on lactation day 14 and from pups on day 4 after birth. As there is no significant difference in the levels of serum total T4 in adult males and day 13 pups hence, blood samples were not processed for further assessment of T4 in blood samples from dams and day 4 pups.
Blood samples from the adult males and day 13 pups were assessed for serum levels of thyroid hormone (T4).
The hormone analysis was carried out using commercially available ELISA kits manufactured by Elabscience, WuHan, P.R.C.

GROSS NECROPSY
All adult animals in the study were humanely sacrificed by exsanguination under deep CO2 anaesthesia and were subjected to a full, detailed gross necropsy which included careful examination of the external surface of the body, all orifices, the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.
All the following tissues from all adult males and females were preserved in 10% neutral buffered formalin: Gross lesions; Brain - cerebrum, cerebellum, midbrain; Spinal cord; Eyes; Thyroid; Parathyroid; Spleen; Thymus; Adrenals; Trachea; Lungs; Heart; Oesophagus; Stomach; Duodenum; Jejunum; Terminal ileum (with peyer's patch); Colon; Rectum; Liver; Kidneys; Urinary bladder; Prostate; Seminal vesicle with coagulating glands; Epididymides; Ovaries; Uterus with cervix; Vagina; Skeletal muscles; Sciatic nerve; Bone marrow (smear); Mesenteric lymph node; Axillary lymph node; Bone-femur; Levator ani plus bulbocavernosus muscle complex; Glans penis; Cowpers gland Testes were collected in Modified Davidson's fluid. The tunica albuginea of the testes was gently punctured at both poles of the organ with a needle to permit rapid penetration of the fixative.
Vaginal smears were examined on the day of necropsy to determine the stage of the oestrous cycle. Dead pups and pups sacrificed on day 13 post-partum were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

ORGAN WEIGHTS
The testes, epididymides, prostate, seminal vesicles with coagulating glands as a whole, levator ani plus bulbocavernosus muscle complex, Cowper’s glands and glans penis of all adult male animals were weighed on termination of treatment (on day 29). Uterus with cervix and ovaries from all adult females were weighed at termination on lactation day 14.
From all adult males and females, thyroid glands were preserved and weighed post fixation. At lactation day 13 the thyroid glands from 1 male and 1 female pup per litter were preserved and weighed post fixation.
Trimming was done very carefully and only after fixation to avoid tissue damage.
In addition, from five adult males and females, randomly selected from each group, the liver, kidneys, adrenals, thymus, spleen, brain and heart were trimmed of any adherent tissue, as appropriate and their weights were taken as soon as possible after dissection to avoid drying.


HISTOPATHOLOGY
Microscopic examination was carried out on tissues as listed below:
On all listed organs and tissues of selected five males and five females of groups G1 (control) and G4 (high dose), sacrificed at termination: Gross lesions; Brain - cerebrum, cerebellum, midbrain; Spinal cord; Eyes; Thyroid; Parathyroid; Spleen; Thymus; Adrenals; Trachea; Lungs; Heart; Oesophagus; Stomach; Duodenum; Jejunum; Terminal ileum (with peyer's patch); Colon; Rectum; Liver; Kidneys; Urinary bladder; Prostate; Seminal vesicle with coagulating glands; Epididymides; Testes; Ovaries; Uterus with cervix; Vagina; Skeletal muscles; Sciatic nerve; Bone marrow (smear); Mesenteric lymph node; Axillary lymph node; Bone-femur; Levator ani plus bulbocavernosus muscle complex; Glans penis; Cowpers gland.
All tissues were fixed in 10% neutral buffered formalin, were embedded in paraffin wax, sectioned at 5 m thickness and stained with haematoxylin and eosin, for microscopic examination.
These examinations were not extended to animals of other dosage groups, as treatment-related changes were not observed in the high dose group.
In absence of any microscopic alteration in the thyroid glands of adult male rats and in absence of any alterations in the values of total T4, microscopic evaluation of thyroid glands from the pups was not carried out.

Statistics:

Statistical analysis was performed using IBM SPSS Statistical Software (version 23). For statistical analysis, the litter was used as the basic sampling unit. Following statistical methods were used to analyse the various parameters:
- One-way ANOVA followed by Dunnett's test
- Kruskal–Wallis followed by Mann–Whitney test
- Chi-Square / fisher test
The results of these statistical analyses were assessed at 5% level of significance (p = 0.05) and designated as significantly higher (S+) / lower (S-) than control values.

Clinical signs:

no effects observed

Description (incidence and severity):

All the animals in the treatment and control groups were examined daily for general clinical signs (cage side observations) and weekly for detailed clinical signs at premating, during mating, gestation and lactation periods.
Treatment with test item for males (28 days) and for females (50 to 60 days involving pregnancy and lactation period) did not induce any adverse clinical signs at any dose levels. No abortion/premature deliveries were observed in any of the dose levels during the study period.

Mortality:

no mortality observed

Description (incidence):

There was no incidence of mortality amongst the rats treated with the test item at any of the dose levels in both males and females. No incidences of moribund or found dead dams were observed during the study period. All animals survived throughout the period of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Test item at and up to the dose of 300 mg/kg body weight/day did not show any remarkable adverse effects on weight gain in treated male and female rats of this study. The values of mean body weights and mean body weight gain for rats treated with test item at and up to 300 mg/kg body weight/day did not differ significantly (p>0.05) from those of the concurrent control group rats during the gestation period and the post-natal lactation period.
During Premating and Mating Period :
The percent body weight gain in the male and female rats treated with the test item at and up to 300 mg/kg body weight/day was found to be comparable to that of the control rats during premating and mating period.
During Gestation Period:
Test item did not show any adverse effect on the body weight gain during the gestation period in female rats.
There was no significant difference (p>0.05) in the mean body weights of control and treated groups of females during the period of gestation. The mean body weight of female rats as measured on gestation days 0, 7, 14 and 20 and the mean body weight changes computed between gestation days 0-7, 7-14 and 14-20 by rats treated with test item did not differ significantly (p>0.05) from those of the control group rats.
During Lactation Period:
The mean body weight of female rats as measured on lactation days 0, 4 and 13 and the mean body weight changes computed between lactation days 0-4 and 4-13 by rats treated with test item did not differ significantly (p>0.05) from those of the control group rats.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Treatment of male and female rats at and up to the dose of 300 mg/kg body weight/day of test item did not induce any remarkable and adverse effects on the daily food intake of animals. The values of mean daily food intake by rats treated with test item did not differ significantly (p>0.05) from those of the concurrent control group females during the premating period, the gestation period and the post-natal lactation period.
During Premating Period:
Mean food consumption of male and female rats from control group and treated dose groups was found to be comparable during the two weeks of the premating period.
During Gestation Period:
The average amount of food consumed per rat per day during gestation period did not differ significantly (p>0.05). Similarly the mean values of food consumption per rat per day during gestational days 0-7, 7-14 and 14-20 by rats treated with test item did not differ significantly (p>0.05) from those of the control group females.
During Lactation Period:
The average amount of food consumed per rat per day during lactation period did not differ significantly (p>0.05) from those of the control group rats. Similarly the mean values of food consumption per rat per day during lactational days 0-4 and 4-13 by rats treated with test item did not differ significantly (p>0.05) from those of the control group rats.

Food efficiency:

no effects observed

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):

Baseline Sampling:
Baseline sampling was performed before start of treatment on randomly selected five male and five female rats. The mean values of haematological parameters such as hemoglobin, haematocrit (PCV), total and differential leucocyte counts, total RBC count, RBC indices, platelet count, reticulocyte count and prothrombin time of male and female rats, were obtained. General blood picture was evaluated on stained blood smear slides.
During Premating Period:
After completion of premating treatment period (day 14 from start of treatment), haematological examinations were made on five males and five females randomly selected from each group. There were no treatment related changes in the above list of haematological parameters as the mean values were comparable to the same from concurrent control animals.
There were sporadic instances of statistically significant changes observed in haematocrit, MCH, MCHC, total RBCs in male rats as well as prothrombin time in female rats. These changes were not dose dependant and hence considered as incidental and not treatment related.
General blood picture evaluation performed on stained blood smear slides revealed no morphological abnormalities and immature cells in red blood cells, white blood cells and platelets in any of the treated rats including the control animals.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item, at and up to the dose level of 300 mg/kg body weight/day, did not induce any changes in the plasma levels of total protein, albumin, alanine aminotransferase, asparate aminotransferase, alkaline phosphatase, glucose, urea, creatinine, blood urea nitrogen (BUN), total bile acids, total cholesterol, sodium, potassium and total T4 in male and female rats, during premating and at termination of the treatment period.
Baseline Sampling:
Baseline sampling was performed before start of treatment on randomly selected five male and five female rats. The mean values of above listed clinical chemistry parameters of male and female rats were obtained.
During Termination Period:
At termination of treatment, above listed clinical chemistry parameters were determined in all males (on day 29) and all females (on lactation day 14).
The test item, at and up to the dose level of 300 mg/kg body weight/day, did not induce any change in clinical chemistry parameters.
No statistically significant changes were observed in any of the parameters in male and female rats.
Estimation of Thyroid Hormones (Total T4)
Serum samples from all males sacrificed at termination on day 29 were assessed for total T4.
The test item, up to the dose level of 300 mg/kg body weight/day, did not induce any change in the total T4 levels. Significant decrease was observed in mean values of G3 and G4 dose groups. This change was not dose dependant and hence considered as incidental and not treatment related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Description (incidence and severity):

Based on the results of haematology especially Total and Differential Leucocyte Counts and general blood picture along with results of organ weights and histopathology of thymus and spleen, there is no evidence of immunological effects.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole, levator ani plus bulbocavernous muscle complex, Cowper’s glands and glans penis of all male adult animals were weighed at termination of treatment (on day 29). While in all female rats, ovaries and uterus with cervix were weighed at termination on lactation day 14.
In addition, from five randomly selected adult male and female rats from each group, the liver, kidneys, adrenals, thymus, spleen, brain, heart and thyroid glands were trimmed off any adherent tissue, as appropriate and their weights were taken as soon as possible after dissection to avoid drying.
The values of absolute and relative organ weights were found to be comparable between the treated and control group of male and female rats.
Significant change was observed in relative organ weight of testes in G3 male rats which was considered as incidental in the absence of dose dependency and not related to the treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item, at and up to the dose level of 300 mg/kg body weight/day, did not induce any remarkable and treatment related gross pathological alterations in any of the tissues of treated rats, as evident by the detailed necropsy examination carried out at termination. However few gross observations recorded were a solitary resorbed embyro in uterus in a female rat from the control group sacrificed on lactation day 14. However it should be noted that the subsequent microscopic examination exhibited no abnormality in the examined uterus.
Similarly a diaphragmatic nodule was observed on liver of one female rat from the control group. It is known that this is a background lesion that occurs sporadically and is not due to treatment, it is a congenital lesion in rats of any age. Similarly one female rat treated at a dose of 75 mg/kg body weight revealed a white foci (4 mm in diameter) on right lateral lobe of liver. Similarly, a male rat treated at a dose of 150 mg/kg body weight revealed undersized testes.
Gross pathological examination of the reproductive organs of male and female rats did not reveal any treatment related morphological alterations.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Systemic effect:
Histopathological examinations of the tissues from five adult male and female rats randomly selected from control group and high dose group did not reveal any significant treatment related histopathological alterations.
Some incidental and spontaneous lesions observed in animals from the control and high dose group (300 mg/kg body weight/day) included perivascular lymphocytic aggregation and foam cells in lungs, cortical vacuolation in adrenal, dilated glands in stomach, sub-mucosal lymphoid hyperplasia in colon and rectum and a diaphragmatic nodule in liver. The microscopic examination of this nodule revealed normal hepatic architecture.
Microscopic examination of liver from one female rat treated at a dose of 75 mg/kg body weight/day revealed solitary granuloma. The granuloma comprised of macrophages, lymphocytes and eosinophils.
All these changes were of minimal severity and are commonly observed in rats of this age and were not considered to be treatment related.
Histopathological examination of the reproductive organs of male and female rats did not reveal any treatment related morphological alterations. However microscopic examination of the testes from one male rat (Rk6128) treated at a dose of 150 mg/kg body weight/day and one male rat (Rk6140) treated at a dose of 300 mg/kg body weight/day revealed mild to moderate degeneration. The degeneration was characterized by number of degenerative features, such as tubular vacuolation, and partial depletion of germ cells. In absence of any other microscopic changes in rest of the reproductive organs examined, this was considered to be incidental and not related to treatment.

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of treatment related effects at highest dose level

Critical effects observed:

no

Conclusions:

Groups of ten male and fifteen female Wistar rats were administered the test item daily by oral gavage at the doses of 75, 150 and 300 mg/kg body weight/day to evaluate its systemic toxicity and effects on reproduction and development. Males were dosed for a period of four weeks and females were dosed for 50 to 60 days (this included two weeks prior to mating, the variable time to conception, the duration of pregnancy and thirteen days after delivery). The findings of this study were as follows:
* no treatment related deaths; no neurotoxic potential;
* no clinical abnormalities;
* no effect on average body weight and body weight gain during premating, mating, gestation and lactation period in females and no effect on average body weight and body weight gain during premating and mating period in males.
* no effect on the average daily food intake in treated rats;
* no effects on the haematological parameters;
* no effects on the blood chemistry parameters; no effects on total T4 levels;
* no alterations in the absolute and relative organ weights;
* no remarkable gross pathological alterations in tissues/organs;
* no histopathological alterations, suggestive of systemic toxicity, at 300 mg/kg body weight/day dose level;
* no effects on organ weight data, gross and histopathological alterations in reproductive organs.
Based on the findings of this study, it is concluded that, the No-Observed-Adverse-Effect-Level (NOAEL) of test item in Wistar rats, following oral administration for a period of four weeks for males and 50 to 60 days for females for systemic toxicity was found to be 300 mg/kg body weight/day.

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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dated 06-05-2019

Limit test:

no

Test material

Test material information

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Details on species / strain selection
The rat was selected for this study because it is a preferred species for toxicity testing as recommended by test guidelines. The Wistar strain was chosen because it is used for non-clinical studies and recommended by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Hylasco Biotechnology (India) Pvt. Ltd., Telangana, India (Under License for Charles River).
- No. of groups: 4 (Vehicle control + three treatment groups)
- No. of rats / group: 10 males and 15 females - Total: 40 males and 60 females
- Age at start of study: 11 to 15 weeks (Healthy virgin animals)
- Body Weight range at start of the treatment:
Male Rats: 366 - 454 g
Female Rats: 231 - 315 g
- Veterinary examination: Prior to final assignment to the study, the animals were subjected to a veterinary examination to ensure that the selected rats were in a good state of health.
- Housing:
Animals were housed in Room No. AR-46 of the experimental animal facility of INTOX.
Animals were housed maximum three per sex / cage in solid [Sized: 42 cm (L) x 29 cm (W) x 19 cm (H)] bottom polypropylene cages with stainless steel grill tops, facilities for food and water bottle, and with bedding of clean and sterilized corn cob. The cages were suspended on stainless steel racks.
Cages were suspended on movable stainless steel racks arranged in such a way that every cage would receive almost same amount of light. For this purpose, cages arranged on the first row were shifted to the last row, those on the second row were shifted to first row. Likewise all the cages were rotated. This kind of cage rotation was carried out every week throughout the treatment and observation period.
Animals were housed in groups or singly, as below:
Pre-mating period: males and females were housed in groups of maximum two or three per cage per sex.
During mating (co-habitation) : one male : one female, per cage.
Post-mating: females were housed individually once they were successfully mated. Males were housed in groups of maximum three per cage. During the post-natal (lactation) period the dams were housed individually along with its litter.
- Acclimatisation: The animals were acclimatised for a period of seven days to the experimental room
in the animal house of INTOX.

DETAILS OF FOOD AND WATER QUALITY:
- 'Altromin' brand pelleted rat feed manufactured by M/s Altromin Spezialfutter GmbH & Co. KG, Ger
many, was provided ad libitum. The diet has been tested and certified to be free from undesired levels
of environmental contaminants.
- Drinking water, passed through ‘Aquaguard’ water filter, and subjected to ultra violet irradiation, was
provided ad libitum in sterilized glass bottles. Water is analysed quarterly for potability and once in a
year for various environmental contaminants.

ENVIRONMENTAL CONDITIONS:
- Air conditioned rooms with 10 to 15 air changes per hour,
- Temperature between 19 to 25 °C,
- Relative humidity 30 to 70%,
- Illumination cycle set to 12 hours light and 12 hours dark.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE:
Test item was miscible in corn oil at the doses levels used in this study, hence corn oil was used as vehicle for dosing formulation.

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations were prepared at appropriate concentrations (i.e. 15, 30 and 60 mg/mL) to meet dosage level requirements. The dosing formulations were prepared freshly each day before dosing. Cyclomixer was used during formulation in order to obtain a homogeneous suspension. Concentrations were adjusted to allow a constant dosage of 5 mL/kg body weight.

Details on mating procedure:

One male to one female (1:1) mating was used in this study. The female was placed with the same male until pregnancy occurs or one week has elapsed. Each morning the females were examined for the presence of sperm. Day 0 of pregnancy was defined as the day a sperm is found. In case pairing was unsuccessful, re-mating of females with proven males of the same group was considered.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity of the test item in the vehicle for highest and lowest concentrations were determined at Analytical Chemistry Section of test facility, in a separate study (Study No. RA2932/AMV-SHA).
Test item formulations (for all concentrations) were subjected for verification of concentration twice (i.e. in first week after initiation of treatment and in last week at termination of treatment) during the study. Formulation analysis was performed at Analytical Chemistry Section of Test Facility. Molybdenum and Boron contents were checked.

Duration of treatment / exposure:

Males: minimum 28 days
Non-pregnant females/pregnant females: approximately 63 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

males and females

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Remarks:

males and females

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

males and females

No. of animals per sex per dose:

10 males and 15 females

Control animals:

yes, concurrent vehicle

Details on study design:

Details on study design
Repeated Dose 14 Days Oral Toxicity Study of test item in Wistar Rat (Dose Range Finding Study) was performed in compliance with the Study Plan (No. P/18697/SOR-14-DRF/19). Groups of five male and five female Wistar rats were administered with test item by oral gavage daily at the doses of 75, 150, 300 and 600 mg/kg body weight for 14 days and were sacrificed on day 15 to evaluate its toxicity. A concurrent vehicle control group received corn oil at the dose of 5 mL/kg.
The rats were examined daily for signs of toxicity, morbidity and mortality. Body weight and food consumption was recorded weekly. All animals sacrificed terminally were subjected to a detailed necropsy and weights of kidneys, liver, adrenals, testes, spleen, brain, ovaries and heart were recorded.
The findings of this study were as follows:
- no mortality in rats treated with test item at and upto the dose of 600 mg/kg body weight;
- no incidence of treatment related clinical abnormalities, at and up to 600 mg/kg body weight;
- no effect on the body weight gain by male and female rats treated at and upto the dose of 300 mg/kg body weight. Reduction in body weight gain on day 14 was observed in male rats at the dose of 600 mg/kg body weight;
- no effect on average daily food consumption by the male and female rats treated at and upto the dose of 300 mg/kg body weight. Reduction in food consumption was observed during last week of tre atment in both male and female rats at the dose of 600 mg/kg body weight;
- dose dependent statistically significant changes (increased adrenal weights) observed in absolute and relative weights of adrenal in male rats;
- no treatment related gross pathological alterations in the tissues of male and female rats at and upto 600 mg/kg body weight.
Based on these findings, the doses selected for the "Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test of test item in Wistar Rat" are 75, 150 and 300 mg/kg body weight/day.

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CLINICAL SIGNS AND MORTALITY
- General Clinical Signs and Mortality:
All signs of illness, together with any behavioural changes or reaction to treatment were observed (cageside observation) once a day for individual animals. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets.
Throughout the study, all animals were checked early on each working day and again in the afternoon to look for dead or moribund animals to allow necropsy examination to be carried out during the working hours of that day.
- Detailed Clinical Examinations:
Once before the first exposure (to allow for within-subject comparisons), and once a week thereafter, detailed clinical observations were made in all parental animals. The rats were subjected to detailed clinical examinations before initiation of the treatment (to allow for within-subject comparisons) and weekly thereafter during the treatment period. These observations were made outside the home cage in a standard arena and preferably at the same time. Signs noted included changes in skin, fur, eyes and mucous membranes, occurrence of secretions, excretions and autonomic activity such as lacrimation, piloerection, pupil size and unusual respiratory pattern. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies, difficult or prolonged parturition or bizarre behavior were also recorded.

FUNCTIONAL OBSERVATION BATTERY
Five males and five females, randomly selected from each group were examined for assessment of sensory reactivity, assessment of grip strength and motor activity. These included the functional observational battery suggested by Moser V. C. (1989). [Animal Behavioral Methods in Neurotoxicity Assessment: SGOMSEC Joint Report; Beverly Kulig et. al, Environ Health Perspect 104 (Suppl 2):193-204 (1996)]
In males, these functional observations were made towards the end of their dosing period, shortly before scheduled sacrifice but before blood sampling for haematology or clinical chemistry. In females these functional tests were made during lactation (e.g., LD 6-13), shortly before scheduled sacrifice.
This neurological examination included :
1. Examinations in home-cage and open field: Posture / Movement, Respiration, Palpebral closure, Lacrimation, Salivation, Skin and hair coat, Urination, Defecation, Locomotor activity, Rearing, Gait
2. Manipulative examination / Responses to stimuli: Tactile (touch) response, Response to non receptive stimuli (tail pinch), Pupil response to light, Proprioception – Righting reflex, Auditory response, Head shaking, Landing foot splay, Grip strength (fore limb and hind limb) - (By ‘Grip Strength Meter, Cat. No. 47200 by UGO Basile Biological Research Apparatus’).

BODY WEIGHT
Males and females were weighed on the first day of dosing, weekly thereafter, and at termination.
During pregnancy, females were weighed on days 0, 7, 14 and 20 and then within 24 hours of parturition (day 1 post-partum), and at day 4 and day 13 post-partum. These observations were reported individually for each adult animal.

FOOD CONSUMPTION
During pre-mating, food consumption was measured weekly (on days 0, 7 and 14). During pregnancy it was measured on gestation days 0, 7, 14 and 20 and during lactation on days 0, 4 and 13. Food consumption during mating period was not measured.
Food consumption was computed as the amount of food consumed in grams per animal per day.The quantity of food consumed by rats in each cage was recorded weekly during premating period. Food consumption during mating period was not be measured.
During pregnancy period the quantity of food consumed by the female in each cage was calculated for the days 0 to 7, 7 to 14 and 14 to 20, whereas during lactation period it was calculated for the days 0 to 4 and 4 to 13.
Food consumption was computed as the amount of food consumed in grams per animal per day.

Oestrous cyclicity (parental animals):

Females were screened for normal oestrous cycles (in two weeks pre-treatment period). Oestrous cycle was monitored before the start of treatment to select the females with regular cyclicity.
Females that failed to exhibit typical 4 or 5-day cycles were not included in the study. Vaginal smears were also monitored daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

The testes, epididymides, prostate, seminal vesicles with coagulating glands as a whole, levator ani plus bulbocavernosus muscle complex, Cowper’s glands and glans penis of all adult male animals were weighed on termination of treatment.

Litter observations:

The duration of gestation was recorded and was calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery to establish the number and sex of pups, still births, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities.
Live pups counted and sexed and litters were weighed within 24 hours of parturition (day 0 or 1 post-partum), on day 4 and day 13 post-partum. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Anogenital Distance
The AGD (Anogenital distance) of each pup was measured daily on the postnatal day (PND) 0 to PND 4. Pup body weight was recorded daily on PND 0 to PND 4 and at termination on day 13.
The AGD was measured by using Dial caliper (Mitutoyo dial caliper, Japan). The arms of the Dial caliper were aligned as follows: for males, the anogenital distance was measured from the cranial (or anterior) edge of the anus to the base (or posterior edge) of the anogenital aperture; and for females, the anogenital distance was measured from the cranial edge of the anus to the base of the urinary aperture (not the base of the vulva). The anogenital distance was recorded in millimeters.

Nipple Retention
The numbers of nipples / areolae in male pups were counted on PND 12.

Dams with Signs of Abortion or Premature Delivery
Dams were observed for signs suggestive of abortion, or of premature delivery.

Dams with No Evidence of Copulation / Mating and those Found Non Pregnant
Females showing no-evidence of copulation / mating were sacrificed 25 days after last day of the mating period.
Dams found sperm positive and failed to deliver by gestation day 24 were weighed and sacrificed by CO2 asphyxiation on their gestation day 25. The dams were subjected to a necropsy examination to observe any gross pathological changes, and the findings were recorded.

Postmortem examinations (parental animals):

GROSS NECROPSY
All adult animals in the study were humanely sacrificed by exsanguination under deep CO2 anaesthesia and were subjected to a full, detailed gross necropsy which included careful examination of the external surface of the body, all orifices, the cranial, thoracic and abdominal cavities and their content
s. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.
All the following tissues from all adult males and females were preserved in 10% neutral buffered formalin: Gross lesions; Brain - cerebrum, cerebellum, midbrain; Spinal cord; Eyes; Thyroid; Parathyroid; Spleen; Thymus; Adrenals; Trachea; Lungs; Heart; Oesophagus; Stomach; Duodenum; Jejunum; Terminal ileum (with peyer's patch); Colon; Rectum; Liver; Kidneys; Urinary bladder; Prostate; Seminal vesicle with coagulating glands; Epididymides; Ovaries; Uterus with cervix; Vagina; Skeletal muscles; Sciatic nerve; Bone marrow (smear); Mesenteric lymph node; Axillary lymph node; Bone-femur; Levator ani plus bulbocavernosus muscle complex; Glans penis; Cowpers gland . Testes were collected in Modified Davidson's fluid. The tunica albuginea of the testes was gently punctured at both poles of the organ with a needle to permit rapid penetration of the fixative.
Vaginal smears were examined on the day of necropsy to determine the stage of the oestrous cycle.

ORGAN WEIGHTS
The testes, epididymides, prostate, seminal vesicles with coagulating glands as a whole, levator aniplus bulbocavernosus muscle complex, Cowper’s glands and glans penis of all adult male animals were weighed on termination of treatment (on day 29). Uterus with cervix and ovaries from all adult
females were weighed at termination on lactation day 14.
From all adult males and females, thyroid glands were preserved and weighed post fixation. At lactation day 13 the thyroid glands from 1 male and 1 female pup per litter were preserved and weighed post fixation.
Trimming was done very carefully and only after fixation to avoid tissue damage.
In addition, from five adult males and females, randomly selected from each group, the liver, kidneys, adrenals, thymus, spleen, brain and heart were trimmed of any adherent tissue, as appropriate and their weights were taken as soon as possible after dissection to avoid drying.

HISTOPATHOLOGY
Microscopic examination was carried out on tissues as listed below:
On all listed organs and tissues of selected five males and five females of groups G1 (control) and G4 (high dose), sacrificed at termination: Gross lesions; Brain - cerebrum, cerebellum, midbrain; Spinal cord; Eyes; Thyroid; Parathyroid; Spleen; Thymus; Adrenals; Trachea; Lungs; Heart; Oesophagus; Stomach; Duodenum; Jejunum; Terminal ileum (with peyer's patch); Colon; Rectum; Liver; Kidneys; Urinary bladder; Prostate; Seminal vesicle with coagulating glands; Epididymides; Testes; Ovaries; Uterus with cervix; Vagina; Skeletal muscles; Sciatic nerve; Bone marrow (smear); Mesenteric lymphnode; Axillary lymph node; Bone-femur; Levator ani plus bulbocavernosus muscle complex; Glans penis; Cowpers gland.
All tissues were fixed in 10% neutral buffered formalin, were embedded in paraffin wax, sectioned at 5 µm thickness and stained with haematoxylin and eosin, for microscopic examination.
These examinations were not extended to animals of other dosage groups, as treatment-related changes were not observed in the high dose group.
In absence of any microscopic alteration in the thyroid glands of adult male rats and in absence of any alterations in the values of total T4, microscopic evaluation of thyroid glands from the pups was not carried out.

Postmortem examinations (offspring):

GROSS NECROPSY
Dead pups and pups sacrificed on day 13 post-partum were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.PY

Statistics:

Statistical analysis was performed using IBM SPSS Statistical Software (version 23). For statistical analysis, the litter was used as the basic sampling unit. Following statistical methods were used to analyse the various parameters:
- One-way ANOVA followed by Dunnett's test
- Kruskal–Wallis followed by Mann–Whitney test
- Chi-Square / fisher test
The results of these statistical analyses were assessed at 5% level of significance (p = 0.05) and designated as significantly higher (S+) / lower (S-) than control values.

Reproductive indices:

- oestrous cycle and cycle legnth before mating,
- pregnancy rate,
- gestational length (duration of pregnancy in days).

Offspring viability indices:

- Live birth index,
- Implantations,
- Post-implantation loss.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

All the animals in the treatment and control groups were examined daily for general clinical signs (cage side observations) and weekly for detailed clinical signs at premating, during mating, gestation and lactation periods.
Treatment with test item for males (28 days) and for females (50 to 60 days involving pregnancy and lactation period) did not induce any adverse clinical signs at any dose levels. No abortion/premature deliveries were observed in any of the dose levels during the study period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There was no incidence of mortality amongst the rats treated with the test item at any of the dose levels in both males and females. No incidences of moribund or found dead dams were observed during the study period. All animals survived throughout the period of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Description (incidence and severity)
Test item at and up to the dose of 300 mg/kg body weight/day did not show any remarkable adverse effects on weight gain in treated male and female rats of this study. The values of mean body weights and mean body weight gain for rats treated with test item at and up to 300 mg/kg body weight/day did not differ significantly (p>0.05) from those of the concurrent control group rats during the gestation period and the post-natal lactation period.
During Premating and Mating Period :
The percent body weight gain in the male and female rats treated with the test item at and up to 300 mg/kg body weight/day was found to be comparable to that of the control rats during premating and mating period.
During Gestation Period:
Test item did not show any adverse effect on the body weight gain during the gestation period in female rats.
There was no significant difference (p>0.05) in the mean body weights of control and treated groups of females during the period of gestation. The mean body weight of female rats as measured on gestation days 0, 7, 14 and 20 and the mean body weight changes computed between gestation days 0-7, 7-14 and 14-20 by rats treated with test item did not differ significantly (p>0.05) from those of the control group rats.
During Lactation Period:
The mean body weight of female rats as measured on lactation days 0, 4 and 13 and the mean body weight changes computed between lactation days 0-4 and 4-13 by rats treated with test item did not differ significantly (p>0.05) from those of the control group rats.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Treatment of male and female rats at and up to the dose of 300 mg/kg body weight/day of test item did not induce any remarkable and adverse effects on the daily food intake of animals. The values of mean daily food intake by rats treated with test item did not differ significantly (p>0.05) from those of the concurrent control group females during the premating period, the gestation period and the post-natal lactation period.
During Premating Period:
Mean food consumption of male and female rats from control group and treated dose groups was fo und to be comparable during the two weeks of the premating period.
During Gestation Period:
The average amount of food consumed per rat per day during gestation period did not differ significantly (p>0.05). Similarly the mean values of food consumption per rat per day during gestational days 0-7, 7-14 and 14-20 by rats treated with test item did not differ significantly (p>0.05) from those of the control group females.
During Lactation Period:
The average amount of food consumed per rat per day during lactation period did not differ significantly (p>0.05) from those of the control group rats. Similarly the mean values of food consumption per rat per day during lactational days 0-4 and 4-13 by rats treated with test item did not differ significantly (p>0.05) from those of the control group rats.

Food efficiency:

no effects observed

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):

Baseline Sampling:
Baseline sampling was performed before start of treatment on randomly selected five male and five female rats. The mean values of haematological parameters such as hemoglobin, haematocrit (PCV), total and differential leucocyte counts, total RBC count, RBC indices, platelet count, reticulocyte count and prothrombin time of male and female rats, were obtained. General blood picture was evaluated on stained blood smear slides.
During Premating Period:
After completion of premating treatment period (day 14 from start of treatment), haematological examinations were made on five males and five females randomly selected from each group. There were no treatment related changes in the above list of haematological parameters as the mean values were comparable to the same from concurrent control animals.
There were sporadic instances of statistically significant changes observed in haematocrit, MCH, MCHC, total RBCs in male rats as well as prothrombin time in female rats. These changes were not dose dependant and hence considered as incidental and not treatment related.
General blood picture evaluation performed on stained blood smear slides revealed no morphological abnormalities and immature cells in red blood cells, white blood cells and platelets in any of the treated rats including the control animals.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item, at and up to the dose level of 300 mg/kg body weight/day, did not induce any changes in the plasma levels of total protein, albumin, alanine aminotransferase, asparate aminotransferase, alkaline phosphatase, glucose, urea, creatinine, blood urea nitrogen (BUN), total bile acids, total cholesterol, sodium, potassium and total T4 in male and female rats, during premating and at termination of the treatment period.
Baseline Sampling:
Baseline sampling was performed before start of treatment on randomly selected five male and five female rats. The mean values of above listed clinical chemistry parameters of male and female rats were obtained.
During Termination Period:
At termination of treatment, above listed clinical chemistry parameters were determined in all males (on day 29) and all females (on lactation day 14).
The test item, at and up to the dose level of 300 mg/kg body weight/day, did not induce any change in clinical chemistry parameters.
No statistically significant changes were observed in any of the parameters in male and female rats.
Estimation of Thyroid Hormones (Total T4)
Serum samples from all males sacrificed at termination on day 29 were assessed for total T4. The test item, up to the dose level of 300 mg/kg body weight/day, did not induce any change in the total T4 levels. Significant decrease was observed in mean values of G3 and G4 dose groups. This change was not dose dependant and hence considered as incidental and not treatment related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Systemic effect:
Histopathological examinations of the tissues from five adult male and female rats randomly selected from control group and high dose group did not reveal any significant treatment related histopathological alterations.
Some incidental and spontaneous lesions observed in animals from the control and high dose group (300 mg/kg body weight/day) included perivascular lymphocytic aggregation and foam cells in lungs, cortical vacuolation in adrenal, dilated glands in stomach, sub-mucosal lymphoid hyperplasia in colon and rectum and a diaphragmatic nodule in liver. The microscopic examination of this nodule revealed normal hepatic architecture.
Microscopic examination of liver from one female rat treated at a dose of 75 mg/kg body weight/day revealed solitary granuloma. The granuloma comprised of macrophages, lymphocytes and eosinophils.
All these changes were of minimal severity and are commonly observed in rats of this age and were not considered to be treatment related.
Histopathological examination of the reproductive organs of male and female rats did not reveal any treatment related morphological alterations. However microscopic examination of the testes from one male rat (Rk6128) treated at a dose of 150 mg/kg body weight/day and one male rat (Rk6140) treated at a dose of 300 mg/kg body weight/day revealed mild to moderate degeneration.
The degeneration was characterized by number of degenerative features, such as tubular vacuolation, and partial depletion of germ cells. In absence of any other microscopic changes in rest of the reproductive organs examined, this was considered to be incidental and not related to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Exposure of female rats to test item at and up to the dose levels of 300 mg/kg body weight/day did not have any adverse effect on the length and frequency of their oestrous cycle.
Total 60 females were screened for normal oestrous cycle for 2 weeks prior to the treatment period. All females exhibited oestrous cycle of 4-5 days. Stages of oestrous cycle were monitored daily in the females randomly assigned to four groups (15 females/group), from the beginning of the treatment period until evidence of mating.
The group mean values of the number of oestrous cycles during the two weeks before the mating period was 2.67, 2.67 and 2.40 among the females exposed to test item at the dose levels of 75, 150 and 300 mg/kg body weight/day respectively. These values were found to be comparable to that (2.53) in the control group.
The length of oestrus cycle was measured in days starting from detection of oestrous stage of the cycle till the oestrous stage of the subsequent cycle. The values of group mean length, in days, of the estrous cycle occurring for two weeks before the mating period were 4.78, 4.88 and 5.38 among the females exposed to test item at the dose levels of 75, 150 and 300 mg/kg body weight/day respectively and were found to be comparable to that (5.07days) of the control group. Thus the test item did not have any adverse effect on the oestrous cycle during the treatment period.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The pregnancy rate was 100% in control and 80%, 80%, 80% in 75, 150 and 300 mg/kg body weight/day dose groups respectively. There was no treatment related adverse effect on pregnancy rate even if there was a slight, but not statistically significant decrease, in mating efficiency.
A comparison of the incidence of various pregnancy related parameters of the control group rats and those treated with test item did not indicate any remarkable differences indicative of any adverse effects due to the treatment. These parameters included incidence of maternal deaths during pregnancy, pregnancy rate (%), mean gestational length and post implantation loss.

Gestational Length
The mean gestational length (duration of pregnancy in days) computed for groups G1 to G4 for all dams was 22.3 ± 0.9 days, 22.1 ± 0.8 days, 22.2 ± 0.9 days and 22.3 ± 0.8 days respectively. The values do not differ from each other significantly (p>0.05).

Live births
The Mean live birth index was 100% in control whereas it was 91.7%, 95.8% and 99.4% in G2, G3 and G4 respectively. This index measures the female’s ability to maintain pregnancy, based on having delivered at least one live pup. The values do not differ from each other significantly (p>0.05) even if there was a slight, but not statistically significant decrease, in live births. This indicated that the test item does not influence the live birth index.

Implantations
The number of uteral implantations in left and right arms of the uterus were counted on day 14 of lactation during terminal sacrifice of the females.
There were no significant differences (p>0.05) in the group mean values of number of implantations, live, dead and total, observed in control group and groups treated with test item. This indicated that the test item did not influence the implantation process.

Total Implants
The total number of implants in control group dams was 202 in control whereas it was 191, 157 and 159 in treated group G2, G3 and G4 respectively. The group mean values of the total number of implants per female were 13.5 (Control-G1), 15.9 (G2), 14.3 (G3) and 14.5 (G4).

Post-implantation Loss
The group mean values of percent post implantation loss was 0.4 (Control-G1), 7.3 (G2) and 9.6 (G4). Post implantation loss was not observed in G3.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of treatment related effects at highest dose level

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Treatment with test item did not induce any abnormal clinical signs, indicative of systemic toxicity, in offsprings during lactation period.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Treatment of dams with test item did not have any adverse effect on the survival of the offsprings during lactation period. The incidences of litters with still-born pups, pups found dead in cage or cannibalized pups were very small and/or comparable across the treated and the control groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Treatment of dams with test item did not have any adverse effect on the offspring's body weight gain during the lactation period.
The values of mean litter body weights and mean litter body weight gain for the offsprings of female rats treated with test item did not differ significantly (p>0.05) from those of the concurrent control group females during the post-natal lactation period.

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:

no effects observed

Description (incidence and severity):

Estimation of Thyroid Hormones (Total T4)
Individual serum samples collected from one or two of the surplus pups per litter on day 13 of lactation were assessed for total T4.
The test item, at and up to the dose level of 300 mg/kg body weight/day, did not induce any changes in the total T4 on day 13 of lactation.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The AGD (Anogenital distance) of each pup was measured daily from the postnatal day (PND) 0 to PND 4. The anogenital distance was recorded in millimeters.
Treatment of dams with test item did not have any adverse effect on the anogenital distance for male and female pups as the average of the anogenital distance (mm) was found to be comparable to that of control male and female pups from the postnatal day (PND) 0 to PND 4.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The number of nipples / areolae in male pups was counted on PND 12 among all male pups from control and treated groups.
The test item did not influence the nipple retention in male pups at post natal day 12.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

On lactation day 13, average absolute weights of thyroid gland from pups of treatment groups i.e. G2 (0.009g), G3 (0.009g) and G4 (0.010g) were found to be comparable with the control group (0.010g).

Gross pathological findings:

no effects observed

Description (incidence and severity):

Dead pups and pups killed on day 13 post-partum were carefully examined for external gross abnormalities.
The total number of foetuses examined for external examination was 109 in the control, 88, 84 and 82 in 75, 150 and 300 mg/kg body weight/day dose groups respectively.
The incidence of normal foetuses and litters observed in this study was 100% in control group and in all treatment groups. There were no any external abnormalities observed in external genitalia or any other organs of pups.

Histopathological findings:

not examined

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of treatment related effects at highest dose level

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Groups of ten male and fifteen female Wistar rats were administered the test item BiLUBE 4131 daily by oral gavage at the doses of 75, 150 and 300 mg/kg body weight/day to evaluate its systemic toxicity and effects on reproduction and development. Males were dosed for a period of four weeks and females were dosed for 50 to 60 days (this included two weeks prior to mating, the variable time to conception, the duration of pregnancy and thirteen days after delivery). The findings of this study were as follows:
* no treatment related deaths; no neurotoxic potential;
* no clinical abnormalities;
* no effect on average body weight and body weight gain during premating, mating, gestation and lactation period in females and no effect on average body weight and body weight gain during premating and mating period in males.
* no effect on the average daily food intake in treated rats;
* no effects on the haematological parameters;
* no effects on the blood chemistry parameters; no effects on total T4 levels;
* no alterations in the absolute and relative organ weights;
* no remarkable gross pathological alterations in tissues/organs;
* no histopathological alterations, suggestive of systemic toxicity, at 300 mg/kg body weight/day dose level;
* no effects on organ weight data, gross and histopathological alterations in reproductive organs;
* no effects on reproductive performance, gestation, parturition, lactation and litter data.
Based on the findings of this study, it is concluded tha the substance is not a primary reprotoxicant. The No-Observed-Adverse-Effect-Level (NOAEL) of BiLUBE 4131 in Wistar rats, following oral administration for a period of four weeks for males and 50 to 60 days for females for systemic toxicity and for reproductive and developmental toxicity was found to be 300 mg/kg body weight/day.