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Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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: 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): 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:
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 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.
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 5: 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: 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 5: sacrifice at the end of the exposure period of 28 days.
(P) Females groups 1 to 5: 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 / 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 adn 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: 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:
Carried out using one control (G1) and three 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 oral administration for 7 days. Control group animals were treated similarly, but with corn oil alone.
Observed for morbidity /mortality and signs of toxicity daily.

Death of one male, two females from G4 and one female from G3.

Bluish perianal staining observed in all animals of all the test substance treated group from day 1 to 7.
Dullness observed in one male and two females from G3 and all animals of G4.
Piloerection noted in one male and one female of G3 and two male and female of G4.
Abdominal distension observed in one female of G3 as well as one male and all females of G4.
Respiratory distress recorded in one male of G4.
Enlarged payers’s patches observed in all animals of test substance treated groups at macroscopic examination.
Gross lesions observed in G3 and G4 animals: 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 enlarged in all animals
Thymus small in size in two male and one female of G4 group.

Selected dose for the main study: 25, 50, 75 and 100 mg/kg b.w
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 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).

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, G5 and all animals from groups G6 and G7: Liver, Kidneys, Adrenals, Thymus, Spleen, Brain and Heart.

Other: see the study in chapter 7.5.1 "Repeated dose toxicity: oral - 28 days"
Oestrous cyclicity (parental animals):
not performed
Sperm parameters (parental animals):
not performed
Litter observations:
Performed for litter produced by females of groups 1 to 5.

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:
effects observed, treatment-related
Description (incidence and severity):
Animals of G4, G5 & G7 exhibited dullness and piloerection at various intervals. Mortability: one dead male in G5, one dead male in G7 and one dead female in G5. Abdominal distension and respiratory distress were observed in those dead animals.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Death of one animal in each groups of high (G5) and high reversal (G7) males and one female in high (G5) dose groups could be considered as treatment related
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 compare to their respective control.
Food consumption and compound intake (if feeding study):
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 compare to their respective control.
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: 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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

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 (PARENTAL ANIMALS):
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 colour 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 (PARENTAL ANIMALS):
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, 17 in 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).


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): G1 to G5
Mating was observed within 4 days, which was normal, in all the groups including control. There were no significant changes attributed to the test substance on the mating period (attached tables 11 and 22 in background material).

No treatment related effect was observed on sex ratio of the pups in any of the treated groups when compared with control group (G1) of animals (attached tables 10, 11, 24 in background material).

Normal gestation length between 21-23 days was observed in females of all the treated groups and was comparable with gestation length of the control group females (attached table 23 in background material).

No statistical significant changes were observed on mean corpora lutea and Implantations in G2, G3, G4 females when compared to the G1. Whereas, in high dose (G5) group a decrease was observed in total corpora lutea and subsequent Implantations with respect to control (G1) group. However these changes were not statistically significant and the mean corpora lutea count was also comparable with respective control group. This decrease is due to the death of one female in this dose group. Hence these changes could not be considered as treatment related (attached tables 9, 10 and 24 in background material).

Mean litter size of the G2, G3 and G4 was comparable with G1. Although not statistically significant, a decrease was observed in the G5 group when compared to G1 due to the death of one animal in G5 group. Hence this change could not be attributed to treatment related (attached table 24 in background material).

Mean litter weight of the G2, G3 and G4 was comparable with G1. The observed decrease in G5 group was not statistically significant when compared with G1 due to the death of one animal in G5 group. Hence this change could not be attributed to treatment related (attached table 10, 24 in background material).

No test substance related effect was observed on the number of dams delivered with live pups in any of the treated group of animals and is comparable with control group (attached tables 10, 11, 24 in background material).

Loss of offspring (pre implantation, post implantation and post natal) in the treated groups (G2, G3 & G4) was comparable with control (G1) group. A statistically insignificant pre implantation loss was observed in G5 group when compared to G1 (attached tables 9, 10, 11, 24 in background material).

ORGAN WEIGHTS (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 document).

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 documents).


Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
not specified
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
no effects observed on sex of live pups

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

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 (attached table 28 in background material).

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

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

G5

Dose (mg/kg b.w.)

0

25

50

75

100

Pairs started (N)

10

10

10

10

10

Females showing evidence of copulation (N)

10

10

10

10

10

Females achieving pregnancy (N)

10

10

10

10

10

Conceiving days 1-5(N)

10

10

10

10

10

Conceiving days ≥6-(N)

0

0

0

0

0

Pregnancy ≤ 21 days(N)

0

3

0

2

3

Pregnancy =22 days(N)

8

7

9

7

6

Pregnancy ≥ 23 days (N)

2

0

1

1

0

Dams with live young born (N)

10

10

10

10

10

Dams with live young at day 4 pp(N)

10

10

10

10

10

Corpora lutea/dam (mean)

14,30

14,40

14,60

15,00

15,33

Implants/dam (mean)

13,60

13,90

14,10

14,70

13,78

Live pups/dam at birth (mean)

12,50

12,70

12,90

13,70

12,89

Live pups/dam at day 4 (mean)

12,30

12,70

12,70

13,70

12,78

Sex ratio (m/f) at birth (mean)

92,31

144,23

115,00

101,47

118,87

Sex ratio (m/f) at day 4 (mean)

92,19

144,23

111,67

101,47

116,98

litter weight at birth (mean)

78,04

74,81

81,96

84,24

79,20

litter weight at day 4 (mean)

125,75

117,43

129,20

133,14

116,04

ABNORMAL PUPS          

Dams with 0 (N)

10

10

10

10

9

dams with 1 ( N)

0

0

0

0

0

Dams with ≥ 2(N)

0

0

0

0

0

LOSS OF OFFSPRING

Pre - implantation (corpora lutea minus implantations)

Females with 0(N)

7

7

7

8

3

Females with 1(N)

0

1

2

1

3

Females with 2(N)

2

2

0

1

0

Females with ≥ 3(N)

1

0

1

0

3

Pre-natal/ post - implantations (implantations minus live Birth)

Females with 0(N)

4

7

3

4

4

Females with 1(N)

3

0

3

3

3

Females with 2(N)

1

1

3

2

1

Females with ≥3(N)

2

2

1

1

1

Post-natal (live births minus alive at post-natal day 4)

Females with 0(N)

8

10

8

10

8

Females with 1(N)

2

0

2

0

1

Females with 2(N)

0

0

0

0

0

Females with ≥3(N)

0

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 the Substance for reproduction/developmental toxicity screening test is considered as 100 mg/kg b.w.
Executive summary:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar ratswas performed to evaluate the Substance

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 andkept 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 in each group 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 colour 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 belong 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.

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. Whereas, in high dose (G5) group a significant decrease was observed in total (but not mean) corpora lutea and subsequent Implantations with respect to control (G1) group. Similarly a decreased litter weight on post-partum day 4 was observed. However these changes were not statistically significant and this decrease is due to the death of one female in this dose group. Hence these changes could not be attributed to treatment related.

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 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. No test substance related gross abnormalities were found in pups of any of treated group.

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.

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 strucutre. 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 the Substance for Reproduction toxicity is considered as 100 mg/kg b.w.

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