Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate
EC Number:
265-449-9
EC Name:
[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate
Cas Number:
65113-55-5
Molecular formula:
C25H30N3.C18H14N3O3S
IUPAC Name:
[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, USA and bred at IIBAT animal house facility
- Age at study initiation: Between 12 and 14 weeks. Females were virgin
- Weight at study initiation: Males: 328 g to 363 g; Females: 222 g to 251 g.
- Fasting period before study: none
- Housing: Males were housed in groups in cages, each cage containing 5 animals during pre-mating and post-mating period. Females were housed in groups of 5 animals per cage during pre-mating. 1 male and 1 female was kept together in a cage until pregnancy occurs. Pregnant females were caged individually. Animals from reversal group were housed group wise and sex wise with 5 animals per cage.
- Diet: Ad libitum - Standard gamma irradiated pellet food.
- Water: Ad libitum - Reverse osmosis water
- Acclimation period: five days in the test room
- Sanitation:
Range finding study: Bedding material, cages, grills were changes once every 3 day and water bottles were changed daily.
Main study: Bedding material, cages, grills and water bottles were changed daily
Cages, grills and water bottles were autoclaved.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 °C to 22.8 °C. Recorded once daily
- Humidity (%): 56% - 64%. Recorded once daily
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light


IN-LIFE DATES:
- (P) Males groups 1 to 5: From July 31, 2012 to August 28, 2012
- (P) Females groups 1 to 5: From July 31, 2012 to September 09 - September 14, 2012
- Males and females groups 6 and 7: From July 31, 2012 to September 22, 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: prepared freshly daily by mixing with corn oil and magnetically stirred to obtain a homogenous suspension just before the administration.

VEHICLE
- Justification for use and choice of vehicle : non toxic vehicle which allows an homogenous solution/suspension.
- Amount of vehicle (if gavage): dose volume maintained at 10 mL/kg b.w.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
(P) Males groups 1 to 5 = 28 days -> 15 days before mating, continously during mating (1 to 5 days) and after mating until the dosing period of 28 days.
(P) Females groups 1 to 5 = From 41 to 44 days -> 15 days before mating, continously during mating (1 to 5 days), during pregnancy (21 to 23 days) and 3 days post partum.

Males and Females from reversal groups (groups 6 and 7): 41 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 and Group 6 (reversal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
Group 4
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 5 and Group 7 (reversal)
No. of animals per sex per dose:
(P) Males from groups 1 to 5: 10/group
(P) Females from groups 1 to 5: 10/group
Males and females from the reversal groups (groups 6 and 7): 5/sex/groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the results of the range finding study carried out
- Rationale for animal assignment: Randomization
- Rationale for selecting satellite groups: the control and the highest dose of the main study were selected for the satellite groups
- Post-exposure recovery period in satellite groups: 14 days

- Range finding study: 4 groups were selected: one control (G1) and 3 dose groups with 3 males and 3 females of each 100 (G2), 300 (G3) and 1000 (G4) mg/kg b.w. of the test substance, daily administered orally for 7 days and observed for morbidity/mortality and signs of toxicity daily. Control group animals were treated similarly, but with corn oil alone.

One male, two females from G4 and one female from G3 was died.
Bluish perianal staining was observed in all animals of all the test substance treated group from day 1 to 7.
Dullness was observed in one male and two females from G3 and all animals of G4.
Piloerection was noted in one male and one female of G3 and two male and female of G4.
Abdominal distension was observed in one female of G3 as well as one male and all females of G4.
Respiratory distress was recorded in one male of G4.
At macroscopic examination Enlarged payers’s patches were observed in all animals of test substance treated groups.
Gross lesions observed in G3 and G4 animals were distended stomach with bluish discoloration of mucosal surface, small intestine and large intestine with bluish discoloration of mucosal surface and seminal vesicles small in size.
Adrenals were enlarged in all animals
Thymus small in size in two male and one female of G4 group.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily general observations for all animals, to record any sign of toxicity.
- Parameters eximaned: changes in skin, fur, eyes, and mucous membranes, occurrence of secretions, excretions (coloration and intensity of faeces and/or urine) and autonomic activity (e.g. lacrimation, piloerection, palpebral closure, palpebral reflex, pupil light response, and unusual respiratory pattern). Changes in gait, mobility, arousal, rearing, posture, vocalizations, activity levels and response to handling, approach response, touch response, click response, tail pinch response, toe pinch as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling), bizarre behavior (e.g. self-mutilation, walking backwards), were also recorded. Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality, were also recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
* (P) Males groups 1 to 5: On pre-mating days 0, 7, mating days 0, 7 and 13.
* (P) Females groups 1 to 5: On pre-mating days 0, 7, mating day 0, pregancy days 0, 7, 14, 20 and on post partym day 4.
* Males and females of the reversal groups: On days 0, 7, 14, 21, 28, 35, 42, 49 and on day 53 (at termination).

BODY WEIGHT: Yes
- Time schedule for examinations:
* (P) Males groups 1 to 5: Prior administration, on pre-mating days 0, 7, mating days 0, 7 and at termination
* (P) Females groups 1 to 5: Prior administration, on pre-mating days 0, 7, mating day 0, Pregnancy days 0, 7, 14, 20, within 24 hours of parturition and on post partum day 4
* Males and Females of the reversal groups: Prior administration, on days 7, 14, 21, 28, 35, 42, 49 and on day 53 (at termination).

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on mating day 0 (i.e. after 15 days of exposure) for all of the 7 groups and on day of scheduled kill of animals for reserval groups 6 and 7.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: on 5 males and 5 females of each of the 7 groups (randomly selected for groups 1 to 5).
- Parameters examined: Erythrocyte (RBC) count, hemoglobin (Hb) concentration, hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), platelet count, Total leucocyte (WBC) count, and differential leucocyte count using Bayer ADVIA 120 fully automated hematology analyzer. Clotting time was evaluated by capillary method.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on mating day 0 (i.e. after 15 days of exposure) for all of the 7 groups and on day of scheduled kill of animals for reserval groups 6 and 7.
- Animals fasted: Yes (overnight)
- How many animals: on 5 males and 5 females of each of the 7 groups (randomly selected for groups 1 to 5).
- Parameters examined: Plasma separated from blood was investigated for glucose, urea, blood urea nitrogen (BUN), creatinine, total cholesterol, triglycerides, albumin, total protein, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), calcium and phosphorus using Dimension plus fully automated biochemistry analyzer. Sodium and potassium were analyzed by Human Humalyte electrolyte analyzer

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
* (P) Males from groups 1 to 5 (on five animals randomly selected) = on mating day 13.
* (P) Females from groups 1 to 5 (on five animals randomly selected) = on post partum day 3.
* Males and Females of the reversal groups 6 and 7 = on a day before terminal blood collection.
- Dose groups that were examined: All
- Battery of functions tested: auditory fonction (Colombus Instrument, USA), grip strength, locomotor activity (Ugo Basile, Italy).

OTHER: see the same field in chapter 7.8.1 "Toxicity to reproduction"
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Macroscopical examination for any abnormalities with special attention to reproductive system's organs. All gross pathological changes were recorded individually for each animal.
- Record of the number of implantation sites and corpora lutea.
- Tissues preserved in 10 % neutral buffer formalin for histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including Peyer’s patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs, uterus, urinary bladder, mesenteric lymph nodes, mandibular lymph nodes, peripheral nerve (sciatic), and a section of bone marrow (sternum).

HISTOPATHOLOGY: Yes
- Dose groups that were examined: groups 1 and 5 (control and 100 mg/kg b.w./day group)
- How many animales: 5 animals per groups, except for histological examination performed on ovaries, testes and epididymis of animals for which all the animals were examined.
- Parameters examined: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including Peyer’s patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea, lungs, uterus, urinary bladder, mesenteric lymph nodes, mandibular lymph nodes, peripheral nerve (sciatic), and a section of bone marrow (sternum). Ovaries, Testes and Epididymis (With special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).
Other examinations:
ORGAN WEIGHT
- Weights of following organs of all male animals were recorded: 1. Testes 2. Epididymis
- Weights of following organs for randomly selected five males and five females of group G1, G2, G3, G4 and all animals from groups G5 and G6: Liver, Kidneys, Adrenals, Thymus, Spleen, Brain and Heart.
Statistics:
Examined parameters were checked for normality:
- normal data was subject to one-way ANOVA
- Non-normal data was subjected to Krustal-Wallis One-Way ANONA on ranks
Student's Newman-Keul's Test was employed for post ANOVA comparison

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of G4 (75 mg/kg/day), G5 (100 mg/kg/day) & G7 (100 mg/kg/day) exhibited dullness and piloerection at various intervals. Mortability observed in the highest dose groups (100 mg/kg/day): one dead male in G5, one dead male in G7 and one dead female in G5. They could be considered as treatment related. Abdominal distension and respiratory distress were observed in those dead animals.
Mortality:
mortality observed, treatment-related
Description (incidence):
Animals of G4 (75 mg/kg/day), G5 (100 mg/kg/day) & G7 (100 mg/kg/day) exhibited dullness and piloerection at various intervals. Mortability observed in the highest dose groups (100 mg/kg/day): one dead male in G5, one dead male in G7 and one dead female in G5. They could be considered as treatment related. Abdominal distension and respiratory distress were observed in those dead animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistical significant decrease in the body weight in males (in G7 from day 14 till the sacrifice and in G4 and G5 on day 28) and in female (in G5 on premating day 14 and gestation day 0) as compared to their respective control.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Significant changes were observed in different groups at various time points. This effect was not dose dependent and these values were well within the historical data of the laboratory. The changes were not considered as treatment related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decrease in grip strength and locomotor activity of G4, G5 and G7 groups as compare to their respective control groups.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological findings observed were either related to agonal or spontaneous, or were indicental and of the type routinely observed in Wistar rats of this stage.
Histopathological findings: neoplastic:
not specified
Details on results:
G1: control
G2: low dose - 25 mg/kg b.w
G3: low intermediate dose - 50 mg/kg b.w
G4: high intermediate dose - 75 mg/kg b.w
G5: high dose - 100 mg/kg b.w
G6: control reversal
G7: high reversal - 100 mg/kg b.w

CLINICAL SIGNS AND MORTALITY:
Mortality: No morbidity/mortality was observed in G1, G2, G3, G4 and G6 animals.
Death of two males: one from G5 on day 21st and one from G7 on day 16th.
Death of one female from G5 on day 27th.

No clinical signs of toxicity were observed in males and females of G1, G2 and G6 group animals.

The bluish perianal staining in animals of G3, G4, G5 & G7 groups was considered due to the color of test substance and this effect was disappeared in G7 group animals on 46th day of observation period in all males or at 49th day of observation in all females after withdrawal of treatment.
Further there were no other relevant changes observed to relate this finding as significant. Hence this effect was not considered as adverse effect.

However, animals belong to G4, G5 & G7 groups exhibited dullness and piloerection at various intervals. These signs were recovered after withdrawal of treatment. In addition, abdominal distension and respiratory distress were observed in dead animals of G5 and G7 (attached Table no. 13 in background material).

Observed weekly individual clinical signs like home cage, handling and standard arena (open field) were similar to daily observations and did not showed any significance (attached Tables no. 6 and 14 in background material).



BODY WEIGHT AND FOOD CONSUMPTION
Observed body weight in G2 and G3 was comparable with respective control group (G1) throughout the observation period.
However, G4, G5 and G7 dose groups showed a decreasing trend from day 14 with respect to their controls (G1 & G6) and continued up to sacrifice in males or until postpartum day4 in females. This decrease was statistically different in high dose reversal from day 14 to till the end, and on day 28 in high intermediate and high dose groups.

Statistical significant decrease was observed in G3 females on day 0 of the gestation with respect to the control (G1) group, this effect was not consistent and was observed only on that day, hence not considered as treatment related. (attached tables no. 1, 2, 3, 15, 16, 17in background material).

Food consumption recorded in treated groups was comparable with respective control groups and there were no significant difference. However, the statistically significant decrease in G2, G3 & G5 females on few days during gestation and postpartum was observed. These changes were not considered as treatment related, since there was no dose dependency and consistency (attached tables 4, 5, 18, 19 in background material).

HAEMATOLOGY
There were no statistical significant changes observed on mating day 0 in females across all the treated groups with respect to their concurrent control groups. However, in males on mating day 0, statistically significant changes were observed in hematology parameters like increase in MCV in G7, MCHC in G3 and decrease in PLT of G5, increase in neutrophils and decrease in leukocytes in G4 group animals when compared to their concurrent controls.

On day 53 in the reversal group there were no statistical significant changes in males, but the hemoglobin and lymphocytes were found to be significantly decreased in females when compared to concurrent control (attached tables 7 and 20 in background material).


CLINICAL CHEMISTRY
No statistically significant changes were observed in biochemistry parameters across the groups in both the time points (attached tables 8, 21 in background material)

NEUROBEHAVIOUR
There was no statistically significant difference recorded in the FOB parameters, grip strength & locomotor activity, of G2 & G3 group animals with respect to their control group G1.
However, in G4, G5 & G7 groups a significant difference was observed in grip strength (both fore limb & hind limb) and locomotor activity (both horizontal and vertical), when compared to respective control groups. This was indicated by observing a statistical significant difference in the fore limb grip strength, horizontal and vertical locomotor activity of G4 and G5 males, in the hind limb grip strength of G5 males as compare to G1.
Though there were no statistical difference in females, there was a decrease in the parameters of G5 group when compared to concurrent control group (attached tables 12 and 25 in background material).

ORGAN WEIGHTS
No treatment related organ weight changes were observed in any of the groups when compared to concurrent control group. The observed statistical significant changes in absolute and relative weights of adrenals in G2 group males and absolute weight of thymus in G4 group females when compared to concurrent control group (G6) was not considered as treatment related, since there was no correlation with either macro or microscopic observations (attached tables 26, 27, 31 and 32 in background material).

GROSS PATHOLOGY
No test substance related gross pathological findings were observed.

Test substance related bluish discoloration at perianal area was recorded in in G3, G4 and G5 group. However, this was considered due to color of test substance which was excreted through feces.
There was no correlating macroscopic or histopathology findings of alimentary tract. Hence, it was not considered to be adverse effect
The macroscopic findings observed were either related to physiological, agonal to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age (attached tables 29 and 33 in background material).

HISTOPATHOLOGY (PARENTAL ANIMALS)
No test substance related histopathological findings were observed.

The histopathological findings observed were either related to, agonal to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age (attached tables 30 and 33 in background material).

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Content of the tables are attached in the field "Attached background material".

Table 1 - Summary of Weekly Body Weight in Male (g)

Table 2 - Summary of Weekly Body Weight in Female (Premating) (g)

Table 3 - Summary of Weekly Body Weight in Female (Gestation & Postpartum) (g)

Table 4 - Summary of Daily Feed Consumption (Premating) (g)

Table 5 - Summary of Daily Feed Consumption in Females (Gestation & Postpartum) (g)

Table 6 - Summary of Detail Signs of Toxicity - Home Cage, Handling & Open Field

Table 7 - Summary of Hematology Parameters

Table 8 - Summary of Biochemistry Parameters

Table 12 - Summary of Functional Observational Battery (FOB) Parameters

Table 13 - Daily Individual Signs of Toxicity

Table 14 - Individual Detail Signs of toxicity- Home cage, Handling, open field & Physiological

Table 15 - Weekly Individual Body Weight in Male (g)

Table 16 - Weekly Individual Body Weight in Female (Premating) (g)

Table 17 - Weekly Individual Body Weight in Female (Gestation & Postpartum) (g)

Table 18 - Daily Cage wise feed consumption (Premating) (g) 

Table 19 - Daily Individual Feed Consumption in Females (Gestation & Postpartum) (g)

Table 20 - Individual Hematology Parameters

Table 21 - Individual Biochemistry Parameters

Table 25 - Individual Functional Observational Battery (FOB) Parameters

Table 26 - Summary of Absolute organ weight (g)

Table 27 - Summary of Relative organ weight (%)

Table 29 - Summary of Gross pathology

Table 30 - Summary of Histopathology

Table 31 - Individual Animal absolute organ weight (g)

Table 32 - Individual Animal relative organ weight (%)

Table 33 - Gross and histopathology findings of individual rats

Applicant's summary and conclusion

Conclusions:
On the basis of the results obtained in the present study, the No Observed Adverse Effect Level (NOAEL) of the tested substance for Repeated Dose Toxicity study is considered as 50 mg/kg b.w.
Executive summary:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats was performed to evaluate the test item

One hundred and twenty rats were randomized into seven groups, five main groups containing 10 rats/group/sexviz.,G1 (Control), G2, G3, G4, G5 (treated groups) and two reversal groups containing 5 rats/group/sexviz.,G6 (control reversal) and G7 (High reversal).

The test substance was suspended in corn oil and administered by gavage daily at the doses of 25, 50, 75 and 100 mg/kg b.w. to the rats belonging to G2 (low), G3 (Low intermediate), G4 (High intermediate), G5 (High) and G7 (High reversal) groups respectively. In males dosing was carried out up to 28 days, where as in females test substance was administered during premating, mating, gestation and up to day 3 post partum.

Animals from reversal group were dosed up to first sacrifice of dams and kept untreated for 14 days to evaluate the reversibility of effects after withdrawal of the treatment. Control and control reversal groups were treated similarly but with corn oil alone. The dose volume was maintained at 10 ml/kg b.w. in all the groups.

Males were sacrificed after completion of four weeks of dosing (Day 28th) and females were sacrificed on day 4 post partum. Rats in reversal groups were sacrificed after 14 days of additional observation period.

Observed body weight in low (G2) and low intermediate (G3) dose groups was comparable with respective control group (G1) throughout the observation period. However, high intermediate (G4), high (G5) and high reversal (G7) dose groups showed a decreasing trend from day 14 with respect to their controls (G1 & G6). This decrease was statistically different in high dose reversal (G7) from day 14 to till the end, and on day 28 in high intermediate and high dose groups.

Food consumption recorded in treated groups was comparable with respective control groups and there was no significant difference. However, the statistically significant decrease in G2, G3 & G5 females on few days was not considered as treatment related, since there was no dose dependency and consistency.

Mortality/morbidity was not observed in low (G2), low intermediate (G3), high intermediate (G4) group of animals. However, the death of one animal each in groups of high (G5), high reversal (G7) males and one female in high (G5) dose groups could be considered as treatment related.

No clinical signs of toxicity were observed in males and females of G1, G2 and G6. The bluish perianal staining in animals of G3, G4, G5 & G7 groups was considered due to the color of test substance, further there were no other relevant changes observed to relate this finding as significant. Hence this effect was not considered as adverse effect. However, animals belonging to G4, G5 and G7 groups exhibited dullness and piloerection at various interval. In addition, abdominal distension and respiratory distress were observed in dead animals of G5 and G7.

Observed weekly individual clinical signs like home cage, handling and standard arena (open field) were similar to daily observations and did not showed any significance.

There was no statistically significant difference recorded in the FOB parameters, grip strength & locomotor activity, of G2 & G3 group animals with respect to their control group. However, in G4, G5 & G7 groups a significant difference was observed in grip strength (both fore limb & hind limb) and locomotor activity (both horizontal and vertical), when compared to respective control groups.

Blood was collected at the end of pre mating period from randomly selected five males and females of main groups and all animals from reversal groups; and on day of scheduled kill of animals from reversal groups, from orbital sinus in heparinized vials (for biochemistry) and in vials containing EDTA (for hematology).

There was no treatment related hematological or biochemical changes observed in any of the treated groups. However, the statistically significant changes of males in various hematological parameters, like MCV in G7, MCHC in G3, PLT in G5, neutrophils & leukocytes in G4 on mating day 0 and Hb, lymphocytes of reversal group (G7) females on day 56 with respect to concurrent controls were not observed in a dose dependant manner. Further, these values were well within the historical data of IIBAT. Hence these changes were not considered as treatment related.

Complete gross pathology was conducted on all adult animals and examined macroscopically for any abnormalities or pathological changes.

No treatment related gross pathological findings were observed. Test substance related bluish discoloration at perianal area was recorded in G3, G4 and G5 group. This was considered due to color of test substance which was excreted through feces and there was no correlating macroscopic or histopathology findings of alimentary tract. Hence, it was not considered as an adverse effect. The macroscopic findings observed were either related to physiological, agonal to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age.

Organ weight of testes and epididymis was recorded from all animals while weight of Liver, Kidneys, Adrenals, Thymus, Spleen, Brain and Heart was recorded in randomly selected five males and five females.

No treatment related organ weight changes were observed in any of the groups when compared to concurrent control group. The observed statistical significant changes in absolute and relative weights of adrenals in G2 group males and absolute weight of thymus in G4 group females when compared to concurrent control group (G6) was not considered as treatment related, since there was no correlation with either macro or microscopic observations.

Full histopathological examination was carried out on the selected five males and five females of control and high dose group.

Treatment related histopathological findings were not observed. The histopathological findings observed were either related to, agonal to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age.

On the basis of the results obtained in the present study, the No Observed Adverse Effect Level (NOAEL) of the test item for Repeated Dose toxicity is considered as 50 mg/kg b.w.

Categories Display