Bis and tris and tetra (4-{bis[4-(dimethylamino)phenyl]methylene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium) [12,21-dihydro-29H,31H-phthalocyanine-bis and tris and tetrasulfonato-k4N29,N30,N31,N32]cuprate

Repeated dose toxicity: oral

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:

other: see 'Remark'

Remarks:

The reliability is rated 1 because the study followed the standard guideline of reference (OECD 422), which describes a procedure designed to evaluate this endpoint, the results were reviewed for reliability and assessed as valid, and the study was conducted under GLP conditions.

Cross-reference

Reason / purpose for cross-reference:

reference to same 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

Test material information

Constituent 1

Reference substance name:

Bis and tris and tetra (4-{bis[4-(dimethylamino)phenyl]methylene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium) [12,21-dihydro-29H,31H-phthalocyanine-bis and tris and tetrasulfonato-k4N29,N30,N31,N32]cuprate

EC Number:

700-615-0

Molecular formula:

BIS: C82H74N14CuS2O6 TRIS: C107H103N17CuS3O9 TETRA: C132H132N20CuS4O12

IUPAC Name:

Bis and tris and tetra (4-{bis[4-(dimethylamino)phenyl]methylene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium) [12,21-dihydro-29H,31H-phthalocyanine-bis and tris and tetrasulfonato-k4N29,N30,N31,N32]cuprate

Constituent 2

Reference substance name:

Sepisol Fast Violet 3B

IUPAC Name:

Sepisol Fast Violet 3B

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Sepisol Fast Violet 3B
- Substance type: Organocopper
- Physical state: Dark violet powder
- Analytical purity: 99.1%
- Purity test date: 2012
- Lot/batch No.:206641
- Expiration date of the lot/batch: 2022
- Stability under test conditions: Stable
- Storage condition of test material: Stored in tightly close container in a dry, cool and well-ventilated area in the utility room

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: 361 g to 400 g; Females: 232 g to 274 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: Bedding material, cages, grills were changes once every 3 day and water bottles were changed daily. Cages, grills and water bottles were autoclaved.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 °C to 22.1 °C. Recorded once daily
- Humidity (%): 51% - 59%. 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 4: From August 16, 2012 to September 13, 2012
- (P) Females groups 1 to 4: From August 16, 2012 to September 26 - September 29, 2012
- Males and females groups 5 and 6: From August 16, 2012 to October 09, 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Sepisol Fast Violet 3B was prepared freshly daily by mixing with corn oil and magnetically stirred to obtain a violet 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 4 = 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 4 = 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: 41 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Groups 1 to 4: 0, 12.5, 25 and 50 mg/kg b.w./day (m/f) respectively
Basis:
other: nominal concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Reversal groups 5 and 6: 0 and 50 mg/kg b.w./day (m/f) respectively
Basis:
other: nominal concentration

No. of animals per sex per dose:

(P) Males from groups 1 to 4: 10/group
(P) Females from groups 1 to 4: 10/group
Males and females from the reversal groups: 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, administered oraly for 7 days and observed for morbidity/mortality and signs of toxicity daily. Control group animals were treated similarly, but with corn oil alone.

On day 3, one male and two females, whereas on day 4 one male and one female from group 4 was found dead.
On day 5, one male from G4 and two males and two females from G3 were found dead.
One male and one female from G3 were found dead on Day 6. Bluish perianal stating was observed in all animals of all test substance treated group from day 1 to 7.
Dullness was observed in two male and one female from G2 and all animals of G3 and G4.
Piloerection was noted in two female and one male of G3 and one male and one female of G4.
Abdominal distension was observed in two males and three females from G3 and three males and two females from G4.
Mouth breathing was recorded in three males and two females of G3 and also in three males on one female of G4.
At macroscopic examination, blue discoloration of stomach and intestine The gross lesions observed in the treated group were blue discoloration of stomach and intestine was observed in all treated groups. Small seminal vesicles were noted in G3 and G4 group, dark red discoloration of liver, enlarged adrenals and mesenteric lymph nodes were observed in G3 animals.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily 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 4: On pre-mating days 0, 7, mating days 0, 7 and 13.
* (P) Females groups 1 to 4: 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 4: Prior administration, on pre-mating days 0, 7, mating days 0, 7 and at termination
* (P) Females groups 1 to 4: 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:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: 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 6 groups and on day of scheduled kill of animals from groups 5 and 6.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: on 5 males and 5 females of each of the 6 groups (randomly selected for groups 1 to 4).
- 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 6 groups and on day of scheduled kill of animals from groups 5 and 6.
- Animals fasted: Yes (overnight)
- How many animals: on 5 males and 5 females of each of the 6 groups (randomly selected for groups 1 to 4).
- 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 4 = on mating day 13.
* (P) Females from groups 1 to 4 = on post partum day.
* Males and Females of the reversal groups 5 and 6 = on a day before terminal blood collection.
- Dose groups that were examined: All
- Battery of functions tested: auditory fonction, grip strength, locomotor activity

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.
- 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 4 (control and 50 mg/kg b.w./day)
- How many animales: 5 animals per groups, exept 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

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In G3 group animals, intermittent dullness was observed in 5 (10) males, 6 (10) females and piloerection was noted in 2(10) males, 3(10) females. In G4 group animals, dullness was observed in 9(10) males, 10(10) females and piloerection was observed in 8 (10) females. In G6 group animals dullness was observed in 4(5) males, 5(5) females and piloerection was observed in 2(5) males, 2(5) females. These signs were recovered after withdrawal of treatment subsequently in G6.

Mortality:

mortality observed, treatment-related

Description (incidence):

In G3 group animals, intermittent dullness was observed in 5 (10) males, 6 (10) females and piloerection was noted in 2(10) males, 3(10) females. In G4 group animals, dullness was observed in 9(10) males, 10(10) females and piloerection was observed in 8 (10) females. In G6 group animals dullness was observed in 4(5) males, 5(5) females and piloerection was observed in 2(5) males, 2(5) females. These signs were recovered after withdrawal of treatment subsequently in G6.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistical significant decrease in group 4 in males from day 7 to the sacrifice and from day 14 in females but not continued during the gestation and post partum.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

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 the locomotor activity of G4 males as compare to G1 males

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY:
No mortality.
No clinical signs of toxicity were observed in males and females of G1, G2 and G5 group animals.
The bluish perianal staining in animals of G3, G4 and G6 groups was considered due to the color of test substance and this effect was disappeared in High reversal (G6) group animals at the end of observation 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.

In G3 group animals, intermittent dullness was observed in 5 (10) males, 6 (10) females and piloerection was noted in 2(10) males, 3(10) females. In G4 group animals, dullness was observed in 9(10) males, 10(10) females and piloerection was observed in 8 (10) females. In G6 group animals dullness was observed in 4(5) males, 5(5) females and piloerection was observed in 2(5) males, 2(5) females. These signs were recovered after withdrawal of treatment subsequently in G6 (Table no. 13 of the final repot).

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 (Tables no. 6, 14 of the final report).


BODY WEIGHT AND FOOD CONSUMPTION
Observed body weight in low (G2) and intermediate (G3) dose groups was comparable with respective control group (G1) throughout the observation period. However, statistical significant decrease was observed in G4 & G6 males from day 7 till the sacrifice as compare to their respective control. G4 females of showed decreasing trend body weight from day 7 and continued till end. Statistical significant decrease in the body weight of G6 females was observed from day 14 to the end of the experiment (Tables no. 1, 2, 3, 15, 16, 17).

Food consumption recorded in treated groups was comparable with respective control groups and there were no significant difference. (Tables no. 4, 5, 18, 19, 20).

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. Although, in males on mating day 0, statistically significant changes were observed in hematology parameters like increase in RBC in G4 males and G3 females, platelet in G4 males when compared to their concurrent controls, not observed in the high reversal and these values were well within the historical data of laboratory. Hence these changes were not considered as treatment related (Tables no. 7, 21).

CLINICAL CHEMISTRY
No statistically significant changes were observed in biochemistry parameters across the groups in both the time points except statistical significant changes in ALT in G4 males, urea, blood urea nitrogen, triglycerides, calcium in G6 males and ALP on mating day 0 and Calcium on day 54 in G6 females as compare to their concurrent controls. These changes were observed in the dose dependant manner and the values are within the historical data of IIBAT, hence not considered test substance related (Tables no. 8, 22).

NEUROBEHAVIOUR
There was no statistically significant difference recorded in the FOB parameters of G2, G3, G4 and G5 group animals with respect to their concurrent control group except statistically significant decrease in the locomotor activity of G4 males as compare to G1 males (Tables no.12, 26).

ORGAN WEIGHTS (PARENTAL ANIMALS)
No test substance related organ weight changes were observed in any of treated group when compared to concurrent control group. (Tables no. 27, 28, 32 and 33 of the final repot)

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test substance related gross pathological findings were observed.

The gross necropsy 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. (Tables no. 30 and 34 of the final report).

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

The histopathologic findings observed were either related to, agonal to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age. (Tables no. 31 and 34 of the final report).

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The tables are attached in the flied "Attached background material".

Content of the attached list:

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)

Table 13 - Daily Individual Signs of Toxicity

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

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 - Daily Individual Feed Consumption in Females For Reversal Groups (g)

Table 21 – Individual Hematology Parameters

Table 22 – Individual Biochemistry Parameters

Table 26 – Individual Functional Observational Battery (FOB)

Table 27 - Summary of Absolute organ weight (g)

Table 28 - Summary of Relative organ weight (%)

Table 30 - Summary of Gross pathology

Table 31 - Summary of Histopathology

Table 32 - Individual Animal absolute organ weight (g)

Table 33 - Individual Animal relative organ weight (%)

Table 34 - 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 Sepisol Fast Violet 3B for Repeated Dose Toxicity study is considered as 25 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 Sepisol Fast Violet 3B.

 

One hundred rats were randomized into six groups, four main groups containing 10 rats/group/sex viz., G1 (Control), G2, G3, G4 (treated groups) and two reversal groups containing 5 rats/group/sex viz., G5 (control reversal) and G6 (High reversal).

 

The test substance Sepisol Fast Violet 3B was suspended in corn oil and administered by gavage daily at the doses of 12.5, 25 and 50 mg/kg b.w. to the rats belonging to G2 (low), G3 (Low intermediate), G4 (High) and G6 (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.

 

No morbidity/mortality was observed in any of the group.

 

No clinical signs of toxicity were observed in males and females of G1, G2 and G5 group animals.

 

The bluish perianal staining observed in G3, G4 and G6 groups was disappeared after withdrawal of treatment in high reversal group (G6).

 

 

In G3 group intermittent dullness and piloerection was observed in few animals, however, in G4 and G6 groups these signs were persisted during the treatment and they were recovered after withdrawal of treatment subsequently.

 

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.

 

Observed body weight in low (G2) and intermediate (G3) dose groups was comparable with respective control group (G1) throughout the observation period. However, statistical significant decrease was observed in G4 & G6 males from day 7 to the sacrifice as compare to their respective control. In G4 females, statistical significant decrease was observed on day 14 but not continued during the gestation and post partum. Statistical significant decrease in the body weight of G6 females was observed from day 14 to the end of the experiment.

No Statistical significant changes were observed in food consumption of treated groups when compared to their respective control groups.

 

There was no statistically significant difference recorded in the FOB parameters viz. Auditory function, grip strength and locomotor activity of G2, G3 and G6 group animals with respect to their concurrent control group, however in G4 group males a statistically significant decrease in the locomotor activity was observed when compared to G1 males.

 

Blood was collected on mating day 0 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 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 RBC in G4 males and G3 females, platelet in G4 males when compared to their concurrent controls. However these changes were not observed in the high reversal group, further these values were well within the historical data of IIBAT. Hence these changes were not considered as treatment related.

 

No statistically significant changes were observed in biochemistry parameters across the groups in both the time points except statistical significant changes in ALT in G4 males, urea, blood urea nitrogen, triglycerides, calcium in G6 males and ALP on mating day 0 and Calcium on day 54 in G6 females as compare to their concurrent controls. These changes were not observed in the dose dependant manner and the values are within the historical data of IIBAT, hence not considered test substance related.

 

All dams were allowed to litter naturally and the size and weight of litters, live births, runts, sex of live pups and the presence of gross abnormalities were recorded within 24 hours of parturition (day 0) and on day 4 post partum.

 

No test substance related effect was observed in parameters like fertility, mating period, gestation length, mean corpora lutea, mean implantation, mean litter size, mean litter/pup weight, implantation losses, and sex ratio of offspring in G2,G3 and G4 groups when compared to control.

 

Gross external examination of live pups sacrificed on day 4 post-partum did not reveal any abnormality that could be attributed to the treatment.

 

Complete gross pathology was conducted on all adult animals and examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.

 

No test substance related gross pathological findings were observed. The gross necropsy 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.

 

Detailed histological examination in all animals of control and high dose group was performed on the Ovaries, Testes, and Epididymis with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure. In addition, 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 Sepisol Fast Violet 3B for Repeated Dose Toxicity study is considered as 25 mg/kg b.w. whereas, the NOAEL of Sepisol Fast Violet 3B for Reproduction/Developmental Toxicity Screening Test is considered as 50 mg/kg b.w.