Tris[4-(diethylamino)phenyl]methylium acetate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Principles of method if other than guideline:

Evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of 2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3yl]benzoxazole-5-sulphonamide (CAS No.: 68427-35-0) in Wistar rats.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazole-5-sulphonamide

EC Number:

270-393-3

EC Name:

2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazole-5-sulphonamide

Cas Number:

68427-35-0

Molecular formula:

C20H19N3O5S

IUPAC Name:

2-[7-(diethylamino)-2-oxo-2H-chromen-3-yl]-1,3-benzoxazole-5-sulfonamide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): 2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazole-5-sulphonamide
- Molecular formula : C20H19N3O5S
- Molecular weight : 413.452 g/mol.
- Smiles :n1c2cc(S(N)(=O)=O)ccc2oc1c1cc2ccc(N(CC)CC)cc2oc1=O
- InChI : 1S/C20H19N3O5S/c1-3-23(4-2)13-6-5-12-9-15(20(24)28-18(12)10-13)19-22-16-11-14(29(21,25)26)7-8-17(16)27-19/h5-11H,3-4H2,1-2H3,(H2,21,25,26)
- Substance type: Organic
- Physical state: Solid
- Analytical purity: 100%

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazole-5-sulphonamide
- Molecular formula : C20H19N3O5S
- Molecular weight : 413.452 g/mol.
- Substance type: Organic
- Physical state: Solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animals were procured from a CPCSEA approved Vendor [Gentox Bio Services Pvt. Ltd. (CPCSEA Registration No.: 1242/RCBT/S/08/CPCSEA)]
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) x wks; (F1) x wks: 12 - 13 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing: Before the animals are brought in, the study room and cages were cleaned and disinfected. During the study, the floor of the experimental room and work tops were swept and mopped with disinfectant solution every day or as on requirement. Cages were cleaned at regular intervals.
A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20 [cm]). Cage rotation was carried out weekly during study period except during mating and during gestation and lactation only for females.
Sterilized corn cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean.
Bedding material of batch No. SPAR-33/2016 and SPAR-34/2016 (Sparconn Life Sciences Bangalore) was used in this study.
All the animals were identified by temporary tail marking with indelible ink and cage cards. Following allocation to the study, each animal was uniquely identified by micro toe pad tattooing and colour coded cage cards labelled with study no., study type, test system, sex, dose, group, animal number, dosing start date, experimental start and completion date. Pups were identified with body marking with permanent non-toxic marker pen
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): conventional laboratory pelleted diet, ad libitum
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum.
- Acclimation period:7 days and 8 days prior to test item administration for Dose Range Finding Study (DRF) and Main Study respectively.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.00 to 24.90 °C
- Humidity (%): 39.90 to 65.50%.
- Air changes (per hr):12 hours light and 12 hours dark
- Photoperiod (hrs dark / hrs light):Air changes were about minimum 12 times per hour and filtered adequately.

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 0, 250, 500 and 1000 mg/kg body weight
- Amount of vehicle (if gavage): 1.0 ml/100g body weight
- Lot/batch no. (if required): Lot nos.: MR020816, A611001 and A1701001
- Purity:

Details on mating procedure:

- M/F ratio per cage:1:1
- Length of cohabitation: until pregnancy occurs or two weeks elapsed.
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Sperm positive vaginal smear was considered as day "0" of gestation.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.; yes
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): : housed individually
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Specificity, Precision (%RSD), Accuracy (% Recovery) and Homogeneity by HPLC-UV

Duration of treatment / exposure:

Approx 63 days

Frequency of treatment:

Daily

Details on study schedule:

not specified

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

Total:124
0 mg/kg bw: 13 male, 13 female
250 mg/kg bw: 13 male, 13 female
500 mg/kg bw: 13 male, 13 female
1000 mg/kg bw: 13 male, 13 female
Recovery Group
0 mg/kg bw: 5 male, 5 female
1000 mg/kg bw: 5 male, 5 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels 250, 500 and 1000 mg/kg body weight were selected for the Main Study based on the results of Dose Range Finding (DRF).
- Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing.
- Other:

Positive control:

not specified

Examinations

Parental animals: Observations and examinations:

Mortality and Morbidity, General clinical sign, motor Activity and Behavioural, Body Weight and Feed Consumption were examined.

Oestrous cyclicity (parental animals):

Estrous cycle were monitored daily

Sperm parameters (parental animals):

not specified

Litter observations:

Live pups, Number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups) and body weight were examined.

Postmortem examinations (parental animals):

Hematology, clinical biochemistry, Organ Weight, gross Pathology and Histopathology were examined.

Postmortem examinations (offspring):

Gross Pathology were examined.

Statistics:

Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks.

Reproductive indices:

Gestational length, Litter size, No. of live births, Post-implantation loss and Pregnancy Index (%) were examined.

Offspring viability indices:

Fetal Survival Index at Post-natal Day 1 and 4 were examined.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Statistically significant decrease were observed in number of rears (250, 500 and 1000 mg/kg bw at pre-treatment and 1000 mg/kg bw at Week 1), number of urine pools (250 mg/kg bw at Week 2) in male as compared to control. In recovery male, statistically significant increase in number of urine pools at week 4 in recovery at 1000 mg/kg bw as compared to recovery control. In female, statistically significant decrease was observed in number of fecal bolus (1000 mg/kg bw recovery at Week 1) as compared to control recovery group.
The above changes observed were inconsistent/ biologically insignificant and not dose dependant hence considered as incidental and not attributed to the effect of test item administration.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

One female was found dead on day 41 at 1000 mg/kg bw due to gavaging error

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Statistically significant decrease was observed in percent body weight change of 250, 500 and 1000 mg/kg bw on day 1-20 as compared to control group.
Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. These changes observed were inconsistent, hence not considered as effect of the test item administration

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Statistically significant decrease in feed consumption was observed in 250, 500 and 1000 mg/kg bw female on gestation day 14-20 as compared to the control. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group.
Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No effect on Eye were observed in treated rats as compared to control.

Haematological findings:

no effects observed

Description (incidence and severity):

At the end of treatment period revealed statistically significant decreased of aPTT in male rats at 1000 mg/kg, statistically significant increased of aPTT in female rats at 250 mg/kg while statistically significant decreased in Lymphocyte Count in female rats at 500 mg/kg.
At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes.
During treatment free recovery period, statistically significant increase was observed in PT of recovery male rats treated at 1000 mg/kg and statistically significant decreased was observed Hematocrit and Hemoglobin in recovery male rats treated at 1000 mg/kg, while statistically significant decreased of Monocyte in female recovery rats at 1000 mg/kg when compared to respective control group. The observed variations in PT, Hematocrit, Hemoglobin and monocyte were considered to be of no toxicological significance, minimal in nature and occurred in the absence of clear dose related response

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

At the end of treatment period revealed, statistically significant increase in Chloride of male rats treated at 500 mg/kg, Sodium of male rats treated at 1000 mg/kg. Statistically significant decrease in bile acid of male rats treated at 250 mg/kg and 1000 mg/kg respectively while statistically significant decrease in Triglyceride of female rats treated at 500 mg/kg.
During treatment-free recovery period revealed statistically significant increase in Potassium in female rats treated at 1000 mg/kg while statistically decrease in Bile acid in female rats treated at 1000 mg/kg was observed when compared to respective control group.
The observed variations in Potassium and Bile acid was considered incidental and not test item related as it was observed only in single sex, inconsistent, not dose dependent and further is not evidenced by histopathological observations.

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes.
Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups.
Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality.
focal mild to multifocal mild mononuclear cell infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration and minimal chronic changes and hydropic degeneration in female liver, Unilateral: focal minimal to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Kidneys, focal mild to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Lungs and focal minimal to multifocal mild GALT hyperplasia in male and focal minimal to multifocal minimal GALT hyperplasia in female intestin at 250 and 1000 mg/kg bw, Vacuolar, degeneration multifocal, mild in male and diffuse vacuolations in female Adrenals at 250 mg/kg bw, minimal cyst in male and mild serosanginous fluid cavity in female Pituitary Gland, focal minimal to multifocal moderate Retention of sperm, focal minimal to focal mild Exfoliation of round spermatid, focal minimal to multifocal mild Seminiferous tubules Vacuolar degeneration, marked diffuse Necrosis of Seminiferous tubules, minimal diffuse Atrophy Seminiferous tubules, minimal to mild Vacuolar degeneration of Leydig cell, marked diffuse Necrosis of Epididymis, marked diffuse decrease in Epididymis sperm and Epididymis of Epididymis in male rat and segmental minimal Vacuolar degeneration of Ovary and minimal Perimetrium and myometrium vacuolations of Uterus in female rats were observed at different doses.
Lesions observed in liver, kidneys, lungs, adrenals, intestine, pituitary gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

One females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period at 250 and 1000 mg/kg bw.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

No statistically significant effect on Gestational length, Litter size, No. of live births, Post-implantation loss, Post-natal loss, Pups sex ratio and Pregnancy index of treated rats were observed as compared to control.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

Remarks on result:

other: No effect observed

Target system / organ toxicity (P0)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

No effect on Survival of pups were observed as compared to control.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effect on weight gain for pups at PND4 were observed as compared to control.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

Pups died during course of study revealed various lesions among the control and treated groups.
Cannibalism in male pups at 250 and 500 mg/kg bw and in female at 250 mg/kg bw, absence of milk in stomach in male at 250 and 500 mg/kg bw and in female at 250 mg/kg bw, reddish discoloration of lungs in female at 250 mg/kg bw and brain haemorrhage in male rat at 250 mg/kg bw.
No gross patholoical changes were observed at 1000 mg/kg bw. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

gross pathology

Remarks on result:

other: No effect observed

Target system / organ toxicity (F1)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Mortality and Morbidity

Sex: Male

Group	Treatment	Dose (mg/kg b.wt.)	No. of Animals/ Group	Observation During Study Period
G1	Control	0	13	NMM
G2	Low	250	13	NMM
G3	Mid	500	13	NMM
G4	High	1000	13	NMM
G1-R	Control- Recovery	0	5	NMM
G4-R	High- Recovery	1000	5	NMM

 

Sex: Female

Group	Treatment	Dose (mg/kg b.wt.)	No. of Animals/ Group	Observation During Study Period
G1	Control	0	13	NMM
G2	Low	250	13	NMM
G3	Mid	500	13	NMM
G4	High	1000	13	01/13*
G1-R	Control -Recovery	0	5	NMM
G4-R	High- Recovery	1000	5	NMM

Keys:NMM = No mortality and morbidity observed, No.= Number, *= One female found dead during clinical sign observation on Gestation Day 24.

Summary of Days of Conception and Pregnancy Index (%)

Group(N)	G1(13)	G2(13)	G3(13)	G4(13)
Dose (mg/kg b.wt.)	0	250	500	1000
No. of females showed evidence of copulation	13	12	13	12
No. of females concieved between Days 1-5 of cohabitation	10	7	10	8
No. of females concieved after Day 5 of cohabitation	3	5	3	4
Females achieved pregnancy	13	12	13	12
Pregnancy Index (%)	100.00	92.31	100.00	92.31

Key:N= number of dams in a group, No. = Number

Post-natal Loss (%) and Pups Survival Index (%)

Group(N)	G1(13)		G2(12)		G3(11)		G4(10)
Dose(mg/kg bwt)	0		250		500		1000
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
No. of Live Births	10.54	1.13	9.67	1.61	10.00	3.00	9.70	1.95
No. of alive pups at Post-natal Day 4	10.31	0.95	8.42	3.15	9.55	2.81	9.50	2.17
Post-natal Loss (%)	1.99	3.80	11.41	29.11	3.99	6.18	2.67	5.84
Fetal Survival Index at Post-natal Day 4 (%)	98.01	3.80	88.59	29.11	96.01	6.18	97.33	5.84

Keys:SD= Standard Deviation, N= number of dams in a group

Note:Group G2 one female was found non-pregnant. Group G3 two females were showed all the pups still births and in Group G4 one female was non-pregnant, one female was found dead and one females showed all the pups still births. 

Mean Gestational Lengthand Litter size

G1(13)		G2(12)		G3(11)		G4(11)
0		250		500		1000
Mean	SD	Mean	SD	Mean	SD	Mean	SD
22.23	0.60	19.69	6.10	22.00	1.53	18.54	8.33
10.54	1.13	9.92	1.78	9.38	3.33	9.27	2.23

Keys:SD= Standard Deviation, N= number of dams in a group

Note:Group G2 one female was found non-pregnant. Group G3 two females were showed all the pups still births and in Group G4 one female was non-pregnant and one female was found dead.

Mean Pups Body Weight, Sex Ratio and Gross Observation

Group	G1			G2			G3			G4
Dose(mg/kg bwt)	0			250			500			1000
Mean Pups Weight	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Day 0/1	5.88	0.25	13	5.68	0.37	12	5.87	0.49	11	5.75	0.25	10
Day 4	8.71	0.41	13	8.48	0.93	11	8.50	1.02	11	8.83	0.78	10
Group(n)	G1(13)			G2(12)			G3(11)			G4(10)
Pups Body Weight gain (%)	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Day 0/1-Day 4	48.09	5.55	13	49.23	15.97	11	44.01	6.89	11	53.52	10.37	10
Group (Number of Litter size)	G1(137)			G2(119)			G3(122)			G4(97)
Sex Ratio at birth (Male/Female)	81/56			56/60			56/54			58/39
Sex Ratio at Day 4 (Male/Female)	80/54			47/55			52/53			57/38
Gross Observations	NAD			NAD			NAD			NAD

Keys:SD= Standard Deviation, N= number of dams with live pups on Day 0/1, n= number of dams with alive pups on Day 4, NAD = No Abnormality Detected

Applicant's summary and conclusion

Conclusions:

No Observed Adverse Effect Level (NOAEL) of the test item 2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3yl]benzoxazole-5-sulphonamide (CAS No.: 68427-35-0) is considered 1000 mg/kg body weight when Wistar male and female rats orally by gavage for Approx 63 days.

Executive summary:

In a Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity study, Wistar male and female rats were treated with 2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3yl]benzoxazole-5-sulphonamide in the concentration of 0, 250, 500 and 1000 mg/kg bw orally by gavage for aprrox 63 days. Statistically significant decrease were observed in number of rears (250, 500 and 1000 mg/kg bw at pre-treatment and 1000 mg/kg bw at Week 1), number of urine pools (250 mg/kg bw at Week 2) in male as compared to control. In recovery male, statistically significant increase in number of urine pools at week 4 in recovery at 1000 mg/kg bw as compared to recovery control. In female, statistically significant decrease was observed in number of fecal bolus (1000 mg/kg bw recovery at Week 1) as compared to control recovery group. The above changes observed were inconsistent/ biologically insignificant and not dose dependant hence considered as incidental and not attributed to the effect of test item administration. One female was found dead on day 41 at 1000 mg/kg bw due to gavaging error. Statistically significant decrease was observed in percent body weight change of 250, 500 and 1000 mg/kg bw on day 1-20 as compared to control group. Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. These changes observed were inconsistent, hence not considered as effect of the test item administration. Statistically significant decrease in feed consumption was observed in 250, 500 and 1000 mg/kg bw female on gestation day 14-20 as compared to the control. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration. No effect on Eye was observed in treated rats as compared to control. Similarly, At the end of treatment period revealed statistically significant decreased of aPTT in male rats at 1000 mg/kg, statistically significant increased of aPTT in female rats at 250 mg/kg while statistically significant decreased in Lymphocyte Count in female rats at 500 mg/kg. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes. During treatment free recovery period, statistically significant increase was observed in PT of recovery male rats treated at 1000 mg/kg and statistically significant decreased was observed Hematocrit and Hemoglobin in recovery male rats treated at 1000 mg/kg, while statistically significant decreased of Monocyte in female recovery rats at 1000 mg/kg when compared to respective control group. The observed variations in PT, Hematocrit, Hemoglobin and monocyte were considered to be of no toxicological significance, minimal in nature and occurred in the absence of clear dose related response. At the end of treatment period revealed, statistically significant increase in Chloride of male rats treated at 500 mg/kg, Sodium of male rats treated at 1000 mg/kg. Statistically significant decrease in bile acid of male rats treated at 250 mg/kg and 1000 mg/kg respectively while statistically significant decrease in Triglyceride of female rats treated at 500 mg/kg. During treatment-free recovery period revealed statistically significant increase in Potassium in female rats treated at 1000 mg/kg while statistically decrease in Bile acid in female rats treated at 1000 mg/kg was observed when compared to respective control group. The observed variations in Potassium and Bile acid was considered incidental and not test item related as it was observed only in single sex, inconsistent, not dose dependent and further is not evidenced by histopathological observations. The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups. Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group. The above changes observed were inconsistent, hence considered as incidental and not attributed to the effect of test item administration. At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of either sex did not differ significantly when compared to respective control group except, statistical significant increase absolute and relative adrenal weight in male rat at 250 mg/kg bw as compared to main control group. Statistical significant decrease relative testes and Epididymis weight in male rat at 1000 mg/kg bw was observed when compared to control rats. Statistical significant increased relative heart weight in male recovery rats was observed when compared to control rats. External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality. Focal mild to multifocal mild mononuclear cell infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration and minimal chronic changes and hydropic degeneration in female liver, Unilateral: focal minimal to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Kidneys, focal mild to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Lungs and focal minimal to multifocal mild GALT hyperplasia in male and focal minimal to multifocal minimal GALT hyperplasia in female intestine at 250 and 1000 mg/kg bw, Vacuolar, degeneration multifocal, mild in male and diffuse vacuolations in female Adrenals at 250 mg/kg bw, minimal cyst in male and mild serosanginous fluid cavity in female Pituitary Gland, focal minimal to multifocal moderate Retention of sperm, focal minimal to focal mild Exfoliation of round spermatid, focal minimal to multifocal mild Seminiferous tubules Vacuolar degeneration, marked diffuse Necrosis of Seminiferous tubules, minimal diffuse Atrophy Seminiferous tubules, minimal to mild Vacuolar degeneration of Leydig cell, marked diffuse Necrosis of Epididymis, marked diffuse decrease in Epididymis sperm and Epididymis of Epididymis in male rat and segmental minimal Vacuolar degeneration of Ovary and minimal Perimetrium and myometrium vacuolations of Uterus in female rats were observed at different doses. Lesions observed in liver, kidneys, lungs, adrenals, intestine, pituitary gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item. In addition, no reproductive toxicity were observed in treated rats such as Gestational length, Litter size, No. of live births, Post-implantation loss, Post-natal loss, Pregnancy index, Pups sex ratio, survival, body weight on PND 4 and gross pathology of treated rats were observed as compared to control. One females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period at 250 and 1000 mg/kg bw. Therefore, No Observed Adverse Effect Level (NOAEL) of the test item 2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3yl] benzoxazole-5-sulphonamide (CAS No.: 68427-35-0) is considered 1000 mg/kg body weight when Wistar male and female rats orally by gavage for Approx 63 days.