Sodium 3-nitrobenzenesulphonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(Adopted on January 22, 2001)

Principles of method if other than guideline:

According to OECD Guideline 414 (Prenatal Developmental Toxicity Study) (Adopted on January 22, 2001)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 3-nitrobenzenesulphonate

EC Number:

204-857-3

EC Name:

Sodium 3-nitrobenzenesulphonate

Cas Number:

127-68-4

Molecular formula:

C6H5NO5S.Na

IUPAC Name:

sodium 3-nitrobenzenesulphonate

Test material form:

solid: granular

Details on test material:

- Name of test material (as cited in study report):hair dye formulation containing 2.25% sodium m-Nitrobenzenesulphonate
- Molecular formula (if other than submission substance):C6H4NNaO5S(sodium m-nitrobenzenesulphonate)
- Molecular weight (if other than submission substance):225.15 (sodium m-nitrobenzenesulphonate)

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana
- Age at study initiation: 13 to 14 weeks
- Weight at study initiation: 214.952 ± 13.13 to 196.16 to 246.01
- Fasting period before study:
- Housing: Rats were housed in standard polysulfone rat cages (size: Length 425 mm x Breadth 266 mm x Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube. Additionally, polycarbonate rat huts were placed inside the cage as environmental enrichment objects which were replaced once a week.

Following was the housing pattern during different periods of the experiment:
i. Pre mating / Acclimatization: Two rats of the same sex per cage were housed per cage.
ii. Mating: Female rats were cohabited with males in a 1:1 ratio in same cage.
iii. Post-mating / Treatment: After mating confirmation, females were housed individually.

Steam sterilized corn cob was used as bedding and was changed along with the cage at least twice a week.
- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet – pellet (Certified), ad libitum
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier, ad libitum
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 65 – 66 %
- Air changes (per hr): adequate fresh air supply (12 to 15 air changes/hour).
- Photoperiod (hrs dark / hrs light):12 hours light and 12 hours dark cycle.

IN-LIFE DATES: From: 27 October 2017
To:29 March 2018

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Remarks:

Milli-Q®

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The dose formulations were freshly prepared daily before dosing and were used within the stability period.

The required quantity of test item was weighed in pre-calibrated beaker*. A small volume (3 to 40 mL) of vehicle (Milli-Q® water) was added and mixed well using a glass rod. The final volume was made up with the vehicle to get the required final concentration. The volume was made up to the upper meniscus during dose formulation preparation.

*Pre-calibration of the beaker to desired volume: Milli-Q® water was measured in a graduated measuring cylinder to the final volume of the batch size (example if batch size was 70 mL, water was taken till 70 mL mark in graduated measuring cylinder). The measured water was transferred into a clean beaker (to be pre-calibrated) and upper and lower meniscus of water were marked on the beaker using a marker. Once these lines were marked, the water was discarded and the beaker was dried.

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): Based on the solubility test conducted under study, the test item is soluble in Milli-Q® water. Hence, Milli-Q® water was used as vehicle for dose formulation preparation.
- Concentration in vehicle: 0, 100, 300 and 1000 mg/kg bw
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and stability of the test chemical in vehicle was established at the concentrations of 0.7 mg/mL and 125 mg/mL. Based on the results, the test item was stable and homogenous in the vehicle up to 48 hours when stored at room temperature.

Details on mating procedure:

- Impregnation procedure: [artificial insemination / purchased timed pregnant / cohoused]: Cohoused
- If cohoused: the female rats were cohabited with males
- M/F ratio per cage: 1:1 ratio
- Length of cohabitation: On the first day, after vaginal smear examination, all the females with proestrous and estrous stages were retained with the males; while the other females were separated from males. Subsequently females were cohabited in batches of required numbers. These females were cohabited with respective males in a 1:1 ratio. The details were recorded in raw data. This procedure was continued until there were sufficient numbers of Day 0 pregnant rats for the study.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. No Data Available
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: [yes, vaginal smear were examination]
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol: Not specified.

Duration of treatment / exposure:

15 days

Frequency of treatment:

Daily

Duration of test:

From GD 5 to GD 19 of presumed gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control Group

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low Dose Group

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid Dose Group

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High Dose Group

No. of animals per sex per dose:

Total: 96
0 mg/kg bw: 24 female
100 mg/kg bw: 24 female
300 mg/kg bw: 24 female
1000 mg/kg bw: 24 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose range finding study was carried out at Test Facility using seven day ‘0’ pregnant rats per group at the doses of 100 (G2), 300 (G3) and 1000 (G4) mg/kg/day along with the concurrent vehicle control (G1). No effect were observed in treated female and Fetal examination. Therefore, based on the results of dose range finding study doses were selected as 0, 100, 300 and 1000 mg/kg bw.
- Rationale for animal assignment (if not random): Rats were randomly distributed to different groups by body weight stratification. Based on the body weight, Day ‘0’ pregnant rats were arranged in the ascending order. These rats were then assigned to the groups starting either from control and treatment group/s in the increasing order of dose or vice-versa on GD 0. As far as possible rats were assigned equally to the study groups, the extra animals remaining were also assigned to groups. This difference was negated on subsequent day/s by assigning rats in such a way that there were equal numbers of rats in all the groups.
- Other: No Data Available

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day - pre dose and post dose (30 minutes to one hour post dose, approx.) during treatment days and once on non-treatment days
- Cage side observations checked in table [No.?] were included. : morbidity and mortality i.e., once in the morning and once in the afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: weighed on gestation days 0, 3, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day # : On gestation day 20
- Organs examined: all visceral organs of dams were examined.

OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Gross evaluation of placenta

Fetal examinations:

- External examinations: Yes:All the fetuses were examined
- Soft tissue examinations: Yes: Approximately one half of the fetuses from each litter were subjected to visceral
- Skeletal examinations: Yes: the remaining half of the fetuses were subjected to skeletal evaluation.
- Head examinations: No data

Statistics:

The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female were analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss, external, visceral and skeletal observations for variations were analyzed using Kruskal wallis test for group comparison.

The incidence of with and without resorptions in dams will be tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.

Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated as * throughout the report.

Indices:

Embryonic resorption index, Fetal resorption index, Implantation index (%), Live fetus index, Dead fetus index were examined.

Historical control data:

Not specified

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed throughout the experimental period at any of the doses tested.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

There was no morbidity or mortality throughout the experimental period in the 100, 300 and 1000 mg/kg/day dose groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The maternal group mean body weights and maternal body weight gain were unaffected by the administration of test chemical at the doses of 100, 300 and 1000 mg/kg/day and were statistically comparable to vehicle control group.

The corrected body weight gain was statistically significantly increased at 1000 mg/kg/day as compared to vehicle control group indicating that body weights of rats were not affected by treatment with test chemical up to the high dose of 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The maternal food consumption was comparable to vehicle control group during different periods of gestation up to the high dose of 1000 mg/kg/day.

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

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effect on gravid uterine weights were observed in treated female rat as compared to control.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological findings in any of the tested doses.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No effect on pre and post-implantation loss rates were observed in treated female rats as compared to control.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No effect on loss rates were observed in treated female rats as compared to control.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No effect on early and late resorptions were observed in treated female rats as compared to control.

Dead fetuses:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in dead fetuses as compaed to control.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

No effect on preganacy duration were observed in treated female rats as compared to control.

Changes in number of pregnant:

not specified

Other effects:

not specified

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

maternal abnormalities

mortality

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in weight of male and female fetuses at 100, 300 and 1000 mg/kg /day.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No reduction in the number of live male and female fetuses were observed due to the administration of the test chemical.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in sex ratio of male and female fetuses at 100, 300 and 1000 mg/kg /day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No adverse effects or changes in the mean litter sizes and weights were observed due to the administration of the test chemical.

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed during external observations of fetuses of dams treated up to 1000 mg/kg/day.

Normal variants: There was no incidence of normal variant in any of the doses tested.

Minor anomalies: There was an incidence of a small fetus each in the vehicle control and low dose group. This finding was not of any toxicological significance as this is commonly observed in rat fetuses.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed during skeletal examination of fetuses of dams treated up to 1000 mg/kg/day.

Normal Variants: Incidences of normal variants were comparable between the vehicle control and the treatment groups.

Minor anomalies: The incidences of minor anomalies were statistically comparable to vehicle control group at all the doses tested.

Major malformations: There were no incidences of major malformations in any of the treatment groups.

Visceral malformations:

no effects observed

Description (incidence and severity):

No changes were observed during visceral examination of fetuses of dams treated up to 1000 mg/kg/day.

Normal Variants, Minor anomalies and Major malformations: There were no incidences of normal variants, minor anomalies and major malformations in any of the doses tested.

Other effects:

not specified

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Treatment related:

no

Any other information on results incl. tables

 Summary of Clinical Signs and Mortality

	Group No.	G1	G2	G3	G4
Observations	Dose (mg/kg/day)	0	100	300	1000
	Total No. of rats found sperm positive	24	24	24	24
Clinical signs		NAD	NAD	NAD	NAD
Mortality		-------------None------------

Summary of maternal group mean body weight (g)

Group No.	Dose (mg/kg/day)	No. of Rats	Group mean body weight (g) on GD
				0	3	5	8	11	14	17	20
G1	0	21	Mean	214.18	222.98	229.95	238.57	252.79	263.87	288.02	314.65
			SD	14.23	14.95	14.68	15.39	16.72	16.62	18.80	22.58
G2	100	23	Mean	216.39	226.08	234.32	242.56	256.20	267.26	291.98	320.04
			SD	15.37	17.51	18.91	20.47	22.54	24.88	27.77	32.57
G3	300	23	Mean	216.68	226.07	233.55	241.12	255.23	266.69	289.97	319.69
			SD	14.73	17.20	18.51	19.99	22.27	22.58	23.32	25.81
G4	1000	24	Mean	214.93	223.73	230.53	239.52	252.39	262.80	285.57	317.15
			SD	13.14	15.71	17.55	20.17	22.05	23.02	27.70	31.98

Summary of maternal body weight gain (g)

Period of treatment (days of gestation)	Group No.		G1	G2	G3	G4
	Dose (mg/kg/day)		0	100	300	1000
	No. of Rats		21	23	23	24
Pre-treatment period (Days 0-5)		Mean	15.77	17.93	16.88	15.60
		SD	3.97	5.36	5.59	5.91
Treatment Period (Days 5-20)		Mean	84.70	85.72	86.13	86.62
		SD	11.05	15.85	11.20	19.02
Entire gestation (Days 0-20)		Mean	100.47	103.65	103.01	102.21
		SD	13.57	19.70	15.14	22.16

 

Summary of Corrected Body Weight and Body Weight Gain (g)

Group No.	Dose (mg/kg/day)	No. of Rats		Gestation Day 5	Terminal body wt on GD 20	Uterine Wt (g)	Carcass weight (corrected body weight on GD 20)	Corrected Bwt gain. (g)
G1	0	21	Mean	58.07	26.63	70.64	244.01	14.06
			SD	7.33	6.78	7.69	20.84	11.60
G2	100	23	Mean	57.65	28.07	64.08	255.97	21.64
			SD	11.05	6.96	12.29	25.60	11.09
G3	300	23	Mean	56.41	29.72	65.39	254.30	20.75
			SD	8.28	6.40	12.49	23.09	10.63
G4	1000	24	Mean	55.04	31.58	60.94	256.21	25.67*
			SD	13.67	7.59	20.30	23.17	9.44

  *: Significantly different from G1                                                 GD: Gestation day          

 

Corrected body weight on gestation day 20 (Carcass weight) = Terminal body weight on
day 20 - unopened uterine weight.

 

Corrected body weight gain = carcass weight - body weight on day 5.

Summary of Maternal Data

	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	100	300	1000
	No. of Rats	21	23	23	24
Gravid uterine weight (g)	Mean	70.64	64.08	65.39	60.94
	SD	7.69	12.29	12.49	20.30
Number of Corpora lutea	Mean	14.38	13.52	13.70	13.13
	SD	1.32	1.50	2.08	2.05
Number of Implantations	Mean	13.43	12.74	12.83	11.42
	SD	1.60	2.32	2.62	3.90
Early Resorptions	Mean	0.43	0.61	0.57	0.25
	SD	0.68	0.89	0.73	0.44
Late Resorptions	Mean	0.05	0.13	0.09	0.13
	SD	0.22	0.34	0.29	0.34
Pre-implantation Loss	Mean	0.95	0.78	0.87	1.71
	SD	1.32	1.31	1.01	2.26
Post-implantation Loss	Mean	0.48	0.74	0.65	0.38
	SD	0.68	0.92	0.71	0.49
Dams with any Resorption	Total	8	11	12	9

	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	100	300	1000
	No. of Rats	21	23	23	24
Early Resorptions (%)	Mean	3.02	5.12	4.20	2.10
	SD	4.76	7.26	5.35	3.77
Late Resorptions (%)	Mean	0.34	0.98	1.30	0.94
	SD	1.56	2.60	4.58	2.54
Pre-implantation Loss (%)	Mean	6.46	6.32	7.06	15.85
	SD	8.78	11.86	9.82	24.98
Post-implantation Loss (%)	Mean	3.36	6.10	5.50	3.03
	SD	4.79	7.43	6.17	4.06
Implantation Index (%)	Mean	93.54	93.68	92.94	84.15
	SD	8.78	11.86	9.82	24.98

 Summary of Litter Data

	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	100	300	1000
	No. of Rats	21	23	23	24
No. of litters		21	23	23	24
Total no. of fetuses		272	276	280	265
Mean litter size		13.0	12.0	12.2	11.0
Total live fetuses
a.  Number		272	276	280	265
b.  Weight (g)	Mean	3.57	3.48	3.49	3.66
	SD	0.24	0.21	0.29	0.47
Live male fetuses
a.  Number		146	134	142	137
b.  Weight (g)	Mean	3.66	3.56	3.58	3.73
	SD	0.26	0.23	0.30	0.48
Live female fetuses
a.  Number		126	142	138	128
b.  Weight (g)	Mean	3.46	3.41	3.41	3.52
	SD	0.22	0.21	0.30	0.23
Sex Ratio - Male : Female		1:0.86	1:1.06	1:0.97	1:0.93
(% of male fetuses)		(53.7%)	(48.6%)	(50.7%)	(51.7%)

 Summary of Gross Pathological Findings

Group No.	G1	G2	G3	G4
Parameters  Dose (mg/kg/day)	0	100	300	1000
1.  No. of rats subjected to caesarean section	24	24	24	24
2.  No. of rats pregnant at caesarean section	21	23	23	24
3.  No. of rats showing gross pathology	0	0	0	0

Summary of Fetal External Observations (Incidence and Percentage)

Group No.	G1				G2				G3				G4
Dose (mg/kg/day)	0				100				300				1000
No. of litters examined	21				23				23				24
No. of fetuses examined	272				276				280				265
	Fetus		Litter		Fetus		Litter		Fetus		Litter		Fetus		Litter
	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%
Normal Variant	None
Minor Anomalies
Small fetus	1	0.37	1	4.76	1	0.36	1	4.35	0	0.00	0	0.00	0	0.00	0	0.00
Major Malformations	None

 Summary of Fetal Visceral Observations (Incidence and Percentage)

Group No.	G1				G2				G3				G4
Dose (mg/kg/day)	0				100				300				1000
No. of litters examined	21				23				23				24
No. of fetuses examined	131				132				132				126
	Fetus		Litter		Fetus		Litter		Fetus		Litter		Fetus		Litter
	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%
Normal Variant	None
Minor Anomalies	None
Major Malformations	None

Applicant's summary and conclusion

Conclusions:

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for Maternal toxicity and fetal developmental toxicity is 1000 mg/kg/day in Wistar rats when the test chemical was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.

Executive summary:

The objective of this study was to evaluate the embryo-fetal developmental toxicity of test chemical when administered to pregnant Wistar rats by oral route during gestation days GD 5 to GD 19. The results of this study helped to establish the No Observed Adverse Effect Level (NOAEL) of the test item for maternal and developmental toxicity in rats. In this study, each group (G1, G2, G3 and G4) consisted of 24 presumed pregnant Wistar rats (gestation day 0). Day '0' of gestation for each individual female rat in the study was considered as the day on which vaginal plug was observed or vaginal smear was found sperm positive. The test chemical was dissolved in vehicle - Milli-Q water and administered orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 100, 300 and 1000 mg/kg/day for low (G2), mid (G3) and high (G4) dose group rats, respectively. The rats in the vehicle control (G1) group received the vehicle alone. A constant dose volume of 10 mL/kg body weight was administered to all groups. The dose formulation solutions were analyzed for active ingredient concentrations at the initiation and termination of treatment. The results of analysis of formulations revealed that the analyzed concentrations were within the acceptable limits. The mated females were observed twice daily for clinical signs, mortality and morbidity. Body weights and food consumption were monitored during the different periods of gestation. The intermittent body weight gain and food consumption was calculated and presented for rats found pregnant at caesarean section. Caesarean section was performed for all the rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the dams was removed (by laparotomy) and the contents were examined. The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. The number of corpora lutea was counted on each ovary. All the fetuses were sexed, weighed and examined for external malformations. Approximately half the number of fetuses from each litter was examined for visceral malformations and the remaining half was evaluated for skeletal malformations. During observations, there were no mortalities and clinical signs at all the doses tested. The maternal body weights and food consumption were comparable to vehicle control group up to the highest dose of 1000 mg/kg/day. The maternal data parameters comprising of mean number of corpora lutea, implantations, number of early and late resorptions, pre and post implantation loss and dams with any resorptions were comparable to vehicle control group at all the doses tested. Gross evaluation of placenta revealed no findings. The litter data parameters comprising of total number of fetuses, mean weights of male and female fetuses and sex ratio were comparable to vehicle control group at all the doses tested. Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs of teratogenicity in any of the tested doses. Therefore, based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for Maternal toxicity and fetal developmental toxicity is 1000 mg/kg/day in Wistar rats when the test chemical was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.