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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13.08.2015 - 04.12.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Department of Science and Technology, Government of India, New Delhi
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
- Strain: HsdHan: WIST rats Conventionally bred (In-house random bred)
- Justification for the selection of species: Rat is the standard laboratory rodent species used for toxicity assessment and recommended globally by many regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10 weeks
- Weight at study initiation: males: 291 - 375 g; females: 176 - 231 g
- Housing:
-- Pre mating: 2 rats of the same sex in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill with Polycarbonate Rat huts as environmental enrichment objects
-- Mating: 1 male and 1 female in standard polysulfone cages with stainless steel top grill
-- Post mating: males were housed in their former cages; females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term.
- Diet: ad libitum; Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories B.V. Maasheseweg 87c PO Box 553, 5800, AN Venray, The Netherlands
- Water: ad libitum; Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: The food and water provided to the animals were tested for contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 -24
- Humidity (%): 59 - 68
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected since it is an exaggerated model of the normal exposure in humans. Gavage was chosen because it is an accurate and reliable method for dosing.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Required quantities of the test item was weighed in pre-calibrated beakers and mixed with a small quantity of corn oil. The volume was filled up with the vehicle to attain desired concentrations of 13, 40 and 120 mg/mL for the low, mid and high groups, respectively. The volume of dose formulation to be prepared was varied depending on the requirement and/or body weights of the rats recorded during experimental period.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was used as vehicle for dose formulation preparation as the same vehicle was used in the dose range finding toxicity study.
- Stability: test item was found to be stable for 24 hours at ambient condition in the vehicle (corn oil).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Active ingredient (A.I.) concentration analysis
- Prepared formulations were sampled on Day 1 and during 2nd month of the treatment period.
- In duplicate sets and analysed In-house.
- For each set, 3 replicates from a composite sample were drawn and analyzed for the test item concentrations. In case of control, two replicates from a composite sample were drawn. The sample analysis was done as per the analytical method validated under Advinus Study No.: G10755.
- Dose formulations were considered acceptable as the overall mean results were within ± 10.0% of the theoretical concentration and the overall relative standard deviation (RSD) was less than 10.0%.
- The second set of samples were analysed during 2nd month as the analysis results of first set were not within the specified range.
Duration of treatment / exposure:
- males: 2 weeks pre-mating, mating and 2 weeks post-mating
- females: 2 weeks pre-mating, mating and post-mating during pregnancy and up to lactation day 4
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
65 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
High dose
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
High dose recovery
No. of animals per sex per dose:
- main groups: 10
- recovery groups: 5
Control animals:
yes, concurrent vehicle
other: vehicle control recovery
Details on study design:
- Dose selection rationale:
The dose levels were selected for this study based on the results of 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No.G10756) and in consultation with the Sponsor.
In the 2-week range finding study in rats no mortality, no clinical signs nor any effects on food intake or body weight gain were observed. At 600 and 1000 mg/kg bw/day liver weights were increased in both sexes and at 1000 mg/kg bw/day kidney weights were increased in both sexes.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CLINICAL SIGNS: Yes
- Time schedule: daily

MORTALITY: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the beginning of study, once per week during treatment and recovery periods

PHYSIOLOGICAL OBSERVATIONS: Yes
- Time schedule: at the end of the functional test

BODY WEIGHT: Yes
- Time schedule for examinations: at the beginning and at weekly intervals. All dams were weighed on Gestation Days (GD) 0, 7, 14 and 20 and on lactation days 0 and 4 and weights were recorded.

FOOD CONSUMPTION: Yes
- Food consumption: using the food consumed at weekly interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food intake/rat/day. Food consumption was not measured during the cohabitation period. Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Day 4 of lactation period.

PARTURITION
The duration of gestation was calculated from day ‘0’ of pregnancy to the day of parturition (Gestation Length).
Females were observed for signs of difficult or prolonged parturition.

LITTERS
- At birth, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.
- The number of pups born (litter size), sex and individual pup body weight of male and female pups on days 0 and 4 post-partum were recorded.
- The litters were observed daily in order to note the number of alive, dead and cannibalized pups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of pre-mating
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes
- How many animals: 5 male and 5 female
- Parameters checked: Coagulation (Prothrombin Time&Activated Partial Thromboplastin Time), Red Blood Corpuscles, Haemoglobin, Haematocrit, Mean Corpuscular Volume, Mean Corpuscular Haemoglobin, Mean Corpuscular Haemoglobin Concentration, Reticulocytes, White Blood Corpuscles, Differential leukocyte count(absolute) and Platelets

CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes
- Parameters checked: Alanine Aminotransferase, Albumin, Alkaline Phosphatase, Aspartate Aminotransferase, Albumin/Globulin ratio [calculated values], Blood urea nitrogen, Bile acids, Calcium, Chloride, Creatine, Gamma Glutamyl Transferase, Glucose, Globulin [calculated values], Inorganic phosphorous, Potassium, Sodium, Total bilirubin, Total cholesterol, Total plasma protein and Triglycerides

NEUROBEHAVIOURAL EXAMINATION: Yes
1. Functional Observation Battery Tests (FOB): at end of the dosing period for males and during lactation period for females, for recovery groups towards the end of recovery period.
2. Observations during Removal of Animal from Home Cage and Handling: whenever an animal was handled
3. Open Field Observation: observation for at least 2 minutes.
4. Functional Tests: motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.
Sacrifice and pathology:
NECROPSY
- How many animals: all, including pups and non-pregnant rats
- Overnight fasting: Yes
- Anaesthetic: isoflurane
- Examinations: weighing, exsanguination and detailed necropsy

HISTOPATHOLOGY
- How many animals: 5 males and 5 females in the control and high dose groups
- Tissues: see table in any other information
- Extra: Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis. Reproductive organs of infertile males and females across the groups were examined microscopically. The tissues were processed for routine paraffin embedding and 4 - 5 micron thickness sections were stained with Mayer’s Haematoxylin Eosin stain. In addition, testes was sectioned at 3-4 μm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues will be archived.
Statistics:
All quantitative variables (body weight, food intake, haematology, clinical chemistry, organ weights and organ weight ratios) were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA is performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment ‘F’ test was found significant.
Pre-implantation loss (%), post implantation loss (%), no. of corpora lutea and implantations, pre-coital interval and gestation length (days) were analysed after suitable transformation (√ x + 1⁄2) of the data. One-way analysis of variance (ANOVA) was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was done when the group differences are found significant.
Z test was performed for testing the differences in proportions for mating and fertility indices.
All analyses and comparisons were evaluated at the 5% (P<0.05) level. Statistically significant differences (P<0.05), indicated by the aforementioned tests were designated by the superscripts throughout the report as stated below: +/-: Significantly higher (+)/lower (-) than the vehicle control group; a+/a-: Significantly higher (a+)/lower (a-) than the vehicle control recovery group.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs in any of the groups during the experimental period.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities in any of the groups during the experimental period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights were not affected by the treatment at any dose tested when compared to vehicle control in both sexes. In the high dose recovery group, the body weights were unaffected both during the treatment and recovery period.
Treatment did not affect the mean body weights at all the tested doses in either sex when compared to vehicle control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption was not altered by the treatment in both the sexes at all the doses tested, when compared to vehicle control. In the high dose recovery group, the food consumption was not affected both during the treatment and recovery period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The test item administration did not reveal any treatment related changes in the hematology or coagulation parameters of both male and female rats.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The test item administration did not reveal any treatment related changes in the clinical chemistry parameters of both male and female rats.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Home cage and Handling observations: No treatment-related abnormalities were observed in all the tested dose groups in both sexes
- Physiological observation: Body temperature: The physiological observation of body temperature was unaffected in both sexes except for significantly higher body temperature at the high dose in main group females. The observation is considered as an incidental finding as no changes were observed in the home cage or open field observations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related changes in the terminal fasting body weights, organ weights and organs weight ratios in both male and female rats.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross and microscopic changes observed in both the sexes. The cause of infertility could not be determined in infertile male and female rats as no significant microscopic changes were observed in the reproductive organs examined.
Neuropathological findings:
no effects observed
Description (incidence and severity):
- Open field observations: No treatment-related abnormalities were observed in any of the doses tested in both sexes.
- Sensory observations: No treatment-related abnormalities were observed in any of the groups in both sexes.
- Motor Activity: The following statistically significant variations were observed in the motor activity of rats when compared to respective vehicle control group:
-- Males: Lower: Stereotypic time at interval 3 at the high dose, ambulatory time at interval 3 at the high dose, horizontal counts at interval 3 at the high dose, ambulatory counts at interval 3 at the high dose in the main groups. Stereotypic time at interval 1, ambulatory time at interval 2, horizontal counts at interval 2 and total counts, ambulatory counts at interval 2 and total counts at the high dose recovery group.
The above observed statistical variations in the motor activity measurement were considered to be incidental as the observed changes did not follow the dose proportion and there were no changes observed in the home cage or open field observations.
-- Females: no significant changes observed at all doses tested.
- Neuromuscular observation: Landing hind limb footsplay: No significant changes were observed at all the tested doses in both sexes. However, an incidence of significantly higher hindlimb footsplay was observed at the high dose in main group males. This change was considered incidental as there were no changes observed in the home cage or open field observations.
- Grip strength: No significant changes were observed at all the tested doses in both sexes.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No significant microscopic changes were observed.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No significant microscopic changes were observed.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No effects observed at highest tested dose.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL for systemic and reproductive toxicity in parental rats is considered to be 600 mg/kg bw/day as there were no adverse effects observed in any parameters up to the highest dose tested.
Executive summary:

A Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test in rats was performed to generate information concerning the effects of the test item on male and female reproductive performance and possible health hazards likely to arise from repeated exposure over a relatively limited period of time. This study was conducted in accordance with OECD Guideline No. 422 for testing of chemicals, “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” adopted on March 22, 1996.

The test item was administered orally at dose levels 65, 200 and 600 mg/kg bw/day corresponding to low, mid and high dose groups of male and female rats. Additional groups for a high dose recovery, a concurrent control and a control recovery were also included in the study.

The males were dosed for a minimum of 4 weeks, up to and including the day before scheduled sacrifice, including minimum 2 weeks prior to mating, during the mating period and approximately 2 weeks post mating. Females were dosed throughout the treatment period, including 2 weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and at least 4 days after delivery, up to and including the day before scheduled sacrifice. Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days post treatment and these animals were not mated and consequently not used for assessment of reproduction/developmental toxicity. The recovery period of the study was started from the day of sacrifice of the first littered animals.

All animals were observed for clinical signs, physical abnormalities and mortality. The body weight and food consumption were measured at periodic intervals. The functional observation battery was done shortly before sacrifice for randomly selected 5 males and 5 females from each group. For recovery groups, functional observation battery was done prior to sacrifice. Laboratory investigations such as haematology and clinical chemistry were performed in randomly selected 5 males and 5 females from each group at the end of the pre-mating period for main groups and at the end of recovery period from all animals of recovery groups. The animals were subjected to detailed necropsy at sacrifice after overnight fasting and study plan specified tissues were collected. Tissues collected from randomly selected 5 males and 5 females in the control and high dose groups were examined microscopically for histopathological changes. Histopathological examination of testes in the high dose group included a qualitative assessment of stages of spermatogenesis. All gross lesions were examined microscopically. The reproductive organs of infertile males and females across the groups were examined microscopically

No clinical signs or mortality were observed during the course of the study. No treatment-related neurological abnormalities/dysfunctions were observed at all doses tested. The body weights and food consumption were unaffected by the treatment at all doses tested. The maternal food consumption during gestation was not affected by the treatment at all doses. The maternal body weights and food consumption during lactation periods were unaffected at all doses tested. Treatment had no effect on pre-coital time and gestation length at all the tested doses. No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested. The survival index was not altered by the treatment at any dose. The test item administration did not reveal any treatment related changes in the hematology, coagulation and clinical chemistry parameters of both males and females. There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females. There were no test item-related changes observed at high dose group and therefore histopathological evaluation was not carried out for the lower and recovery dose groups. Gross examination of pups on lactation day 4 did not reveal any changes. There were no test item related gross and microscopic changes in both males and females.

The No Observed Adverse Effect Level (NOAEL) for systemic and reproductive toxicity in parental rats is considered to be 600 mg/kg bw/day as there were no adverse effects observed in any parameters up to the highest dose tested.