Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 6-7 weeks
- Weight at study initiation: Males: 184.83 to 210.53 g ; Females: 142.63 to 165.38 g

- Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in each group and sex was housed individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week. During the experimental period, animals were housed in a single experimental room of barrier area.
- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet – Pellet (Certified) manufactured by Envigo, P.O.Box 44220, Madison WI 53744-4220, was provided ad libitum to the rats.
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: Start: 28 May 2017 ; End: 01 June 2017

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 25°C
- Humidity (%): 59 to 68 %
- Air changes (per hr): 13.3 – 14.3
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 02 June 2017 To: 29 June 2017

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Route of test item administration was through oral gavage. The oral route was chosen because it will provide an exaggerated model of the normal exposure in humans.
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Required quantities of the test item was weighed in to a pre-calibrated beaker* and the vehicle [0.5 % (w/v) of carboxymethyl cellulose in Milli-Q® water] was added up to the pre-mark and mixed using glass rod. The final concentration achieved was 11, 33 and 100 mg/mL for the G2, G3 and G4/G4R groups, respectively. The suspensions were mixed well by stirring using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5 % (w/v) Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q® Water was used to prepare the dose formulations as the same vehicle was used in the acute oral toxicity study (Study No. G12734) and 14-day repeated dose oral toxicity study in rats (Study No. N3254).

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during week 4 of the treatment period and analysed in-house. For each set, duplicate sample was drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Advinus Study No. G12742. One set of samples were analysed for concentration (a.i) analysis.
Dose formulations were considered acceptable as the overall mean results were within ± 15.0% of the claimed concentration and the overall relative standard deviation (RSD) was less than 10.0%.
The unused back up samples were disposed as analysis results of the first set of samples were within the acceptable limits.
Duration of treatment / exposure:
28 day
Frequency of treatment:
Daily once
Doses / concentrationsopen allclose all
Dose / conc.:
110 mg/kg bw/day (nominal)
Dose / conc.:
330 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males + 5 females
Total = 60 (30 males + 30 females)
Control animals:
yes
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
The dose levels of 110 (G2), 330 (G3) and 1000 (G4/G4R) mg/kg/day were selected for this study based on the results of 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No.: N3254) and in consultation with the Sponsor.
In addition to the test doses, vehicle control and vehicle control recovery groups were included. Animals in the vehicle control and vehicle control recovery were handled in a manner similar to the treatment groups except for test item administration. No correction for purity was used during dose formulation preparation.

- Rationale for animal assignment (if not random): Animals were randomly distributed to different groups by body weight stratification method using ProvantisTM software. Rats with extreme body weights were discarded. Grouping was done one day prior to start of treatment during acclimatization.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily once
- Cage side observations checked in table [No.2] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination of all animals was performed with an ophthalmoscope prior to start of the treatment, at the end of the treatment period for main groups and at the end of recovery period for recovery groups.
- Dose groups that were examined: All groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period for main groups and at the end of the recovery period for recovery groups
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in Main report [Section 8.7.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period for main groups and at the end of the recovery period for recovery groups
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in Main report [Section 8.7.4] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the treatment period for main groups and at the end of the recovery period for recovery groups
- Animals fasted: Yes
- Parameters checked in Main report [Section 8.8] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 4th week of treatment period for main groups and towards the end of recovery period for recovery period.
- Dose groups that were examined: All groups
- Battery of functions tested: sensory activity / open field observation / grip strength / motor activity : Yes

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see in Main report Section 8.9)

HISTOPATHOLOGY: Yes (see in Main report Section 8.10)
Statistics:
Data captured using Provantis™ for the parameters body weight and organ weights; laboratory Investigations - Haematology (Coagulation tests PT and APTT which will be entered retrospectively in Provantis™) and Clinical Chemistry were analyzed using built-in statistical tests.
Derived data like net body weight change, food consumption and organ weight ratios were also analyzed using above mentioned methods.
The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like neurological observations (neuromuscular observation/body temperature/body weights) 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 is found to be significant.
The data pertaining to males and female rats was evaluated separately.
All analyses and comparisons was evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated by the following symbols throughout the report:
*: Significantly higher/lower than the respective control group

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight bluish discolouration of the skin was observed at 110 and 330 mg/kg bwt/day doses after test item administration in both sexes. Moderate bluish discolouration of the skin was observed at 1000 mg/kg bwt/day dose after test item administration and became slight prior to next dose (pre-dose) on each day of observation. Light bluish colour faeces was observed at 100 mg/kg bwt/day dose, whereas at 330 and 1000 mg/kg bwt/day doses, the faeces colour observed was dark bluish colour in both sexes. In addition, light coloured faeces was observed on Day 26 in animals numbers Ru1321 and Ru1322 at 330 mg/kg bwt/day. The bluish discolouration of the skin/faeces observed could be due to physical nature of the test item. This effect was considered non adverse and due to the physical presence of the test item without toxic activity.
Mortality:
no mortality observed
Description (incidence):
No mortalities were observed at any of the tested doses in both sexes.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight was significantly lower on Day 28 (-7.1%) and net body weight gain during Days 1-8 at 110 and 330 mg/kg/day doses in males. This significant difference was considered incidental because of isolated occurrence. Further, the decrease is not consistant through out the treatment period.
At 1000 mg/kg/day, the treatment resulted in decreased mean body weight on Days 15, 22 and 28 (-6.5 to -8.4%) and net body weight gain during Days 1-8 in the main group males. On Day 8, the mean body weight was apparently lower (-5.2%) without statistical significance in males. In the high dose recovery group males, the mean body weights were apparently lower (-3.9 to 6.9%) during the treatment period. During the recovery period, the net body weight gains were apparently higher when compared to vehicle control recovery group indicating recovery of the changes observed during treatment period.
In females, the mean body weights and net body weight gains were not significantly different from the vehicle control both during the treatment and recovery periods.
Thus, the treatment resulted in decreased the body weight at 1000 mg/kg/day dose in males.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption was significantly lower during Days 1-8, 15-22 and 22-28 at 110 mg/kg/day, during Days 15-22 at 330 mg/kg/day and dose in males. This statistically significant difference was considered incidental as the body weights were not altered by the treatment.
At 1000 mg/kg/day, the treatment resulted in decrease food consumption during Days 1-8, 15-22 and 22-28 in the main group males and during Days 8-15 in recovery group males during the treatment period. In females, the decreased food consumption during Days 1-8 in recovery group was observed during the treatment period. This single incidence was considered incidental as there was no corresponding changes in body weights..
Thus, the treatment resulted in decrease in the food consumption, associated with decreased body weights at 1000 mg/kg/day dose males.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmological examination carried out with an ophthalmoscope prior to start of treatment, at the end of the treatment and recovery period did not reveal any abnormalities in the eyes of the experimental rats.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There was an increase in reticulocyte count at 1000 mg/kg/day in both males and females (33% and 68% respectively). This increase was associated with the microscopic finding of increased extramedullary hematopoiesis in spleen and was reversible only in females.

The blood smear examination did not reveal presence of Heinz bodies at all the doses tested.

All the other observed intergroup differences including the statistical significances which attained were considered incidental as either the differences were very minimal or the changes were not dose related
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes in clinical chemistry parameters. The intergroup differences noted in bilirubin in females only values were not dose related and also the values were within the range of normal variation.
The statistical significance attained for the bile acid concentration in different treatment groups was attributed to the lower values in concurrent control groups and the individual animal values were comparable to historical control data range. Thus, this finding was not related to test item administration.
All the other differences were also considered incidental as the changes were either very minimal or they were toxicologically insignificant.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The urine colour change (red, orange, brown and blue ≥ 110 mg/kg/day males, ≥ 330 mg/kg/day females) was related to test item administration. The blue urine may have been noticed in the fresh sample whereas red, brown and orange would have developed over the period of overnight urine collection.

The urine samples from 1000 mg/kg/day groups were positive for bilirubin and urobilinogen concentration. This finding was considered as a false positive reaction attributed to the discolouration of urine and thus not related to an test item administration on hemoglobin metabolism. Further, there were no other biochemical changes to associate with these findings.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related abnormalities were observed in any of the tested dose groups in both sexes.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The mean terminal fasting body weight in 1000 mg/kg day males was lower when compared to controls. This minimal weight change was also associated with the test item-related reduction in food consumption and lower body weight gain at this dose group and thus considered as test item-related. The weight reduction noted at 110 mg/kg/day was considered as an incidental finding as there were no corresponding changes in the body weight gain and food consumption. The terminal fasting body weights were not affected in females.
An increase in liver weight was present in ≥ 330 mg/kg/day males and females. This increased liver weight had the microscopic correlate of hepatocellular hypertrophy and considered as an adaptive response to the test item administration.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The test item-related discolouration (blue) was present in liver, stomach and intestine contents and attributed to the physical nature of the test item.

Blue discolouration of liver surface was present in ≥ 330 mg/kg/day dose groups. In stomach, the mucosal discolouration was noted only at 1000 mg/kg/day whereas the discoloured (blue) stomach contents were noted in ≥ 110 mg/kg/day males and ≥ 330 mg/kg/day females. Microscopic examination of these organs did not reveal any associated morphological changes at 1000 mg/kg/day. The intestinal contents were blue in ≥ 110 mg/kg/day males and females. The findings in stomach and intestinal segments reversed whereas liver surface discolouration was present at the end of recovery period.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic changes were noted in liver, kidneys and spleen.
In liver, hepatocellular hypertrophy in ≥ 330 mg/kg/day dose group males and females was considered as test item- related, reversible finding. This finding was of minimal to mild in severity with diffuse distribution. In the absence of liver enzyme profile changes, this change was considered as an adaptive response to test item administration.
In kidneys, the cortical tubules showed presence of droplets at 1000 mg/kg/day in males. These droplets were also noted in the lumen of the cortical tubules with minimal to mild severity, considered as test item related reversible change.
In spleen, increased extramedullary hematopoiesis at 1000 mg/kg/day in both males and females was related to test item administration and was reversible in females. In males, this finding was present at the end of recovery period.
All the other findings in different groups were considered as incidental/spontaneous lesions common for this age group rats.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 330 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
kidney
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

 

TABLE 1.            Summary of Body Weights (g) – Males

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                      Day numbers relative to Start Date                                                      

                                         Group Sex              1         8        15        22        28  

                                          --------------------------------------------------------------------------

                                         G1     m  Mean    195.892   227.666   254.378   281.364   299.244 

                                                    S.D.      8.503    11.409    12.723    13.800    13.683 

                                                     N         5         5         5         5         5     

                                                    ------  -------    -------   -------   -------   ------- 

                                         G2     m  Mean    196.936   221.498   243.058   265.306   278.084*

                                                    S.D.      4.561     4.213     3.815      7.483     8.201 

                                                     N         5         5         5         5         5     

                                                    ------  -------   -------   -------   -------   ------- 

                                         G3     m  Mean    194.888   219.454   243.252   266.434   278.082*

                                                    S.D.      5.222     6.928     8.417    10.350     6.584 

                                                     N         5         5         5         5         5     

                                                    ------  -------   -------   -------   -------   ------- 

                                         G4     m  Mean    196.912   215.862   237.176*  258.902*  274.232*

                                                    S.D.      6.946     7.589     7.320     6.584     6.435 

                                                    N         5          5         5         5         5     

---------------------------------------------------------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                       

                                     Group Sex              1       8      15      22      28      35      42 

                                     ----------------------------------------------------------------------------------

                                      G1R    m  Mean    201.508 234.716 262.710 287.952 307.688 327.860 340.854

                                                 S.D.      8.353  11.085  10.829  11.468  14.206  18.059  17.607

                                                  N         5       5       5       5       5       5       5   

                                                 ------  ------- ------- ------- ------- ------- ------- -------

                                      G4R    m  Mean    200.506 225.610 247.782 268.214 288.168 313.380 333.956

                                                 S.D.      6.255   9.802  11.388   9.681  10.704  13.660  17.361

                                                 N         5       5       5       5       5       5       5   

---------------------------------------------------------------------------------------------------------------------------------------------------------

Statistics Test: 1 Way Analysis of Variance: * - Significance different from vehicle control group at 5% level

Group G1 - 0 mg/kg/day      Group G2 - 110 mg/kg/day     Group G3 - 330 mg/kg/day     Group G4 - 1000 mg/kg/day                                                                              

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day

 

 

 


TABLE 2.Summary of Body Weights (g) – Females

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                      

                                          Group Sex              1         8        15        22        28   

                                         --------------------------------------------------------------------------

                                         G1     f  Mean    155.450   170.112   182.524   189.950   196.944 

                                                    S.D.      3.854     5.513     8.057     9.436     8.082 

                                                     N         5         5          5         5         5     

                                                    ------  -------   -------   -------   -------   ------- 

                                         G2     f  Mean    154.474   165.688   180.890   194.862   198.352 

                                                    S.D.      6.835     5.553    12.562    15.108     8.449 

                                                     N         5         5         5         5         5     

                                                    ------  -------   -------   -------   -------   ------- 

                                         G3     f  Mean    153.952   164.380   178.846   186.330   188.910 

                                                    S.D.      6.607     8.202    13.189    14.226    12.778 

                                                     N         5         5         5         5         5     

                                                    ------  -------   -------   -------   -------   ------- 

                                         G4     f  Mean    155.222   171.556   183.338   191.332   198.436 

                                                    S.D.      1.678      5.631     7.957     7.145     6.549 

                                                     N         5         5         5         5         5     

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                      

                                     Group Sex              1       8      15      22      28      35      42 

                                     ----------------------------------------------------------------------------------

                                     G1R    f  Mean    155.556 171.464 184.560 195.094 202.898 211.640 215.952

                                                S.D.      6.522  11.118  15.073  18.654  20.355  21.157  23.221

                                                 N         5        5       5       5       5       5       5   

                                                ------  ------- ------- ------- ------- ------- ------- -------

                                     G4R    f  Mean    150.540 164.524 174.690 183.814 190.030 200.488 204.134

                                                S.D.      6.172   5.545   8.278   5.584   7.861   7.106   6.145

                                                 N         5       5       5       5        5       5       5   

---------------------------------------------------------------------------------------------------------------------------------------------------------

Statistics Test: 1 Way Analysis of Variance

Group G1 - 0 mg/kg/day      Group G2 - 110 mg/kg/day     Group G3 - 330 mg/kg/day     Group G4 - 1000 mg/kg/day                                                                             

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day                                                                                                                    

 

 

 

 


 

TABLE 3.            Summary of Net Body Weight Gain (g) – Males

---------------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                      

                                                 Base                                                             Abs        %  

                                               Weight                                                            Gain     Gain 

                                                 Day     From:      1          8         15         22         1        1 

                          Group   Sex         1        To:       8          15         22         28        28       28  

                           --------------------------------------------------------------------------------------------------------

                          G1       m         195.892  Mean     31.774     26.712     26.986     17.880    103.352   52.77

                                                 8.503  S.D.      3.745      1.882      3.215      1.477      6.807    2.83

                                                  5      N         5          5           5           5           5         5  

                          -----   ---        -------  ----    ---------  -------    -------    -------    -------  ------

                          G2       m         196.936  Mean     24.562*    21.560     22.248     12.778      81.148   41.21

                                                 4.561  S.D.      1.377      2.095      3.919      2.497      6.201    3.14

                                                  5      N          5          5           5           5           5        5  

                          -----   ---        -------  ----    ---------  -------    -------    -------    -------  ------

                          G3       m         194.888  Mean     24.566*    23.798     23.182     11.648     83.194   42.71

                                                 5.222  S.D.      6.590      2.761      2.915     11.000      4.297    2.50

                                                  5       N         5          5           5          5          5        5  

                          -----   ---        -------  ----    ---------  -------    -------    -------    -------  ------

                          G4       m         196.912  Mean     18.950*    21.314     21.726     15.330     77.320   39.35

                                                 6.946  S.D.      3.359      6.220      3.256      2.870      5.715    3.79

                                                  5      N          5           5          5          5          5        5  

---------------------------------------------------------------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                       

                                                    Base                                                     Abs        %  

                                                   Weight                                                  Gain     Gain 

                                                    Day     From:     1        8       15       22        1        1 

                              Group   Sex         1        To:       8       15       22        28      28       28  

                              ------------------------------------------------------------------------------------------------

                              G1R      m         201.508  Mean     33.208   27.994   25.242    19.736  106.180   52.70

                                                     8.353  S.D.      4.994    3.605    3.056    4.598    8.281    3.70

                                                     5      N          5        5         5          5         5         5  

                              -----   ---        -------  ----    -------  -------  -------  -------  -------  ------

                              G4R      m         200.506  Mean     25.104   22.172   20.432    19.954   87.662   43.82

                                                     6.255  S.D.      7.924    5.335    4.845    3.455   11.712    6.60

                                                      5      N          5         5         5         5        5         5  

---------------------------------------------------------------------------------------------------------------------------------------------------------------

Statistics Test: 1 Way Analysis of Variance       * - Significance different from vehicle control group at 5% level

Abs Gain = absolute bodyweightgainbetween base period and end of the analysis period                                                                        

% Gain = percentage bodyweightgainbetween base period and end of the analysis period                                                                        

Group G1 - 0 mg/kg/day      Group G2 - 110 mg/kg/day     Group G3 - 330 mg/kg/day     Group G4 - 1000 mg/kg/day                                                                              

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day                                                                                                                   

 

 


 

 

TABLE 3 contd. Summary of Net Body Weight Gain (g) – Males

 

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                      Day numbers relative to Start Date                                                      

                                                              Base                                 Abs        %  

                                                              Weight                                Gain     Gain 

                                                              Day     From:     28       35       28       28 

                                         Group   Sex         28       To:      35       42        42       42  

                                        ----------------------------------------------------------------------------

                                        G1R      m         307.688  Mean     20.172   12.994   33.166   10.77

                                                              14.206  S.D.      7.526    1.211    7.234    2.22

                                                               5      N         5        5        5        5  

                                        -----   ---        -------  ----    -------  -------  -------  ------

                                        G4R      m         288.168  Mean     25.212   20.576   45.788   15.84

                                                              10.704  S.D.      3.742    4.605    7.194    2.04

                                                               5      N         5        5        5         5  

---------------------------------------------------------------------------------------------------------------------------------------------------------

Statistics Test: 1 Way Analysis of Variance

Abs Gain = absolute bodyweightgainbetween base period and end of the analysis period                                                                        

% Gain = percentage bodyweightgainbetween base period and end of the analysis period                                                                         

                                                                               

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day                                                                                                                   

 

 

 


 

TABLE 4.            Summary of Net Body Weight Gain (g) – Females

---------------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                      

                                                Base                                                              Abs        %  

                                               Weight                                                            Gain     Gain 

                                                Day     From:      1           8         15         22          1        1 

                          Group   Sex         1        To:       8         15         22         28          28       28  

                          --------------------------------------------------------------------------------------------------------

                          G1       f         155.450  Mean     14.662     12.412      7.426      6.994     41.494   26.70

                                                 3.854  S.D.      3.997      4.424      2.933      7.502      6.851    4.35

                                                 5      N         5          5          5          5          5        5  

                          -----   ---        -------  ----    ---------  -------    -------    -------    -------  ------

                          G2       f         154.474  Mean     11.214     15.202     13.972      3.490     43.878   28.55

                                                  6.835  S.D.      3.126      7.912     14.143      8.906      9.088    6.80

                                                 5      N         5          5          5          5          5        5  

                          -----   ---        -------  ----    ---------  -------    -------    -------    -------  ------

                          G3       f         153.952  Mean     10.428     14.466      7.484      2.580     34.958   22.61

                                                  6.607  S.D.      3.529      5.463      3.979      4.069      6.505    3.38

                                                 5      N         5          5          5          5          5        5  

                           -----   ---        -------  ----    ---------  -------    -------    -------    -------  ------

                          G4       f         155.222  Mean     16.334     11.782      7.994      7.104     43.214   27.83

                                                  1.678  S.D.      4.887      4.458      4.026      3.711      5.540    3.42

                                                 5      N         5          5          5          5          5        5  

---------------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                      

                                                    Base                                                     Abs        %  

                                                   Weight                                                     Gain     Gain 

                                                    Day     From:      1        8       15       22        1        1 

                              Group   Sex         1        To:        8       15       22       28      28       28  

                              ------------------------------------------------------------------------------------------------

                              G1R      f         155.556  Mean      15.908   13.096   10.534    7.804   47.342   30.28

                                                     6.522  S.D.      6.268    7.058    4.437    3.485   15.848    9.49

                                                     5      N          5        5        5        5        5        5  

                              -----   ---        -------  ----    -------  -------  -------  -------  -------  ------

                              G4R      f         150.540  Mean      13.984   10.166    9.124    6.216   39.490   26.32

                                                     6.172  S.D.      3.896    3.302    4.946    3.535    7.190    5.27

                                                     5      N          5        5        5        5        5        5  

---------------------------------------------------------------------------------------------------------------------------------------------------------------

Statistics Test: 1 Way Analysis of Variance:

Abs Gain = absolute bodyweightgainbetween base period and end of the analysis period                                                                        

% Gain = percentage bodyweightgainbetween base period and end of the analysis period                                                                        

Group G1 - 0 mg/kg/day      Group G2 - 110 mg/kg/day     Group G3 - 330 mg/kg/day     Group G4 - 1000 mg/kg/day

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day                                                                                                                    

 


 

TABLE 4 contd. Summary of Net Body Weight Gain (g) – Females

 

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                     Day numbers relative to Start Date                                                       

                                                              Base                                 Abs        %  

                                                             Weight                                Gain     Gain 

                                                               Day     From:     28       35       28       28 

                                        Group   Sex         28       To:      35       42        42       42  

                                         ----------------------------------------------------------------------------

                                                                                                                    

                                        G1R      f         202.898  Mean      8.742    4.312   13.054    6.38

                                                              20.355  S.D.      3.135    3.403    2.980    0.84

                                                               5      N         5        5        5        5  

                                        -----   ---        -------  ----    -------  -------  -------  ------

                                        G4R      f         190.030  Mean     10.458    3.646   14.104    7.47

                                                               7.861  S.D.      1.550    2.547    3.008    1.78

                                                               5      N         5        5        5        5  

---------------------------------------------------------------------------------------------------------------------------------------------------------

Statistics Test: 1 Way Analysis of Variance

Abs Gain = absolute bodyweightgainbetween base period and end of the analysis period                                                                        

% Gain = percentage bodyweightgainbetween base period and end of the analysis period                                                                         

                                                                               

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day                                                                                                         

                                                                             


 

 

TABLE 5.            Summary ofCagewiseAverage Food Consumption (g/rat/day) – Males

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                        Day numbers relative to Start Date

                                                             From:      1         8        15        22  

                                                Group Sex    To:      8        15        22        28  

                                                -------------------------------------------------------------

                                                G1    m   Mean   20.707    20.956    22.310    21.743 

                                                            S.D.    0.758     0.924     1.252     0.887 

                                                             N        3         3         3         3    

                                                                    ------    ------    ------    ------ 

                                                 G2    m   Mean   18.538*   18.883    18.961*   19.781*

                                                            S.D.    0.208     0.481     0.947     0.601 

                                                            N        3          3         3         3    

                                                                    ------    ------    ------    ------ 

                                                G3    m   Mean   19.825    19.807    19.372*   19.981 

                                                            S.D.    1.222     1.469     1.273     0.811 

                                                            N        3         3         3         3    

                                                                     ------    ------    ------    ------ 

                                                G4    m   Mean   17.488*   18.692    18.706*   19.341*

                                                            S.D.     0.280     1.286     1.095     0.932 

                                                            N        3         3         3         3    

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                             Day numbers relative to Start Date

                                                    From:      1        8       15       22       28       35 

                                        Group Sex    To:      8       15       22       28       35       42 

                                        -----------------------------------------------------------------------------

                                         G1R   m   Mean   20.989   22.600   22.583   20.397   18.662   22.176

                                                    S.D.    1.001    1.641    1.175    4.614    2.718    1.454

                                                     N        3        3        3        3        3        3   

                                                            ------   ------   ------   ------   ------   ------

                                         G4R   m   Mean   18.679   19.564   19.275*  20.466   22.579   23.012

                                                    S.D.    1.728    1.316    1.535    1.008    0.277    1.096

                                                    N         3        3        3        3        3        3   

---------------------------------------------------------------------------------------------------------------------------------------------------------

Statistics Test: 1 Way Analysis of Variance: * - Significance different from vehicle control / vehicle control recovery group at 5% level

Group G1 - 0 mg/kg/day      Group G2 - 110 mg/kg/day     Group G3 - 330 mg/kg/day     Group G4 - 1000 mg/kg/day                                                                              

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day                                                                                                                   

N = No. of cages

 


 

TABLE 6.            Summary ofCagewiseAverage Food Consumption (g/rat/day) – Females

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                        Day numbers relative to Start Date

                                                            From:      1         8        15        22  

                                                Group Sex    To:      8        15        22        28  

                                                -------------------------------------------------------------

                                                G1    f   Mean   14.199    14.791    14.820    15.104 

                                                            S.D.    0.850     1.167     1.216     1.158 

                                                            N        3         3         3         3    

                                                                     ------    ------    ------    ------ 

                                                G2    f   Mean   13.587    13.868    14.479    13.964 

                                                             S.D.    0.856     2.261     1.168     1.017 

                                                            N        3         3         3         3    

                                                                    ------    ------    ------    ------ 

                                                G3    f   Mean   13.892    15.185    14.047    14.141 

                                                            S.D.    1.518     1.704     1.123     1.629 

                                                             N        3         3         3         3    

                                                                    ------    ------    ------    ------ 

                                                 G4    f   Mean   14.040    14.554    14.074    14.665 

                                                            S.D.    0.495     0.778     1.384     0.496 

                                                            N        3         3         3         3    

---------------------------------------------------------------------------------------------------------------------------------------------------------

                                                                           Day numbers relative to Start Date

                                                    From:      1        8       15       22       28       35 

                                        Group Sex    To:      8       15       22        28       35       42 

                                        -----------------------------------------------------------------------------

                                        G1R   f   Mean   14.656   15.436   15.960   15.780   16.081   16.017

                                                    S.D.    0.629    1.818    1.925    1.865    2.066    1.125

                                                    N        3        3        3        3        3        3   

                                                             ------   ------   ------   ------   ------   ------

                                        G4R   f   Mean   12.802*  13.159   13.471   13.890   15.298   16.024

                                                     S.D.    0.338    0.445    0.240    0.559    0.885    1.270

                                                    N        3        3        3        3        3        3   

---------------------------------------------------------------------------------------------------------------------------------------------------------

Group G1R - 0 mg/kg/day    Group G4R - 1000 mg/kg/day       * - Significance different from vehicle control recovery group at 5% level                                                                                                            

Group G1 - 0 mg/kg/day      Group G2 - 110 mg/kg/day     Group G3 - 330 mg/kg/day     Group G4 - 1000 mg/kg/day                                                                              

N = No. of cages

 

Applicant's summary and conclusion

Conclusions:
To summarise, the results of the study indicate that the oral administration of test item for 28 days in Wistar rats at dose level of 110 mg/kg/day dose did not cause any toxicological effect on general health, body weights, food consumption, haematology, clinical chemistry, coagulation parameters, organ weights and gross pathology at all the tested doses in both sexes. Bluish discolouration of the skin (slight to moderate), bluish coloured faeces (light to dark), discolouration of urine, grossly observed blue discolouration of stomach contents and intestinal contents were attributed to physical nature of the test item.
At 330 mg/kg/day dose, the treatment did not cause any toxicological effect on general health, body weights, food consumption, clinical chemistry, coagulation parameters and terminal fasting body weight. Bluish discolouration of the skin (slight to moderate), bluish colour faeces (light to dark), discolouration of urine, grossly observed blue discolouration of stomach contents and intestinal contents were attributed to the physical nature of the test item.

The treatment resulted in an increase in liver weight in males and females associated with hepatocellular hypertrophy, which is considered as an adaptive response to the test item administration and hence, a test item related non-adverse effect. Grossly blue discolouration of liver lobes, stomach and intestinal contents was attributed to the colour of the test item.

At 1000 mg/kg bwt/day, the treatment decreased the body weight, net body weight gain and food consumption in males. Methemoglobin concentration was higher in females associated with increase in reticulocyte count. Treatment resulted in increased reticulocyte count in males and females associated with increased extramedullary hematopoiesis in spleen. Bluish discolouration of the skin (slight to moderate), bluish colour faeces (light to dark) and bluish urine discolouration was present both in males and females were attributed to the physical nature of test item and considered as test item-related non-adverse change. The treatment resulted in increased in liver weight in males and females associated with hepatocellular hypertrophy. This was considered adaptive response to the test item administration and hence, considered test item related non-adverse effect.

Grossly blue discolouration of liver surface, stomach mucosa, contents in stomach and intestinal segments were attributed to the colour of the test item. In kidneys, cortical tubules showed the presence of the droplets in cytoplasm and in lumen in males. This change was considered a test item related non-adverse effect as the there were no associated changes in biochemical parameters. All these changes had reversed at the end of recovery period except for the increased reticulocyte count and increased extramedullary hematopoiesis of spleen in males. The gross finding of liver discolouration was also present at the end of recovery period indicating retention of the test material in the liver for more than 14 days at this dose.
Considering the changes in body weight and food consumption in males, higher methemoglobin concentration in females, increased reticulocyte count associated with extramedullary hematopoiesis in spleen at 1000 mg/kg Bwt/day dose group, the evaluated No Observed Adverse Effect Level (NOAEL) is considered to be 330 mg/kg/day following oral gavage administration for 28 consecutive days to wistar rats under the test conditions and doses employed.
Executive summary:

The purpose of this repeated dose toxicity study was to evaluate the systemic toxicity profile of the test item,in wistar rats when administered orally by gavage for 28 consecutive daysand to assess the reversibilityof any effects during a subsequent 14days recovery period. This study was also intended to provide the information on major toxic effects, target organs and an estimation of a No Observed Adverse Effect Level (NOAEL).

The test item was weighed and dissolved in vehiclei.e.,0.5% Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q®Water and administered to rats at the graduated dose levels of 110, 330, and 1000 mg/kg/day for low dose (G2), mid dose (G3) and high dose (G4 ) / high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1)/ vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 10 mL/kg body weight. Each group in the experiment was comprised of five male and five female rats.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not done at the test facility. The stability of test item in the vehicle was carried out separately under Advinus Study No. G12742 at 1 and 250 mg/mL concentrations. Based on the results, the test item was found to be stable for up to 4 days when stored at room temperature.The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during Week 4 (Day 26) of the treatment period. The results indicated that the analysed concentrations were within ± 15% of variations from the theoretical concentrations. 

Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and and at the end of recovery period for recovery groups. The body weights and food consumption was measured during in-life phase of the experiment. Neurological examinations were conducted towards the end of treatment for main groups and towards the end of recovery period for recovery groups.The clinical laboratory investigations such as haematology, coagulation, clinical chemistry and urine analysis were performed at termination.

All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratio were derived as percent fasting body weights. Histopathology examination was carried out on the preserved organs of the vehicle control (G1) and high dose group animals (G4). In addition, all tissues with gross lesions were examined microscopically. Liver and spleen in males and females and kidneys in males were examined from the lower dose and recovery groups as higher incidence of microscopic lesions were noted in these organs at high dose.

Salient findings are provided below:

· Clinical Signs, Mortalities and Ophthalmological Examination:Clinical Signs and Mortality:Bluish discolouration of the skin (slight to moderate) and bluish colour faeces (light to dark) were observed at all the tested doses in both sexes. This could be due to physical nature of the test item. There was no mortality observed at any of the doses tested in both sexes. Ophthalmological examination did not reveal any ocular abnormalities.

· Neurological Findings:No treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested.

· Body Weights:The treatment decreased the body weights and net body weight gains at 1000 mg/kg/day in males.

· Food Consumption:The treatment decreased the food consumption at 1000 mg/kg/day in males.

· Haematology:Treatment resulted in increase in reticulocyte count at 1000 mg/kg/day in both males and females (33% and 68% respectively) associated with the microscopic finding of increased extramedullary hematopoiesis in spleen and was reversible only in females.

· Methemoglobin:In females, the higher methemoglobin concentration at 1000 mg/kg dose groups was associated with a minimal increase in reticulocyte count at 1000 mg/kg/day.

· Coagulation and Clinical chemistry:No test item-related changes were observed in the coagulation, clinical chemistry and urine parameters.

·  Urine Parameters:Discolouration of urine (red, orange, brown and blue ≥ 110 mg/kg/day males, ≥ 330 mg/kg/day females) was related to test item administration attributed to the physical nature of the test item.

· Terminal fasting body weight, organ weights and organ weight ratios:The terminal fasting body weight was lower in males. An increase in liver weight was observed in ≥ 330 mg/kg/day males and females associated with hepatocellular hypertrophy and considered as an adaptive response to the test item administration.

· Gross and Histopathology:At 110 mg/kg/day, grossly observed blue discolouration of stomach and intestinal contents were attributed to the colour of the test item.

At 330 mg/kg/day dose, grossly blue discolouration of stomach, liver lobes and intestinal contents were attributed to the physical nature of the test item. Microscopically, hepatocellular hypertrophy of liver was observed in males and females and considered adaptive response to the test item administration and non-adverse effect.

At 1000 mg/kg bwt/day, grossly blue discolouration of liver surface, stomach mucosa, stomach content and intestinal segments were attributed to the colour of the test item. Grossly blue discolouration of liver surface, stomach mucosa, contents in stomach and intestinal segments were attributed to the colour of the test item. Microscopically, hepatocellular hypertrophy of liver was observed in males and females. In the absence of liver enzyme profile changes, this change was considered as an adaptive response to test item administration and considered test item related non-adverse effect. In kidneys, cortical tubules showed the presence of the droplets in cytoplasm and in lumen in males. This change was considered test item related non-adverse effect as the there were associated changes in biochemical parameters. In spleen, increased extramedullary hematopoiesis in both males and females was related to test item administration. All these changes were reversible except for the increased reticulocyte count and increased extramedullary hematopoiesis in spleen in males which change on a longer time scale. The gross finding of liver discolouration was also present at the end of recovery period indicating retention of the test material for more than 14 days at this dose. Considering the changes in body weight and food consumption in males, higher methemoglobin concentration in females, increased reticulocyte count associated with extramedullary hematopoiesis in spleen at 1000 mg/kg Bwt/day dose group, the evaluated No Observed Adverse Effect Level (NOAEL) is considered to be 330 mg/kg/day following oral gavage administration for 28 consecutive days to wistar rats under the test conditions and doses employed.