Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 May - 22 Sept 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)
Remarks:
Government of India, Department of Science and Technology, National Good Laboratory Practice (GLP) Compliance Monitoring Authority (NGCMA)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
recommended by regulatory agencies
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Biosciences, Inc., USA, through its representative Vivo Bio Tech Ltd., Telangana, India
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation:males: 303 - 389 g (mean 356.30 g) / females: 210 - 242 g (mean: 226.33 g)
- Housing: Maximum three animals per sex and cage in solid bottom polypropylene cages with stainless steel grill tops, facilities for food and water bottle, and with bedding of clean and sterilized corn cob.
- Diet (ad libitum): 'Altromin' brand extruded pelleted rat feed manufactured by Altromin Spezialfutter GmbH & Co. KG, Germany
- Water (ad libitum): Potable water, passed through 'Aquaguard' water filter, and subjected to ultra violet irradiation
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: daily
Dosing formulations were prepared freshly each day before dosing. Corn oil was used as vehicle control item. The test item was completely miscible in corn oil. Required amount of test item was weighed on an analytical balance, then corn oil was added gradually to achieve appropriate concentrations i.e. 20, 60 and 200 mg/mL to meet dosage level requirements.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no.: 040516
- Date of Manufacture: 4 May 2016
- Date of Expiry: 3 May 2018
- Storage Conditions: room temperature (27 +/- 9 °C)
- Manufacturer: Medcraft International Pvt. Ltd. New Delhi, India
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item in the vehicle for the highest and lowest concentrations were determined at the Analytical Chemistry Section of the test facility in a separate study. A validated HPLC-UV method was used for identification and quantification of the test item in the vehicle. The HPLC method has been developed in the test facility. Test item formulations were subjected to verification for highest and lowest concentrations twice (i.e. in first week after initiation of treatment and in last week at termination of treatment) during the study.
Duration of treatment / exposure:
Males: minimum four weeks (minimum of two weeks prior to mating, during the mating period and, approximately, two weeks post-mating).
Females: throughout the study (two weeks prior to mating, variable time to conception, during pregnancy and thirteen days after delivery).
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Males: 10
Females: 15
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a 14-day dose range finder study. Groups of five male and five female Wistar rats were administered the test item by oral gavage daily at the doses of 125, 500 and 1000 mg/kg bw for 14 days and were sacrificed on day 15 to evaluate its toxicity. A concurrent vehicle control group receiving corn oil at the dose of 5 mL/kg was also maintained. The test item did not induce any mortality and treatment-related clinical abnormalities in rats treated at and up to the dose of 1000 mg/kg bw/day. No mortality or abnormal clinical signs were observed in vehicle control group animals. Hence, dose levels of 100, 300, and 1000 mg/kg bw/day were selected for the main study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow for within-subject comparisons), and once a week thereafter
- Detailed clinical 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.

BODY WEIGHT: Yes
- Time schedule for examinations: 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 and at day 4 and day 13 post-partum.

FOOD CONSUMPTION: Yes
- During pre-mating, food consumption was measured weekly (on days 0, 7 and 14). During pregnancy it was measured on gestation day 0, 7, 14 and 20 and in lactation on days 4 and 13. Food consumption during mating period was not measured.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken before start of treatment and after completion of pre-mating period
- Anaesthetic used for blood collection: Yes, light CO2 anaesthesia
- Animals fasted: Yes, overnight but with access to water ad libitum
- How many animals: 5 males and 5 females randomly selected from each group before start of treatment and 5 males and 5 females randomly selected from each group after completion of pre-mating period
- Parameters checked: Haemoglobin (Hb), Haematocrit (PCV), Erythrocyte Count, Total (Total RBC), Reticulocytes, Total Leukocyte Count (Total WBC), Platelet Count, Total (Platelet), Prothrombin time as a measure of blood clotting (seconds), Differential Leukocyte (WBC) Count (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: Yes
- Time schedule for collection of blood: after termination
- Animals fasted: Not specified
- How many animals: 5 males and 5 females randomly selected from each group
- Parameters checked: Sodium, Potassium, Glucose, Total Cholesterol, Urea, Creatinine, Total Protein, Albumin, Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Bile acids, Thyroid Hormone (total), Urea nitrogen

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at one time during the study following the daily dose administration (males shortly before the scheduled sacrifice and females during the last week of lactation shortly before scheduled sacrifice)
- Dose groups that were examined: five P generation males and five P generation females randomly selected from each group
- Battery of functions tested (Examinations in home-cage and open field): Posture / Movement, Respiration, Palpebral closure, Lacrimation, Salivation, Skin and hair coat, Urination, Defecation, Locomotor activity, Rearing, Gait
- Battery of functions tested (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)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Tissues and organs examined: 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
- Further details: 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 tissues listed from five adult males and females, randomly selected from each group, were preserved in 10% neutral buffered formalin. 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 killed 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.

HISTOPATHOLOGY: Yes
- Tissues and organs examined: 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
- Further details: From five adult males and females, randomly selected of groups G1 (control) and G4 (high dose), sacrificed at termination, the 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 and female 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. The 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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Significantly low (p < 0.05) mean percentage PCV in high dose males and significantly high (p < 0.05) mean reticulocyte count percentage in high dose females were considered incidental because there was no dose-dependant change and the values were within respective historical control range.
Clinical biochemistry findings:
no effects observed
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
Description (incidence and severity):
A few instances of statistically significant (p < 0.05) changes compared to that of control organ weights were noticed in males and were considered incidental as they were within historical control ranges. Observations for males include:
- Mean absolute weight of adrenals 0.044 ± 0.001 g; Historical data range 0.030 to 0.072 g
- Mean absolute weight of spleen 0.73 ± 0.07 g; Historical data range 0.43 to 1.09 g
- Mean relative weight of spleen 0.20 ± 0.02 g; Historical data range 0.16 to 0.29 g
Moreover, a few instances of statistically significant (p < 0.05) changes were noticed in female rats and were considered incidental. They were, increase in absolute and relative weight of adrenals in (G2) low dose group and increase in only relative weight of adrenals in (G3) mid dose group rats. The effect was not evident in the high dose group rats and hence, these changes were not considered to be treatment-related as there were no dose-related responses. Also the relative and absolute weights of adrenals were well within the historical control range of female rats of similar age group from the test facility, though not from reproduction study.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Some incidental and spontaneous lesions observed in animals from the high dose group (1000 mg/kg bw/day) comprised of bile duct hyperplasia in liver; ectopic thymus in thyroid and lymphocytic cell infiltration in prostate. All these changes were of minimal severity and were solitary in nature. These changes are commonly observed in rats and as they are known to be spontaneous in nature 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.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS:
Treatment 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.

MORTALITIY:
No mortality occurred during the study period that was considered to be related to treatment with the test substance. All animals survived throughout the treatment period of the study.

FUNCTIONAL OBSERVATIONS (NEUROLOGICAL EXAMINATION):
The functional observations did not reveal any remarkable and treatment-related incidence of neurological abnormalities. In addition, no findings indicative of a neurotoxic potential of the test item were observed.

BODY WEIGHT AND WEIGHT GAIN:
The test item at and up to the highest dose of 1000 mg/kg bw/day did not have any remarkable and adverse effects on the weight in male and female rats.

FOOD CONSUMPTION:
Treatment of male and female rats at and up to the highest dose of 1000 mg/kg bw/day 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 pre-mating period, the gestation period and the post-natal lactation period.

HAEMATOLOGY:
No toxicologically relevant changes occurred in haematological parameters of treated rats. Few changes like significantly lower (p < 0.05) mean percentage PCV in high dose males and significantly higher (p < 0.05) mean reticulocyte count percentage in high dose females were considered incidental as there was no dose-dependent change and the values were within respective historical control range: Mean PCV of high dose males was 42.32 ± 2.19%, which is well within historical control range (41.11 to 48%) and the mean reticulocyte count percentage in high dose females was 4.42 ± 1.36% which is well within historical control range (1.83 to 4.58%).

CLINICAL CHEMISTRY:
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological relevance as these occurred in the absence of a treatment-related trend. At the end of treatment these changes consisted of lower potassium and alkaline phosphatase in males at 300 mg/kg bw/day, and higher urea levels in females at 300 mg/kg bw/day.

ORGAN WEIGHTS:
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios. A few instances of statistically significant (p < 0.05) changes compared to that of control organ weights were noticed in males and were considered incidental as they were within historical control ranges. Differences in organ weights were observed in mean absolute weights of adrenals and spleen and the mean relative weight of spleen. Moreover, a few instances of statistically significant (p < 0.05) changes were noticed in female rats and were considered incidental. They were, increase in absolute and relative weight of adrenals in (G2) low dose group and increase in only relative weight of adrenals in (G3) mid dose group rats. The effect was not evident in the high dose group rats and hence, these changes were not considered to be treatment-related as there were no dose-related responses. Also the relative and absolute weights of adrenals were well within the historical control range of female rats of similar age group from the test facility, though not from reproduction study.

GROSS PATHOLOGY:
Macroscopic observations at necropsy did not reveal any toxicologically relevant alterations in tissues of treated rats. Moreover, examination of the reproductive organs of male and female rats did not reveal any treatment-related morphological changes.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Histopathological examinations of adult male and female rats randomly selected from control group and high dose groups did not reveal any significant and treatment-related histopathological alterations. Some incidental and spontaneous lesions observed in animals from the high dose group (1000 mg/kg bw/day) include bile duct hyperplasia in liver; ectopic thymus in thyroid and lymphocytic cell infiltration in prostate. All changes observed were of minimal severity and were solitary in nature. These changes are commonly observed in rats and as they are known to be spontaneous in nature 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. Based on the results of the haematology investigation, especially of 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 any immunological effects.

Effect levels

Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical signs, functional observations, body weights, food consumption, clinical pathology, macroscopy, organ weights, and histopathology.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion