Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 April - 06 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD 408 Guideline without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: MEXORYL SCK
- Physical state: Beige powder
- Analytical purity: saponines content (determined by HPLC assay) 57.0 % w/w
- Lot/batch No.: R0069579A 008 X 001
- Expiration date of the lot/batch: September 2012
- Storage condition of test material: At room temperature away from light

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd, Jerusalem, Israel.
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: (G1: 239.526 ± 13.712 g; G2: 238.559 ± 15.221 g; G3: 238.389 ± 15.285 g; G4: 238.660 ± 14.613 g); females (G1: 180.868 ± 12.133 g; G2: 182.655 ± 11.327 g; G3: 181.481 ± 12.806 g; G4: 181.995 ± 9.034 g)
- Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 175 mm), with stainless steel top grill.
- Diet: Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories B.V., The Netherlands, ad libitum
- Water: 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, India, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-24 °C
- Humidity: 58-67 %
- Air changes: 12-15 air changes/hour
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Milli Q water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The required quantity of test item was weighed in pre-calibrated amber coloured beakers and dissolved in the Milli-Q water by using glass rod. Then required volume of Milli-Q water was added till final volume was obtained so as to attain the desired concentration. Dose solutions were prepared one day prior to commencement of treatment, the same was used on Day 1 of treatment and thereafter dose solutions were prepared daily, afresh and used within the prescribed stability (24 h) period.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

STABILITY AND HOMOGENEITY OF THE TEST ITEM IN THE VEHICLE:
- Stability and homogeneity test was determined at 1 and 200 mg/mL prior to start of treatment and the test solutions was stable for 24 hours at ambient and refrigerated conditions.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and concentration (A.I.) analysis, the prepared dose solutions were sampled one day before treatment and during months 2 and 3 of treatment period in duplicate sets (two replicates were drawn from top, middle and bottom layer each). For control, duplicate samples from only middle layer were drawn.
The results indicated that the mean analysed concentration of the active ingredient was within the acceptable limits (15% and relative standard deviation (RSD) is less than 10%) of variation and was homogeneously mixed in the vehicle. The analyzed control (0 mg/mL) samples did not show any interference.
Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
Once daily for 90 consecutive days
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Three dose levels of 100, 300 and 1000 mg/kg bw/day were selected based on the results of 14-Day Repeated Dose Oral Toxicity Study of MEXORYL SCK in Wistar Rats by Oral Route (Study No.: G8174) and in consultation with the Sponsor wherein the highest dose tested, 1000 mg/kg bw/day, did not induce any toxicological effect. In addition to the test doses, a vehicle control group was included.
- Rationale for animal assignment: Animals were randomly distributed to different groups by body weight stratification method using Provantis™ software. The grouping was done two day prior to initiation of treatment.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed for clinical signs (pre and post dose) daily. Observations for morbidity and mortality were carried out twice daily. As there were no clinical signs of concern the observations for morbidity and mortality were carried out once during public holidays and weekends (except for a week end on treatment Days 4 and 5, wherein, observations for morbidity and mortality were carried out twice).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examination was done prior to the test item administration on Day 1 and once a week thereafter except on Week 13 wherein detailed clinical examination was performed on Day 6 of that week for all the rats.
- During detailed clinical examination, all rats were observed cage-inside/outside for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g. self mutilation, walking backwards).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded before the administration of test item (on Day 1 of treatment) and at weekly intervals thereafter except on Week 13 of treatment wherein the body weight was measured on Day 6 of that week. Additionally, fasting body weight was recorded prior to terminal sacrifice on Day 91 of the in-life phase of the experiment.

FOOD CONSUMPTION AND FOOD EFFICIENCY:
- Food consumption was measured at weekly intervals except on Week 13 wherein the food consumption was measured on Day 6 of that week during in-life phase of the experiment. The food spillage was weighed and captured online at each food leftover recording session and during cage change and this was taken into consideration for derivation of food intake (g/rat/day). Cagewise food consumption was calculated by 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 interval period to determine the food intake/rat/day.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination of all animals was performed with an ophthalmoscope, one day prior to start of treatment and for control and high dose group on treatment Day 90.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 91; blood was collected from retro-orbital sinus plexus with fine capillary tube
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- Animals fasted: Yes; all rats were fasted overnight (water allowed)
- How many animals: 10 animals/sex/dose
- An aliquot of blood was collected in tubes containing 3.2 % sodium citrate for determination of Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT). The remaining blood was collected into K2EDTA tubes for haematology and heparinised tubes for clinical chemistry.
- Parameters checked for haematology: Haematocrit, Hemoglobin, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Mean corpuscular volume, Mean platelet volume, Platelets, RBC, WBC, Reticulocytes, Differential white cell count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), PT and APTT.
- Parameters checked for clinical chemistry: Alanine aminotransferase, Alkaline phosphatase, Aspartate aminotransferase, Albumin, Albumin/Globulin ratio, Blood urea nitrogen, Calcium, Chloride, Creatine kinase, Creatinine, Gamma glutamyl transpeptidase, Globulin, Glucose, Inorganic phosphorous, Lactate dehydrogenase, Potassium, Sodium, Total bilirubin, Direct Bilirubin, Indirect Bilirubin (calculated), Total cholesterol, Total plasma protein and Triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from all rats prior to sacrifice in urine collection tubes.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes; For urine collection, each rat was placed in specially fabricated cages overnight (water allowed) and the next morning the collected urine was sent for analysis.
- Parameters checked: Specific gravity, Nitrite, pH, Proteins, Glucose, Ketone bodies, Urobilinogen, Bilirubin, Appearance (colour and clarity) and Volume. Urine was also subjected to microscopic examination for sediments such as crystals, epithelial cells, erythrocytes, leukocytes and casts.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 12th week of treatment period
- Dose groups that were examined: All groups
- Home cage observations: Each rat was observed in the home cage for posture and for presence and absence of abnormal vocalizations and convulsions.
- Observations during removal of rat from home cage and handling: ease of removal from home cage, handling reactivity, palpebral closure, eye examination, piloerection, lacrimation, salivation, skin/fur examination, perineum wetness, respiration, muscle tone and extensor thrust response
- Open field observation: gait, posture, mobility score, arousal level, clonic and tonic movements, stereotypic behaviours, bizarre behaviour, urination, defecation, rearing and vocalizations
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Landing Hindlimbs Footsplay
-Physiological Observations: The body (rectal) temperature (°C) was measured and recorded.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; At the end of treatment period (Day 90) all rats were fasted overnight (water allowed). On the following day (Day 91) all rats were weighed, anaesthetized with isoflurane, exsanguinated and were subjected to detailed necropsy.
HISTOPATHOLOGY: Yes; Histopathological examination was carried out on all the preserved tissues/organs from all animals in the control group (G1) and the high dose (G4) group.
- On completion of the gross pathology examination, the tissues and organs noted in table no. 7.5.1/1 were collected, weighed and preserved in 10 % neutral buffered formalin except for the testes and eyes. The paired organs were weighed together and combined weight was presented.
- The tissues were processed for routine paraffin embedding and 5 µm sections were stained with Mayer’s Haematoxylin Eosin stain.
Other examinations:
None
Statistics:
- The Data captured using Provantis™ for the parameters namely body weights, food consumption and organ weights were analyzed using built-in statistical tests.
- Derived data like net body weight change and organ weight ratios were also analyzed using above mentioned methods.
- The statistical analysis of the experimental data captured other than Provantis™, was carried out using the developed and validated package in Excel and using licensed copies of SYSTAT Statistical package Ver.12.0. All quantitative variables like neurological observations (neuromuscular observation and body temperature) and clinical pathology (haematology, coagulation and clinical chemistry) data was tested for normality and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Comparison of means between treatment groups and control group was done using Dunnett’s test where the overall treatment, ‘F’ test is found to be significant.
- 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: +/-: Significantly higher (+)/lower (-) than the vehicle control group

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:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- No treatment related mortality or treatment related clinical signs were observed at any of the dose levels tested.
- The incidence of hair thinning with hair regrowth bilaterally on the neck region was observed in one male belonging to control group on Day 91 and the same was observed in one male (from Day 42 to 86) and one female (from Days 42 to 91) belonging to 300 mg/kg bw/day dose group. This is a common observation in this species and hence considered incidental and not related to treatment.

BODY WEIGHT AND WEIGHT GAIN
- In the 1000 mg/kg bw/day dose group males, significantly lower mean body weights were observed on Days 22, 29, 36, 43, 50, 57, 64, 71, 78, 85 and 90 and significantly lower net body weight gains during Days 1-8, 15-22 and 57-64, when compared to the vehicle control group. This decrease in the mean body weight was of low magnitude (7.79-9.53 %), within the normal historical range and also was not associated with clinical pathology, gross and histopathological changes of the organs or tissues examined and hence, considered not test item related.
- No treatment related changes were observed in the mean body weights at 100 and 300 mg/kg bw/day doses in males and all the doses tested in females, when compared to the vehicle control group.
- Significant increase in the net body weight gain during Days 64-71 in males at 100 and 300 mg/kg bw/day doses and significant decrease observed during Days 36-43 in females 100 mg/kg bw/day dose were considered to be incidental because of lack of dose relationship and also since these changes were not observed in the succeeding weeks.

FOOD CONSUMPTION
- No changes were observed in the food consumption in either sex at any of the dose levels tested when compared to the vehicle control group.

OPHTHALMOSCOPIC EXAMINATION
- No treatment - related ocular abnormalities observed in any of the treated groups.

HAEMATOLOGY
- Decreased hemoglobin concentration in all treated groups in males, increased MCV at 1000 mg/kg bw/day dose males and decreased MCHC in all treated groups in males and 1000 mg/kg bw/day dose females were considered toxicologically insignificant as the magnitude of change was very minimal (≤ 5 %) and these changes were within the historical control range.
- Decreased absolute monocyte counts in males at 300 (44 %) and 1000 (28 %) mg/kg bw/day dose were considered as toxicologically insignificant as there was no dose correlation and these changes were within the historical control range. An increased platelet count in 300 mg/kg bw/day dose females was considered as incidental change as there was no dose progression and the change was within the historical control range.
- Decreased Prothrombin time (8 %) in females at 1000 mg/kg bw/day dose was considered as incidental change as magnitude of change was minimal and the change was likely due to random biological variation.

CLINICAL CHEMISTRY
- Decreased triglycerides concentration was observed in all treated groups in males. The decrease in triglycerides at 1000 mg/kg bw/day dose was considered as treatment-related non adverse change as it was likely due to decreased body weight. Decreased triglycerides at 100 and 300 mg/kg bw/day doses were considered toxicologically insignificant as it was not associated with any changes in body weight gain and these changes were within the historical control range.
- Decreased BUN concentration in males at 1000 mg/kg bw/day dose was considered toxicologically insignificant as decreased blood urea nitrogen concentration has no biological significance and the change was within historical control range.
- At 1000 mg/kg bw/day dose, increased sodium (1 %) and chloride (2 %) in males and decreased calcium concentration in females was considered as incidental changes as the magnitude of change was minimal. Increased inorganic phosphorous concentration in 1000 mg/kg bw/day dose group females was considered incidental change as it was within historical control range and was likely due to random biological variation.

URINALYSIS
- There were no treatment-related changes in any of the urinalysis parameters analyzed.

NEUROBEHAVIOUR
- No treatment related neurological manifestations/abnormalities observed in the functional observational battery tests at any of the doses tested.

ORGAN WEIGHTS
- Decreased terminal fasting body weight (10 %) in males at 1000 mg/kg bw/day dose was considered as treatment-related non adverse change.
- Decreased absolute weight of liver in males at 1000 mg/kg bw/day dose was considered toxicologically insignificant as there was no change in relative weights.
- Increased absolute and relative weight of lungs in females at 100 and 1000 mg/kg bw/day doses was considered toxicologically insignificant as there was no clear dose response.

GROSS AND HISTOPATHOLOGY
- A single incidence of mesenteric mass observed in 300 mg/kg bw/day dose male was microscopically confirmed as sarcoma-nos (not otherwise specified) type. This change was considered incidental tumor of spontaneous origin without any relation to test item administration.
- Enlarged atrium observed in heart of 100 mg/kg bw/day dose male was considered as incidental change of no toxicological significance as it was not associated with any microscopic change.
- Epididymal abscess observed in control male was microscopically associated with sperm granuloma and was considered as incidental change.
- Distended urinary bladder observed in 1000 mg/kg bw/day dose male was considered incidental change as it was not associated with any microscopic change.
- Dilated uterus observed in different groups was considered as physiological change as it is cyclical change observed in different stages of estrous cycle.
- There were no test item-related changes in all the tissues/organs examined. The few incidences of microscopic findings observed in different groups were considered incidental and not related to test item as incidences were comparable to control group and did not show any dose wise trend or patterns.

OTHERS:
Homogeneity and Stability of the Test Item
- The test item, MEXORYL SCK at 1 mg/mL and at 200 mg/mL in the test solutions was stable for 24 h at ambient and refrigerated conditions and found to be homogenous in the vehicle.

Dose Formulation Analysis of the Test Item:
- The dose formulations (at 20, 60 and 200 mg/mL) were analysed for active ingredient (A.I.) concentration and homogeneity one day prior to commencement of treatment and during second and third month of the treatment period. The results indicated that the mean analysed concentration of the active ingredient was within the acceptable limits (15 % and RSD is less than 10 %) of variation and was homogeneously mixed in the vehicle. The analyzed control (0 mg/mL) samples did not show any interference.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicity effects were observed

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of MEXORYL SCK was considered to be 1000 mg/kg bw/day in Wistar rats.
Executive summary:

In a repeated dose oral toxicity study conducted according to the OECD Guideline 408 and in compliance with GLP, MEXORYL SCK was administered daily by oral gavage to groups of Wistar rats (10/sex/dose) at the dose-levels of 0 (vehicle), 100, 300 and 1000 mg/kg bw/day in the vehicle (Milli Q water) with the dose volume of 5 mL/kg bw/day for 90 days. Examinations during the study included: mortality, clinical signs, functional observation battery (including motor activity), body weight change, food consumption, ophthalmology, laboratory investigations: hematology, blood clinical chemistry, urinalysis, macroscopic examination of main organs, measurement of organ weights and histopathology.

 

No treatment - related clinical signs or mortality observed in any of the treated groups. No treatment - related ocular abnormalities observed in any of the treated groups. No treatment related neurological manifestations/abnormalities observed in the functional observational battery tests at any of the doses tested. The treatment resulted in significantly lower mean body weights and net body weight gains in male rats at 1000 mg/kg bw/day compared to vehicle control group. The decrease in the mean body weight was of low magnitude (7.79-9.53 %), within historical control range and was not associated with clinical pathology, gross and histopathological changes of the organs or tissues examined and hence, considered non-adverse. No treatment - related change in the feed consumption observed in any of the treated groups. There were no test item-related changes in hematology and coagulation parameters, organ weights, gross and microscopic changes in both males and females except for decreased triglycerides concentration in males of all treated groups and decreased terminal fasting body weight observed in males at 1000 mg/kg bw/day which was considered as treatment-related but a non-adverse change. No treatment - related gross or histopathological changes observed in any of the treated groups.

 

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of MEXORYL SCK was considered to be 1000 mg/kg bw/day in Wistar rats.