Registration Dossier
Registration Dossier
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EC number: 807-560-2 | CAS number: 123944-63-8
Repeated dose toxicity: oral
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:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- 1,4-bis({3-[2-(2-hydroxyethoxy)ethoxy]propyl}amino)-9,10-dihydroanthracene-9,10-dione
- EC Number:
- 807-560-2
- Cas Number:
- 123944-63-8
- Molecular formula:
- C28H38N2O8
- IUPAC Name:
- 1,4-bis({3-[2-(2-hydroxyethoxy)ethoxy]propyl}amino)-9,10-dihydroanthracene-9,10-dione
- Test material form:
- solid: particulate/powder
Constituent 1
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 or test system 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:
- behaviour (functional findings)
- body weight and weight gain
- clinical biochemistry
- clinical signs
- gross pathology
- haematology
- histopathology: non-neoplastic
- mortality
- ophthalmological examination
- organ weights and organ / body weight ratios
- urinalysis
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.
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