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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

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.

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 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.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until proof of pregnancy (maximum 5 days).
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- No unsuccessful attemps.
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

(P) Males groups 1 to 4: 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: 15 days before mating, continously during mating (1 to 5 days), during pregnancy (21 to 23 days) and 3 days post partum (Total: from 41 to 44 days)

Males and Females from reversal groups: 41 days (until first scheduled kill of dams)

Frequency of treatment:

daily

Details on study schedule:

(P) Males groups 1 to 4: sacrifice at the end of the exposure period of 28 days.
(P) Females groups 1 to 4: sacrifice at the end of the exposure period of 41 to 44 days

Males and Females of the reversal groups: Maintained for 14 days after dosing period prior sacrifice (sacrifice at day 54). No mating.

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:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Reversal group 0 and 50 mg/kg b.w./day (m/f) respectively
Basis:
nominal conc.
Groups G5 (control reversal) and G6 (high reversal)

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/group

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 (if not random): Randomization

- 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

Parental animals: Observations and examinations:

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).

GROSS PATHOLOGY
- 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).

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.

Other: see the same study in chapter 7.5.1 "Repeated dose toxicity: oral".

Oestrous cyclicity (parental animals):

not performed

Sperm parameters (parental animals):

not performed

Litter observations:

Performed for litter produced by females of groups 1 to 4.

Examined as earliest after delivery.
Observation made:
* Numbers and sex
* still and live births
* runts and presence of gross abnormalities
* weighed within 24 hours of parturition and on post partum day 4.

Postmortem examinations (parental animals):

SACRIFICE
- (P) Male animals: All surviving animals were sacrificed at the end of the dosing period, i.e. on day 28.
- (P) Maternal animals: All surviving animals were sacrificed at the end of the dosing period, i.e. on post partum day 4.
- Male and female animals of the reversal groups: All surviving animals were sacrified at the end of the 14 days observations period, following the end of the dosing period, i.e. on day 53.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [2] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

External examinations for gross abnormalities.

Statistics:

Examined parameters were checked for normality:
- Normal data was subjected to one-way ANOVA
- Non-normal data was subjected to Kruskal-Wallis One-Way ANOVA on Ranks

Student's Newman-Keul's Test was employed for post ANOVA comparison.

Reproductive indices:

sex ratio: (Number of male pups/ Number of female pups) x 100

Offspring viability indices:

not applicable

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

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 consumption and compound intake (if feeding study):

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.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

no treatment related changes observed when compared to concurrent control group

Other effects:

no effects observed

Description (incidence and severity):

Test substance intake: No statistical sigificant changes when compared to concurrent control group

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No effect 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 when compared to control.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
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 (table no. 6, 14 of the final report).


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
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 (Table 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. (Table 4, 5, 18, 19, 20).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating was observed within 5 days, which was normal, in all the groups including control. There were no significant changes attributed to the test substance on the mating period (Table 11, 23 of the final report).

No treatment related effect was observed on sex ratio of the pups in any of the treated groups (G2, G3 & G4) when compared with control group (G1) of animals (Table 10, 11, 25 of the final report).

Normal gestation length between 21-23 days was observed in all the treated groups (G2, G3 and G4) when compared with the control group (G1) (Table 24 of the final report).

No statistical significant changes were observed on mean Corpora lutea and Implantations in G2, G3and G4 females when compared to the G1. (Table 9, 10 and 25 of the final report).

Mean litter size of the G2, G3 and G4 was comparable with G1 (Table 25 of the final report).

Mean litter weight of the G2, G3 and G4 was comparable with G1 (Table 10, 25 of the final report).

No test substance related effect was observed on the number of dams delivered with live pups in any of the treated group (G2, G3 and G4) of animals and is comparable with control group (G1) (Tables 10, 11, 25 of the final report).

Loss of offspring (pre implantation, post implantation and post natal) in the treated groups (G2, G3 & G4) was comparable with control (G1) group (Table 9, 10, 11, 25 of the final report).

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 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 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 31 and 34 of the final report).

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

> 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Details on results (F1)

GROSS PATHOLOGY (OFFSPRING)
Gross external examination of live pups sacrificed on day 4 post-partum did not reveal any abnormality that could be attributed to the treatment (Tables 11 and 29 of the final report).

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

The tables are attached in the field "Attached background material", exept for the table 11 below.

Table 11 - Summary of effects on reproduction/development tabular report.

OBSERVATIONS	Values
Group	G1	G2	G3	G4
Dose (mg/kg b.w.)	0	12.5	25	50
Pairs started (N)	10	10	10	10
Females showing evidence of copulation (N)	10	10	10	10
Females achieving pregnancy (N)	10	10	9	10
Conceiving days 1-5(N)	10	10	10	10
Conceiving days ≥6-(N)	0	0	0	0
Pregnancy ≤ 21 days(N)	1	3	3	3
Pregnancy =22 days(N)	9	6	6	6
Pregnancy ≥ 23 days (N)	0	1	0	1
Dams with live young born (N)	10	10	9	10
Dams with live young at day 4 pp(N)	10	10	9	10
Corpora lutea/dam (mean)	14.40	13.30	14.10	14.70
Implants/dam (mean)	13.60	12.40	12.00	13.70
Live pups/dam at birth (mean)	12.30	11.20	11.67	12.40
Live pups/dam at day 4 (mean)	12.20	11.20	11.56	12.40
Sex ratio (m/f) at birth (mean)	103.08	98.52	150.83	144.87
Sex ratio (m/f) at day 4 (mean)	101.42	98.52	132.49	144.87
litter weight at birth (mean)	82.56	72.55	73.41	78.25
litter weight at day 4 (mean)	121.94	117.09	112.58	119.53
ABNORMAL PUPS
Dams with 0 (N)	8	9	8	9
dams with 1 ( N)	2	1	1	1
Dams with ≥ 2(N)	0	0	0	0
LOSS OF OFFSPRING
Pre - implantation (corpora lutea minus implantations)
Females with 0(N)	4	6	4	5
Females with 1(N)	4	2	1	3
Females with 2(N)	2	0	3	1
Females with ≥ 3(N)	0	2	2	1
Pre-natal/ post - implantations (implantations minus live Birth)
Females with 0(N)	2	6	1	5
Females with 1(N)	4	3	4	1
Females with 2(N)	3	0	2	3
Females with ≥3(N)	1	1	2	1
Post-natal (live births minus alive at post-natal day 4)
Females with 0(N)	9	10	8	10
Females with 1(N)	0	1	0	0
Females with 2(N)	0	0	0	0
Females with ≥3(N)	0	0	0	0

 

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 reproduction/developmental toxicity screening test 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 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.