5,12-dihydro-2,9-dimethylquino[2,3-b]acridine-7,14-dione

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 April 2021 to 08 November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

As per OECD Test Guideline No. 414, “Prenatal Developmental Toxicity Study”, adopted on 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5,12-dihydro-2,9-dimethylquino[2,3-b]acridine-7,14-dione

EC Number:

213-561-3

EC Name:

5,12-dihydro-2,9-dimethylquino[2,3-b]acridine-7,14-dione

Cas Number:

980-26-7

Molecular formula:

C22H16N2O2

IUPAC Name:

2,9-dimethyl-5,7,12,14-tetrahydro-5,12-diazapentacene-7,14-dione

Test material form:

solid: nanoform

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility and stability of the test substance in the solvent/vehicle: The test item was insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxymethyl Cellulose at the concentration of 100 mg/mL, the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight as per in-house solubility/suspendibility test results.
Hence, 0.5% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations. Also, the prepared test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxymethyl Cellulose within the mean percent recovery range of 85 to 115%.

FORM AS APPLIED IN THE TEST: The test item/vehicle were administered to animals through oral gavage using stainless steel intubation cannula attached to a disposable syringe once daily.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology (India) Pvt. Ltd., Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) Registration No.: 1808/PO/RcBt/S/15/CPCSEA.
- Age at study initiation: 10 to 12 weeks
- Weight at study initiation: Males: 259.08 to 298.31 g; Females: 210.61 to 269.75 g
- Fasting period before study: No
- Housing:
i. Pre-mating/Acclimatization
Maximum of three animals of same sex per cage were housed in sterilized standard polypropylene cage (Size: L 430 × B 285 × H 150 mm). Steam sterilized clean paddy husk were used as bedding material and were changed along with the cage twice a week.
ii. Mating
During mating, three rats (one male and two females) were housed in standard polypropylene cages.
iii. Post mating
After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polypropylene cages.
- Diet: Altromin Spezialfutter GmbH & Co. KG
- Water: Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 17 April 2021 to 21 April 2021 [for the first batch of animals left for cohabitation] / 09 May 2021 [for the last batch of animals left for cohabitation].

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 to 24.8oC
- Humidity (%): 49 to 62%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 17 April 2021 To: 10 June 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose samples of the test item were analyzed for Homogeneity and dose concentration formulation by UV-Vis method. The results are found to be within the acceptable range of ±15% to the nominal concentration and relative standard deviation (%RSD) was ˂ 10%.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:2
- Length of cohabitation: 14 Days
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility: Yes
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

The test item/vehicle was administered to respective group of female rats once daily from gestation day 5 to 19.

Frequency of treatment:

Once Daily

Duration of test:

15 days for each presumed pregnant female.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle Control

Doses / concentrations 2

Dose / conc.:

111 mg/kg bw/day

Remarks:

Low Dose

Doses / concentrations 3

Dose / conc.:

333 mg/kg bw/day

Remarks:

Mid Dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High Dose

No. of animals per sex per dose:

25 presumed pregnant females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A dose range finding study for prenatal developmental toxicity study with the test item by oral gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 was conducted with the doses of 0, 111, 333 and 1000 mg/kg body weight/day for vehicle control, low, mid and high dose groups, respectively in consultation with the sponsor. The study included the general and maternal end point assessments such as, maternal body weights, feed consumption, corrected body weight for maternal body weight gain, gravid uterus weight, uterine content observations, implantations and gross pathological examinations. The dose range finding study also included the fetal/prenatal developmental assessments such as, fetal weights (sex wise) per litter, fetal external examinations and fetal gross soft tissue examinations. The results of this range finding study did not produce any indication of maternal and fetal (prenatal developmental) toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Hence, the same dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid and high dose groups, respectively in consultation with the sponsor.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once Daily
- Cage side observations checked in table [No.1] were included.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on gestation days (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Ovaries, Uterus with cervix, thyroid and parathyroid glands.

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: one-half of fetuses from each litter (even numbered)
- Skeletal examinations: Yes: one-half of fetuses from each litter (odd numbered)
- Head examinations: Yes: one-half of fetuses from each litter (even numbered)

Statistics:

The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data. After verification, the data was subjected to various statistical analysis using SPSS software version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests was designated by the superscripts throughout the report as stated below:
* Statistically significant (P<0.05) change than the control group.

Note: Data of non-pregnant females and dams with total implantation loss was included for mean calculation and statistical analysis of systemic end points, but excluded for maternal end points. However, the individual animal data is presented in the appendices.

The parameter / end point and type of statistical analysis test followed is as mentioned below:

Parametric: One-way ANOVA with Dunnett’s post test:
• Maternal body weight
• Percent change in maternal body weight
• Corrected body weight for maternal increase
• Gravid uterus weight
• Maternal Feed consumption
• Mean fetal weight per dam
• Mean fetal crown rump length per dam
• Mean fetal anogenital distance per dam
• Serum T3, T4 and TSH levels
• Organ weight

Non-Parametric: Kruskal-Wallis followed by the Mann-Whitney test:
• No. of corpora lutea per dam
• No. of implantations per dam
• Litter size per dam
• No. of live/dead fetuses per dam
• Percent of live/dead fetuses per dam
• No. of early/late resorptions (if any) per dam
• Percent of early/late resorptions (if any) per dam
• Sex ratio (m/f) per dam
• Pre/Post implantation losses (%) per dam
• Fetal external / visceral / skeletal anomalies per dam

Frequencies Comparison: Cross Tabs - Chi-square test:
• Frequencies comparison,
- No. of pregnant / non-pregnant
- No. of dams with / without live fetuses
- No. of dams with / without dead fetuses
- No. of dams with / without resorptions

Indices:

Corrected Body Weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) - Gravid uterus weight
Pre-implantation Loss (%) = Total Number of Corpora lutea - Number of Implantation sites / Total Number of Corpora lutea x 100
Post-implantation Loss (%) = Number of Implantation sites - Number of Viable Fetuses / Number of Implantation Sites x 100
Anogenital Distance (AGD) Ratio = Anogenital Distance (mm) / Cube root of fetal weight x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity noted in any of the animals from vehicle control and all the tested dose groups throughout the experimental period.

Mortality:

no mortality observed

Description (incidence):

There were no morbidity/mortality noted in any of the animals from vehicle control and all the tested dose groups throughout the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean gestational (maternal) body weight and percent change in mean gestational (maternal) body weight gain during pregnancy in all the tested dose groups throughout the experimental period when compared with vehicle control group.

There were no changes noted in mean maternal body weight change corrected for gravid uterus weight in all the tested dose groups when compared with vehicle control group.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no changes noted in mean gestational (maternal) feed consumption in all the tested dose groups when compared with vehicle control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The thyroid along with parathyroid was collected, preserved and weighed post fixation from all the dams of each group. There were no changes or statistically significant differences noted for mean absolute and relative weight of thyroid along with parathyroid in all the tested dose groups when compared to the vehicle control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes noted in any of the animals from all the tested dose groups and vehicle control group during conduct of the necropsy.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related histopathological findings noted in thyroid and parathyroid glands from any of the animals of all the tested dose groups.
However, ultimobranchial cysts and ectopic thymus in thyroid gland were noted in this study during microscopic examination. These changes were considered as incidental findings as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats. (Elizabeth McInnes, 2012).

Other effects:

no effects observed

Description (incidence and severity):

There were no changes or statistically significant differences noted for mean serum T3 levels (ng/mL), T4 levels (ng/mL) and TSH levels (µIU/mL) in any of the tested dose groups when compared to the vehicle control group.

Details on results:

There were no systemic/general toxicity effects noted in all the tested dose groups under the experiemntal conditional employed.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

no abortions were noted in any of the tested dose group dams.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

a. Pre-Implantation Loss
The mean percentage of pre-implantation loss per litter was 6.34, 5.48, 5.48 and 5.64 for groups G1, G2, G3 and G4 respectively. There were no changes or statistically significant differences noted for percentage of pre-implantation loss per litter across dose groups when compared to the vehicle control group.

b. Post-Implantation Loss
The mean percentage of post-implantation loss per litter was 6.23, 8.52, 4.37 and 6.60 for G1, G2, G3 and G4 respectively. There were no changes or statistically significant differences noted for mean percentage of post-implantation loss per litter across dose groups when compared to the vehicle control group.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One incidental finding with total implantation/litter loss was noted in 1 out of 25 females from group G3. This occurence is an alone and incidental finding.

Early or late resorptions:

no effects observed

Description (incidence and severity):

a.Very Early Resorptions/Empty Implantation Sites per Litter:
The mean number of very early resorptions per litter was 0.14, 0.09, 0.10 and 0.00 with a percentage of was 1.42, 1.01, 1.59 and 0.00 for groups G1, G2, G3 and G4 respectively. There were no changes or statistically significant differences noted for mean number and percentage of very early resorptions per dam across dose groups when compared to the vehicle control group.


b.Early Resorptions per Litter:
The mean number of early resorptions per litter was 0.48, 0.59, 0.38 and 0.45 with a percentage of 4.81, 5.92, 2.78 and 4.80 for groups G1, G2, G3 and G4 respectively. There were no changes or statistically significant differences noted for mean number and percentage of early resorptions per dam across dose groups when compared to the vehicle control group.

c.Late Resorptions per Litter:
The mean number of late resorptions per litter was 0.00, 0.14, 0.00 and 0.14 with a percentage of 0.00, 1.59, 0.00 and 1.80 for groups G1, G2, G3 and G4 respectively.

There were no changes or statistically significant differences noted for mean number and percentage of late resorptions per dam across dose groups when compared to the vehicle control group.

Dead fetuses:

no effects observed

Description (incidence and severity):

no dead fetuses noted in any of the dams from all the dose groups.

Description (incidence and severity):

no effects observed
No animals were littered within gestation day 20 and all the pregnant females were subjected to caesarean section.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A total of 21 (out of 25), 22 (out of 25), 21 (out of 25) and 22 (out of 25) females out were found with live fetuses yielding to pregnancy with a pregnancy rates of 84%, 88%, 84% and 88% respectively from group G1 (0 mg/kg body weight/day), G2 (111 mg/kg body weight/day), G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day). One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G3.

Details on maternal toxic effects:

The oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 - ca. 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food efficiency

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

other: thyroid hormonal [T3/T4 and TSH] levels

Maternal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

uterus

Description (incidence and severity):

One incidental finding with total implantation loss (with empty implantation sites) was noted in 1 out of 25 females from group G3.

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean male fetal weight per litter was 4.27 g, 4.36 g, 4.31 g and 4.36 g and the mean female fetal weight per litter was 4.21g, 4.37g, 4.25g and 4.34 g for groups G1, G2, G3 and G4 respectively. There were no changes or statistically significant differences noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no changes or statistically significant differences noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean litter sizes, assessed as the total number of fetuses in uteri [live plus dead] per dam, was 9.90, 10.05, 11.48 and 10.82 for groups G1, G2, G3 and G4, respectively. There were no changes or no statistically significant differences noted in all the tested dose groups when compared with vehicle control group for mean litter size.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio per litter calculated as the mean of male/female fetuses per litter was 1.16, 1.28, 1.09 and 1.13 for G1, G2, G3 and G4 respectively. There were no changes or statistically significant differences noted for mean male/female sex ratio across the dose groups when compared to the control group.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no changes or no statistically significant differences noted in all the tested dose groups when compared with vehicle control group for mean litter size.

There were no changes or statistically significant differences noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.

External malformations:

no effects observed

Description (incidence and severity):

A total of 208 (21), 221 (22), 241 (21) and 238 (22) fetuses (litters) were available for gross external examination from groups G1, G2, G3 and G4 respectively.
There were no test item-related fetal structural and developmental external malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were 108 (21), 116 (22), 127 (21), and 123 (22) fetuses (litters) for skeletal examination from groups G1, G2, G3 and G4 respectively.
There were no test item-related fetal skeletal malformations or skeletal developmental variations for any of the fetuses examined from all the tested dose group litters.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were 100 (21), 105 (22), 114 (21) and 115 (22) fetuses (litters) for visceral/soft tissue examination from groups G1, G2, G3 and G4 respectively.
There were no test item-related fetal fetal structural and developmental visceral/soft tissue malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred.

Other effects:

no effects observed

Description (incidence and severity):

The mean male fetal crown rump length per litter was 38.01 mm, 38.17 mm, 38.12 mm and 38.04 mm and the female mean crown rump length per litter was 37.82 mm,
37.89 mm, 37.83 mm and 37.82 mm for groups G1, G2, G3 and G4 respectively. There were no changes or statistically significant differences noted in mean fetal crown rump length of either sex per litter across the dose groups when compared with the vehicle control group.

The mean male fetal anogenital distance per litter was 4.46 mm, 4.47 mm, 4.50 mm and 4.47 mm and the mean female anogenital distance per litter was 2.50 mm, 2.48 mm, 2.48 mm and 2.49 mm for groups G1, G2, G3 and G4 respectively. The mean male fetal anogenital distance ratio per litter was 2.76, 2.74, 2.77 and
2.74 and the mean female anogenital distance ratio per litter was 1.55, 1.52, 1.53 and 1.53 for groups G1, G2, G3 and G4 respectively. There were no changes or statistically significant difference noted in mean fetal anogenital distance and mean fetal anogenital distance ratio per litter in either sex across the dose groups when compared with the vehicle control group.

Details on embryotoxic / teratogenic effects:

The oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of fetal/developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

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

other: anogenital distance and fetal crown rump length

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

The fetal developmental and or structural alterations are considered incidental as these observations are comparable with the vehicle control group and also these alterations are common for this species and strain.

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

TABLE 1. SUMMARY OF PREGNANCY STATUS RECORD

Group & Dose (mg/kg body weight/day)	Pregnancy Status
	No. of presumed pregnant females / Group	No. of females confirmed with pregnancy	No. of females with total implantation loss	No. of females confirmed as non-pregnant	Rate of Pregnancy (%)
G1 & 0	25	21	0	4	84.0
G2 & 111	25	22	0	3	88.0
G3 & 333	25	21	1	3	84.0
G4 & 1000	25	22	0	3	88.0

%: Percentage.

TABLE 2. SUMMARY OF CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD

Group & Dose (mg/kg body weight/day)	Clinical Signs and Mortality Record
	Clinical Signs of Toxicity (No. of Animals revealed)	No. of Mortalities / Total No. of animals
G1 & 0	N (25)	0/25
G2 & 111	N (25)	0/25
G3 & 333	N (25)	0/25
G4 & 1000	N (25)	0/25

N: Normal.

TABLE 3. SUMMARY OF GESTATION BODY WEIGHT (g) RECORD

Group & Dose (mg/kg body weight/day)		Body Weight (g) on Gestation Day (GD)
		0	3	5	8	11	14	17	19	20
G1 & 0	Mean	257.53	262.09	267.77	276.16	290.02	306.16	330.67	349.43	357.59
	±SD	13.51	13.31	12.19	11.60	13.79	14.23	20.15	23.64	23.11
	n	21	21	21	21	21	21	21	21	21
G2 & 111	Mean	259.94	264.46	269.36	277.70	290.19	306.10	329.59	350.19	359.64
	±SD	13.57	13.45	13.25	14.34	14.39	16.27	18.79	22.86	21.45
	n	22	22	22	22	22	22	22	22	22
G3 & 333	Mean	260.60	265.12	270.47	279.49	292.28	310.46	337.17	358.29	366.43
	±SD	11.03	11.35	11.59	12.82	13.07	15.40	19.95	24.72	24.84
	n	21	21	21	21	21	21	21	21	21
G4 & 1000	Mean	258.20	263.18	268.41	277.82	291.93	309.08	332.99	351.60	360.82
	±SD	13.18	13.31	13.16	13.46	13.12	15.15	17.75	20.50	20.38
	n	22	22	22	22	22	22	22	22	22

SD: Standard Deviation; n: Number of Dams.             

Note: The data of 4, 3, 3 & 3 non-pregnant animals from groups G1, G2, G3 and G4 respectively, 1 animal with total implantation loss from group G3 are excluded for mean calculations, but presented in individual animal data. 

TABLE 4. SUMMARY OF PERCENT CHANGE IN GESTATION BODY WEIGHT GAIN (%) RECORD

Group & Dose (mg/kg body weight/day)		Percent Change in Body Weight gain (%) during Gestation Day (GD)
		0 to 3	3 to 5	5 to 8	8 to 11	11 to 14	14 to 17	17 to 19	19 to 20
G1 & 0	Mean	1.78	2.20	3.15	5.01	5.58	7.98	5.66	2.36
	±SD	0.43	0.77	0.93	1.85	1.76	3.43	2.52	1.39
	n	21	21	21	21	21	21	21	21
G2 & 111	Mean	1.74	1.86	3.09	4.52	5.49	7.69	6.22	2.76
	±SD	0.50	0.50	0.89	1.48	2.18	3.03	2.38	2.01
	n	22	22	22	22	22	22	22	22
G3 & 333	Mean	1.73	2.02	3.33	4.59	6.22	8.57	6.23	2.29
	±SD	0.53	0.74	0.88	1.43	2.06	2.24	2.55	1.79
	n	21	21	21	21	21	21	21	21
G4 & 1000	Mean	1.93	2.00	3.51	5.11	5.88	7.74	5.58	2.64
	±SD	0.47	1.00	0.97	1.45	2.07	2.65	2.02	1.02
	n	22	22	22	22	22	22	22	22

SD: Standard Deviation; n: Number of Dams.             

Note: The data of 4, 3, 3 & 3 non-pregnant animals from groups G1, G2, G3 and G4 respectively, 1 animal with total implantation loss from group G3 are excluded for mean calculations, but presented in individual animal data. 

TABLE 5. SUMMARY OF GRAVID UTERUS WEIGHT (g) AND MATERNAL BODY WEIGHT CHANGE CORRECTED FOR GRAVID UTERINE WEIGHT (g)

Group & Dose (mg/kg body weight/day) Body Weight Change (g) from GD 5 to GD 20 Gravid Uterus Weight (g) Corrected Body weight (Gram) Corrected Body weight (Percentage) G1 & 0 Mean 89.81 62.82 27.00 10.11 ±SD 20.75 17.98 9.85 3.83 n 21 21 21 21 G2 & 111 Mean 90.28 62.27 28.01 10.52 ±SD 20.76 19.05 9.97 4.02 n 22 22 22 22 G3 & 333 Mean 95.96 68.91 27.05 10.05 ±SD 20.48 18.78 7.51 2.88 n 21 21 21 21 G4 & 1000 Mean 92.41 66.74 25.67 9.60 ±SD 16.23 16.99 9.88 3.73 n 22 22 22 22

SD: Standard Deviation; n: Number of Dams; GD: Gestation Day.

Corrected Body Weight (g)

=

(Gestation day 20 body weight - Gestation day 5 body weight) - Gravid uterus weight

TABLE 6. SUMMARY OF AVERAGE GESTATION FEED CONSUMPTION (g/animal/day) RECORD

Group & Dose (mg/kg body weight/day)		Feed Consumption (gm/animal/day) during Gestation Day (GD)
		0 to 3	3 to 5	5 to 8	8 to 11	11 to 14	14 to 17	17 to 19	19 to 20
G1 & 0	Mean	14.55	19.84	17.58	19.36	21.07	23.81	26.08	28.66
	±SD	0.94	3.32	0.79	1.01	1.74	1.30	3.14	3.38
	n	21	21	21	21	21	21	21	21
G2 & 111	Mean	14.80	19.81	17.30	19.23	20.74	23.85	26.59	28.00
	±SD	1.19	3.00	0.99	0.77	1.66	2.56	3.47	3.28
	n	22	22	22	22	22	22	22	22
G3 & 333	Mean	15.14	19.08	17.33	19.79	21.56	24.67	25.43	29.32
	±SD	1.12	2.58	1.14	0.84	1.00	0.98	1.75	3.40
	n	21	21	21	21	21	21	21	21
G4 & 1000	Mean	14.60	19.01	16.85	19.38	21.25	24.25	25.36	29.47
	±SD	0.99	2.53	1.32	0.86	1.38	1.26	1.97	4.10
	n	22	22	22	22	22	22	22	22

SD: Standard Deviation; n: Number of Dams.

Note: The data of 4, 3, 3 & 3 non-pregnant animals from groups G1, G2, G3 and G4 respectively, 1 animal with total implantation loss from group G3 are excluded for mean calculations, but presented in individual animal data. 

TABLE 7. SUMMARY OF UTERINE OBSERVATIONS PER LITTER RECORD

Group & Dose (mg/kg body weight/day)		Corpora luetea (No.)	Implantation sites (No.)	Litter Size (No.)	Live Fetuses			Dead Fetuses (No.)	Very Early Resorptions/ Empty Implantation sites (No.)	Early Resorptions (No.)	Late Resorptions (No.)
					Total (No.)	Male (No.)	Female (No.)
G1 & 0	Mean	11.10	10.52	9.90	9.90	4.95	4.95	0.00	0.14	0.48	0.00
	±SD	2.79	3.22	3.35	3.35	2.44	2.01	0.00	0.48	0.81	0.00
	n	21	21	21	21	21	21	21	21	21	21
G2 & 111	Mean	11.36	10.86	10.05	10.05	5.36	4.68	0.00	0.09	0.59	0.14
	±SD	3.09	3.52	3.99	3.99	2.74	1.89	0.00	0.43	0.96	0.47
	n	22	22	22	22	22	22	22	22	22	22
G3 & 333	Mean	12.48	11.95	11.48	11.48	5.76	5.71	0.00	0.10	0.38	0.00
	±SD	3.30	3.75	3.82	3.82	2.79	2.10	0.00	0.44	0.80	0.00
	n	21	21	21	21	21	21	21	21	21	21
G4 & 1000	Mean	11.95	11.41	10.82	10.82	5.23	5.59	0.00	0.00	0.45	0.14
	±SD	2.26	2.91	3.39	3.39	2.29	2.34	0.00	0.00	0.96	0.47
	n	22	22	22	22	22	22	22	22	22	22

SD: Standard Deviation; n: Number of Dams.

TABLE 8. SUMMARY OF MATERNAL RECORD PER LITTER

Group & Dose (mg/kg body weight/day)		Pre- Implantation Loss (%)	Post- Implantation Loss (%)	Live Fetuses (%)	Dead Fetuses (%)	Very Early Resorptions/ Empty Implantation sites (%)	Early Resorptions (%)	Late Resorptions (%)	Male/Female Sex ratio	Male Fetuses (%)	Female Fetuses (%)
G1 & 0	Mean	6.34	6.23	100.00	0.00	1.42	4.81	0.00	1.16	48.31	51.69
	±SD	11.41	12.50	0.00	0.00	5.05	9.16	0.00	0.87	14.90	14.90
	n	21	21	21	21	21	21	21	21	21	21
G2 & 111	Mean	5.48	8.52	100.00	0.00	1.01	5.92	1.59	1.28	52.53	47.47
	±SD	10.67	16.27	0.00	0.00	4.74	10.31	5.65	0.74	12.21	12.21
	n	22	22	22	22	22	22	22	22	22	22
G3 & 333	Mean	5.48	4.37	100.00	0.00	1.59	2.78	0.00	1.09	48.86	51.14
	±SD	11.07	8.79	0.00	0.00	7.27	5.80	0.00	0.58	12.94	12.94
	n	21	21	21	21	21	21	21	21	21	21
G4 & 1000	Mean	5.64	6.60	100.00	0.00	0.00	4.80	1.80	1.13	48.09	51.91
	±SD	11.21	16.09	0.00	0.00	0.00	10.78	7.17	0.88	14.57	14.57
	n	22	22	22	22	22	22	22	22	22	22

SD: Standard Deviation; n: Number of Dams.

TABLE 9. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group & Dose (mg/kg body weight/day)		Thyroid along with parathyroid^#
G1 & 0	Mean	0.0222
	±SD	0.0019
	n	25
G2 & 111	Mean	0.0215
	±SD	0.0022
	n	25
G3 & 333	Mean	0.0210
	±SD	0.0022
	n	25
G4 & 1000	Mean	0.0220
	±SD	0.0028
	n	25

SD: Standard Deviation; n: Number of Dams;^#: Weighed post fixation. 

TABLE 10. SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TERMINAL BODY WEIGHT RECORD

Group & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Thyroid along with parathyroid
G1 & 0	Mean	346.11	0.0065
	±SD	34.21	0.0008
	n	25	25
G2 & 111	Mean	351.71	0.0061
	±SD	29.85	0.0008
	n	25	25
G3 & 333	Mean	353.27	0.0060
	±SD	38.52	0.0008
	n	25	25
G4 & 1000	Mean	351.04	0.0063
	±SD	33.19	0.0009
	n	25	25

SD: Standard Deviation; n: Number of Dams.

TABLE 11. SUMMARY OF SERUM TRIIODOTHYRONINE (T3) LEVELS (ng/mL) RECORD           

Group & Dose (mg/kg body weight/day)		Serum T3 Levels(ng/mL)
G1 & 0	Mean	2.305
	±SD	0.367
	n	25
G2 & 111	Mean	2.141
	±SD	0.358
	n	25
G3 & 333	Mean	2.119
	±SD	0.302
	n	25
G4 & 1000	Mean	2.202
	±SD	0.302
	n	25

SD: Standard Deviation; n: Number of Dams; ng/mL: Nano gram per milliliter.

TABLE 12. SUMMARY OF SERUM THYROXINE (T4) LEVELS (ng/mL) RECORD

Group & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1 & 0	Mean	63.879
	±SD	10.371
	n	25
G2 & 111	Mean	57.858
	±SD	8.790
	n	25
G3 & 333	Mean	62.972
	±SD	14.263
	n	25
G4 & 1000	Mean	57.050
	±SD	9.921
	n	25

SD: Standard Deviation; n: Number of Dams; ng/mL: Nano gram per milliliter.

TABLE 13. SUMMARY OF SERUM THYROID STIMULATING HORMONE (TSH) LEVELS (µIU/mL) RECORD     

Group & Dose (mg/kg body weight/day)		Serum TSH Levels(µIU/mL)
G1 & 0	Mean	4.331
	±SD	4.325
	n	25
G2 & 111	Mean	6.165
	±SD	7.547
	n	25
G3 & 333	Mean	3.726
	±SD	4.368
	n	25
G4 & 1000	Mean	6.888
	±SD	8.626
	n	25

  SD: Standard Deviation; n: Number of Dams considered for mean calculations; µIU/mL: Micro International Unit per milliliter.

TABLE 14. SUMMARY OF MEAN FETAL WEIGHT, MEAN FETAL CROWN RUMP LENGTH, MEAN FETAL ANOGENITAL DISTANCE AND RATIO RECORD

Group & Dose (mg/kg body weight/day)		Fetal Weight (g)			Crown rump Length (mm)			Anogenital Distance Measurement (mm)			Anogenital Distance Ratio
		Male	Female		Male	Female		Male	Female		Male	Female
G1 & 0	Mean	4.27	4.21		38.01	37.82		4.46	2.50		2.76	1.55
	±SD	0.22	0.29		0.38	0.50		0.12	0.06		0.10	0.06
	n	21	21		21	21		21	21		21	21
G2 & 111	Mean	4.36	4.37		38.17	37.89		4.47	2.48		2.74	1.52
	±SD	0.30	0.32		0.47	0.61		0.15	0.11		0.10	0.06
	n	22	22		22	22		22	22		22	22
G3 & 333	Mean	4.31	4.25		38.12	37.83		4.50	2.48		2.77	1.53
	±SD	0.25	0.32		0.27	0.90		0.14	0.09		0.09	0.08
	n	21	21		21	21		21	21		21	21
G4 & 1000	Mean	4.36	4.34		38.04	37.82		4.47	2.49		2.74	1.53
	±SD	0.21	0.27		0.43	0.55		0.11	0.09		0.07	0.05
	n	22	22		22	22		22	22		22	22

SD: Standard Deviation; n: Number of Dams.

Note: The presented mean values are group mean of all litters from each group which was calculated from mean of individual fetal values obtained from each litter sex wise. 

TABLE 15. SUMMARY RECORD OF FETAL EXTERNAL EXAMINATION PER LITTER

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		111		333		1000
Litters Evaluated for External Examination		No.	21		22		21		22
Fetuses Evaluated for External Examination		No.	208		221		241		238
VARIATIONS
Region/Tissue↓	Alteration↓
General	Subcutaneous haemorrhagic spot beneath the skin
	Litter Incidences	No. (%)	3	14.29	2	9.09	5	23.81	1	4.55
	Fetal Incidences	No. (%)	6	2.88	3	1.36	8	3.32	2	0.84

No.: Number; %: Percentage.

TABLE 15 (Contd…). SUMMARY RECORD OF FETAL EXTERNAL EXAMINATION PER LITTER

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		111		333		1000
Litters Evaluated for External Examination		No.	21		22		21		22
Fetuses Evaluated for External Examination		No.	208		221		241		238
MALFORMATIONS
Region/Tissue↓	Alteration↓
General	Runt
	Litter Incidences	No. (%)	0	0.00	0	0.00	1	4.76	0	0.00
	Fetal Incidences	No. (%)	0	0.00	0	0.00	1	0.41	0	0.00
Head	Meningo-encaphalacele
	Litter Incidences	No. (%)	0	0.00	0	0.00	1	4.76	0	0.00
	Fetal Incidences	No. (%)	0	0.00	0	0.00	1	0.41	0	0.00
Pelvic region	Spina bifida/Spina meningocele
	Litter Incidences	No. (%)	0	0.00	0	0.00	0	0.00	1	4.55
	Fetal Incidences	No. (%)	0	0.00	0	0.00	0	0.00	1	0.42

No.: Number; %: Percentage.

TABLE 16. SUMMARY OF FETAL VISCERAL EXAMINATION PER LITTER RECORD

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		111		333		1000
Litters Evaluated for Visceral Examination		No.	21		22		21		22
Fetuses Evaluated for Visceral Examination		No.	100		105		114		115
VARIATIONS
Region/Tissue↓	Alteration↓
Liver	Discoloured (Pale coloured)
	Litter Incidences	No. (%)	2	9.52	2	9.09	0	0.00	2	9.09
	Fetal Incidences	No. (%)	2	2.00	2	1.90	0	0.00	2	1.74
Kidneys	Renal pelvis dilation (bilateral)
	Litter Incidences	No. (%)	2	9.52	1	4.55	2	9.52	2	9.09
	Fetal Incidences	No. (%)	2	2.00	1	0.95	2	1.75	2	1.74
Ureters	Dilated (bilateral)
	Litter Incidences	No. (%)	4	19.05	1	4.55	5	23.81	4	18.18
	Fetal Incidences	No. (%)	4	4.00	1	0.95	5	4.39	5	4.35

No.: Number; %: Percentage.

TABLE 16 (Contd…). SUMMARY RECORD OF FETAL VISCERAL EXAMINATION PER LITTER

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		111		333		1000
Litters Evaluated for Visceral Examination		No.	21		22		21		22
Fetuses Evaluated for Visceral Examination		No.	100		105		114		115
MALFORMATIONS
Region/Tissue↓	Alteration↓
Brain	Dilation of lateral ventricles (slight)
	Litter Incidences	No. (%)	1	4.76	2	9.09	0	0.00	0	0.00
	Fetal Incidences	No. (%)	2	2.00	2	1.90	0	0.00	0	0.00
Brain	Third ventricle dialtion (slight)
	Litter Incidences	No. (%)	0	0.00	1	4.55	0	0.00	0	0.00
	Fetal Incidences	No. (%)	0	0.00	1	0.95	0	0.00	0	0.00

No.: Number; %: Percentage.

TABLE 17. SUMMARY OF SKELETAL EXAMINATION OF FETUSES PER LITTER RECORD

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		111		333		1000
Litters Evaluated for Skeletal Examination		No.	21		22		21		22
Fetuses Evaluated for Skeletal Examination		No.	108		116		127		123
VARIATIONS
Region/Tissue↓	Alteration↓
Skull Bone	Parietal - Incomplete ossification
	Litter Incidences	No. (%)	1	4.76	0	0.00	1	4.76	1	4.55
	Fetal Incidences	No. (%)	1	0.93	0	0.00	1	0.79	2	1.63
Skull Bone	Interparietal - Incomplete ossification
	Litter Incidences	No. (%)	1	4.76	1	4.55	1	4.76	1	4.55
	Fetal Incidences	No. (%)	1	0.93	1	0.86	1	0.79	3	2.44
Skull Bone	Supraoccipital - Incomplete ossification
	Litter Incidences	No. (%)	0	0.00	0	0.00	0	0.00	1	4.55
	Fetal Incidences	No. (%)	0	0.00	0	0.00	0	0.00	1	0.81
Skull Bone	Exooccipital - Incomplete ossification
	Litter Incidences	No. (%)	0	0.00	0	0.00	0	0.00	1	4.55
	Fetal Incidences	No. (%)	0	0.00	0	0.00	0	0.00	1	0.81
Skull Bone	Hyoid body - Poor ossification
	Litter Incidences	No. (%)	1	4.76	0	0.00	0	0.00	0	0.00
	Fetal Incidences	No. (%)	1	0.93	0	0.00	0	0.00	0	0.00
Sternum	Sternebra No. 1 - Unossified
	Litter Incidences	No. (%)	0	0.00	0	0.00	1	4.76	0	0.00
	Fetal Incidences	No. (%)	0	0.00	0	0.00	1	0.79	0	0.00
Sternum	Sternebra No. 2 - Incomplete ossification
	Litter Incidences	No. (%)	1	4.76	0	0.00	0	0.00	1	4.55
	Fetal Incidences	No. (%)	1	0.93	0	0.00	0	0.00	1	0.81
Sternum	Sternebra No. 5 - Unossified
	Litter Incidences	No. (%)	7	33.33	4	18.18	6	28.57	4	18.18
	Fetal Incidences	No. (%)	7	6.48	6	5.17	9	7.09	5	4.07
Sternum	Sternebra No. 5 - Incomplete ossification
	Litter Incidences	No. (%)	10	47.62	4	18.18	3	14.29	2	9.09
	Fetal Incidences	No. (%)	13	12.04	5*	4.31	4*	3.15*	2*	1.63*
Sternum	Sternebra No. 5 - Poor ossification
	Litter Incidences	No. (%)	2	9.52	4	18.18	3	14.29	6	27.27
	Fetal Incidences	No. (%)	2	1.85	4	3.45	4	3.15	6	4.88
Sternum	Sternebra No. 5 - Asymmetrical ossification
	Litter Incidences	No. (%)	2	9.52	0	0.00	0	0.00	2	9.09
	Fetal Incidences	No. (%)	2	1.85	0	0.00	0	0.00	2	1.63
Sternum	Sternebra No. 6 - Unossified
	Litter Incidences	No. (%)	5	23.81	2	9.09	6	28.57	4	18.18
	Fetal Incidences	No. (%)	6	5.56	2	1.72	7	5.51	5	4.07

No.: Number; %: Percentage;*: Statistically significant (P<0.05) change than the control group.

TABLE 17 (Contd…). SUMMARY OF SKELETAL EXAMINATION OF FETUSES PER LITTER RECORD

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		111		333		1000
Litters Evaluated for Skeletal Examination		No.	21		22		21		22
Fetuses Evaluated for Skeletal Examination		No.	108		116		127		123
VARIATIONS
Region/Tissue↓	Alteration↓
Sternum	Sternebra No. 6 - Incomplete ossification
	Litter Incidences	No. (%)	11	52.38	8	36.36	9	42.86	8	36.36
	Fetal Incidences	No. (%)	20	18.52	15	12.93	11	8.66	13	10.57
Sternum	Sternebra No. 6 - Poor ossification
	Litter Incidences	No. (%)	2	9.52	5	22.73	2	9.52	6	27.27
	Fetal Incidences	No. (%)	2	1.85	6	5.17	2	1.57	7	5.69
Sternum	Sternebra No. 4 - Assymetrical ossification
	Litter Incidences	No. (%)	0	0.00	0	0.00	2	9.52	0	0.00
	Fetal Incidences	No. (%)	0	0.00	0	0.00	2	1.57	0	0.00
Rib	Rib No. 3 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	2	9.52	2	9.09
	Fetal Incidences	No. (%)	0	0.00	0	0.00	2	1.57	2	1.63
Rib	Rib No. 4 - Wavy Bilateral
	Litter Incidences	No. (%)	0	0.00	0	0.00	2	9.52	2	9.09
	Fetal Incidences	No. (%)	0	0.00	0	0.00	2	1.57	2	1.63
Rib	Rib No. 5 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	2	9.52	2	9.09
	Fetal Incidences	No. (%)	0	0.00	0	0.00	2	1.57	2	1.63
Rib	Rib No. 6 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	2	9.52	2	9.09
	Fetal Incidences	No. (%)	0	0.00	0	0.00	2	1.57	2	1.63
Rib	Rib No. 7 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	2	9.52	2	9.09
	Fetal Incidences	No. (%)	0	0.00	0	0.00	2	1.57	2	1.63
Rib	Rib No. 8 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	2	9.52	2	9.09
	Fetal Incidences	No. (%)	0	0.00	0	0.00	2	1.57	2	1.63
Rib	Rib No. 9 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	2	9.52	2	9.09
	Fetal Incidences	No. (%)	0	0.00	0	0.00	2	1.57	2	1.63
Rib	Rib No. 10 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	2	9.52	2	9.09
	Fetal Incidences	No. (%)	0	0.00	0	0.00	2	1.57	2	1.63
Rib	Rib No. 11 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	2	9.52	2	9.09
	Fetal Incidences	No. (%)	0	0.00	0	0.00	2	1.57	2	1.63

No.: Number; %: Percentage.

TABLE 17 (Contd…). SUMMARY OF SKELETAL EXAMINATION OF FETUSES PER LITTER RECORD

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		111		333		1000
Litters Evaluated for Skeletal Examination		No.	21		22		21		22
Fetuses Evaluated for Skeletal Examination		No.	108		116		127		123
VARIATIONS
Region/Tissue↓	Alteration↓
Rib	Rib No. 12 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	2	9.52	2	9.09
	Fetal Incidences	No. (%)	0	0.00	0	0.00	2	1.57	2	1.63
Rib	Rib No. 13 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	2	9.52	2	9.09
	Fetal Incidences	No. (%)	0	0.00	0	0.00	2	1.57	2	1.63
Rib	Supernumerary rib (14^thRib) ossification site (unilateral - left)
	Litter Incidences	No. (%)	1	4.76	2	9.09	1	4.76	2	9.09
	Fetal Incidences	No. (%)	1	0.93	2	1.72	1	0.79	2	1.63
Rib	Supernumerary rib (14^thRib) ossification site (unilateral right)
	Litter Incidences	No. (%)	1	4.76	1	4.55	0	0.00	1	4.55
	Fetal Incidences	No. (%)	1	0.93	2	1.72	0	0.00	1	0.81
Rib	Supernumerary rib (14^thRib) ossification site (Bilateral)
	Litter Incidences	No. (%)	1	4.76	0	0.00	0	0.00	0	0.00
	Fetal Incidences	No. (%)	1	0.93	0	0.00	0	0.00	0	0.00
Rib	Supernumerary rib (14^thRib) rudimentary (Unilateral-left)
	Litter Incidences	No. (%)	1	4.76	0	0.00	0	0.00	0	0.00
	Fetal Incidences	No. (%)	1	0.93	0	0.00	0	0.00	0	0.00
Rib	Supernumerary rib (14^thRib) rudimentary (unilateral-right)
	Litter Incidences	No. (%)	0	0.00	1	4.55	0	0.00	0	0.00
	Fetal Incidences	No. (%)	0	0.00	1	0.86	0	0.00	0	0.00
Rib	Supernumerary (14^thRib) rudimentary (Bilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	0	0.00	1	4.55
	Fetal Incidences	No. (%)	0	0.00	0	0.00	0	0.00	1	0.81
Rib	Supernumerary (14^thRib) full/supernumerary (Unilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	0	0.00	1	4.55
	Fetal Incidences	No. (%)	0	0.00	0	0.00	0	0.00	1	0.81
Thoracic Vertebra	Centrum No. 8 - Dumbbell Shaped
	Litter Incidences	No. (%)	1	4.76	0	0.00	0	0.00	0	0.00
	Fetal Incidences	No. (%)	1	0.93	0	0.00	0	0.00	0	0.00
Thoracic Vertebra	Centrum No. 9 - Dumbbell Shaped
	Litter Incidences	No. (%)	0	0.00	1	4.55	3	14.29	0	0.00
	Fetal Incidences	No. (%)	0	0.00	1	0.86	3	2.36	0	0.00
Thoracic Vertebra	Centrum No. 10 - Dumbbell Shaped
	Litter Incidences	No. (%)	5	23.81	3	13.64	4	19.05	7	31.82
	Fetal Incidences	No. (%)	5	4.63	6	5.17	5	3.94	10	8.13
Thoracic Vertebra	Centrum No. 10 - Asymmetrical ossification
	Litter Incidences	No. (%)	0	0.00	1	4.55	0	0.00	1	4.55
	Fetal Incidences	No. (%)	0	0.00	1	0.86	0	0.00	1	0.81

No.: Number; %: Percentage.

TABLE 17 (Contd…). SUMMARY OF SKELETAL EXAMINATION OF FETUSES PER LITTER RECORD

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		111		333		1000
Litters Evaluated for Skeletal Examination		No.	21		22		21		22
Fetuses Evaluated for Skeletal Examination		No.	108		116		127		123
VARIATIONS
Region/Tissue↓	Alteration↓
Thoracic Vertebra	Centrum No. 11 - Dumbbell Shaped
	Litter Incidences	No. (%)	6	28.57	7	31.82	5	23.81	11	50.00
	Fetal Incidences	No. (%)	6	5.56	14	12.07	9	7.09	17	13.82
Thoracic Vertebra	Centrum No. 12 - Dumbbell Shaped
	Litter Incidences	No. (%)	9	42.86	7	31.82	6	28.57	11	50.00
	Fetal Incidences	No. (%)	10	9.26	12	10.34	11	8.66	19	15.45
Thoracic Vertebra	Centrum No. 12 - Asymmetrical ossification
	Litter Incidences	No. (%)	0	0.00	1	4.55	0	0.00	0	0.00
	Fetal Incidences	No. (%)	0	0.00	1	0.86	0	0.00	0	0.00
Thoracic Vertebra	Centrum No. 13 - Dumbbell shaped
	Litter Incidences	No. (%)	0	0.00	2	9.09	0	0.00	3	13.64
	Fetal Incidences	No. (%)	0	0.00	2	1.72	0	0.00	4	3.25
Thoracic Vertebra	Centrum No. 1 - Dumbbell Shaped
	Litter Incidences	No. (%)	0	0.00	1	4.55	0	0.00	1	4.55
	Fetal Incidences	No. (%)	0	0.00	1	0.86	0	0.00	1	0.81
Lumbar Vertebra	Centrum No. 1 - Dumbbell Shaped
	Litter Incidences	No. (%)	0	0.00	1	4.55	0	0.00	1	4.55
	Fetal Incidences	No. (%)	0	0.00	1	0.86	0	0.00	1	0.81
Forelimb	Proximal Phalanges - Unossified (Bilateral)
	Litter Incidences	No. (%)	1	4.76	3	13.64	8	38.10	6	27.27
	Fetal Incidences	No. (%)	2	1.85	5	4.31	13*	10.24	12*	9.76
Forelimb	Proximal Phalanges - Poor ossification (Bilateral)
	Litter Incidences	No. (%)	1	4.76	1	4.55	4	19.05	2	9.09
	Fetal Incidences	No. (%)	1	0.93	1	0.86	5	3.94	2	1.63
Forelimb	Metacarpel No. 4 - Poor ossification (Bilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	2	9.52	0	0.00
	Fetal Incidences	No. (%)	0	0.00	0	0.00	2	1.57	0	0.00
Forelimb	Metacarpel No. 5 - Unossified (Bilateral)
	Litter Incidences	No. (%)	0	0.00	0	0.00	1	4.76	0	0.00
	Fetal Incidences	No. (%)	0	0.00	0	0.00	1	0.79	0	0.00

No.: Number; %: Percentage;*: Statistically significant (P<0.05) change than the control group.

TABLE 17 (Contd…). SUMMARY OF SKELETAL EXAMINATION OF FETUSES PER LITTER RECORD

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		111		333		1000
Litters Evaluated for Skeletal Examination		No.	21		22		21		22
Fetuses Evaluated for Skeletal Examination		No.	108		116		127		123
MALFORMATIONS
Region/Tissue↓	Alteration↓
Thoracic Vertebra	Centrum No. 6 - Split
	Litter Incidences	No. (%)	1	4.76	0	0.00	0	0.00	0	0.00
	Fetal Incidences	No. (%)	1	0.93	0	0.00	0	0.00	0	0.00
Thoracic Vertebra	Centrum No. 9 - Split
	Litter Incidences	No. (%)	0	0.00	0	0.00	1	4.76	0	0.00
	Fetal Incidences	No. (%)	0	0.00	0	0.00	1	0.79	0	0.00
Thoracic Vertebra	Centrum No. 10 Split
	Litter Incidences	No. (%)	1	4.76	0	0.00	0	0.00	3	13.64
	Fetal Incidences	No. (%)	1	0.93	0	0.00	0	0.00	3	2.44
Thoracic Vertebra	Centrum No. 11 - Split
	Litter Incidences	No. (%)	1	4.76	0	0.00	1	4.76	1	4.55
	Fetal Incidences	No. (%)	1	0.93	0	0.00	1	0.79	1	0.81
Thoracic Vertebra	Centrum No. 12 - Split
	Litter Incidences	No. (%)	1	4.76	2	9.09	1	4.76	1	4.55
	Fetal Incidences	No. (%)	1	0.93	3	2.59	1	0.79	2	1.63

No.: Number; %: Percentage.

Applicant's summary and conclusion

Conclusions:

In conclusion, the oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed. Based on the obtained results from this pre-natal developmental toxicity study conducted in Sprague Dawley Rats, the NOAEL (No observed adverse effect level) of the test item for both maternal and fetal toxicity is estimated as 1000 mg/kg body weight/day under experimental conditions employed.

Executive summary:

The objective of this pre-natal developmental toxicity study conducted as per OECD test guideline 414 was to provide general information concerning the effects of prenatal exposure of test item on the presumed pregnant females and in the developing organisms. This study was also conducted to assess the maternal toxicity as well as death, structural abnormalities, or altered growth in the fetuses. The aim of this study was to estimate no-observed-adverse-effect-level (NOAEL) of the test item for both maternal and fetal toxicity under the experimental conditions employed.

 

A total of 100 mated female Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant females. The animals in group G1 were administered with vehicle [0.5% w/v Carboxymethyl Cellulose], the animals in groups G2, G3 and G4 were administered with test item at the dose levels of 111, 333 and 1000 mg/kg body weight with dose concentrations of 11.1, 33.3 and 100 mg/mL in 0.5% w/v Carboxymethyl Cellulose for low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 5 to 19. The end points of assessment for dams were maternal death, maternal body weight and clinical signs of maternal toxicity and the end points of assessment for fetuses were fetal weights, growth and development, structural variations and malformations or altered growth.

 

Homogeneity and dose formulation analysis for dose concentration verification was performed during the first and last week of the treatment. The analysis results of test samples were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

 

A total of 21, 22, 21 and 22 females from group G1, G2, G3 and were found with implantations yielding to pregnancy with rates of 84%, 88%, 84% and 88% respectively.

 

All the dams from each group were observed for clinical signs of toxicity once daily, for mortalities twice daily during the experimental period. The body weight was recorded on Gestation Day (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section). The feed consumption was measured from GD 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17, 17 to 19 and 19 to 20. All the dams were euthanized on gestation Day 20 by exposing to CO₂ asphyxiation and subjected to detailed gross pathological examination. All the females were observed for status of pregnancy and the gravid uterus weight for all the dams was recorded on the day of caesarean section. Each dam was observed for number of live fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The ovaries of all the dams were observed for number of corpora lutea. The pre-and post-implantation losses per dam were calculated based on number of corpora lutea and number of implantation sites. The weight of thyroid along with the parathyroid was recorded post-fixation for all the group animals. The assessment of thyroid hormones, thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) was conducted for all the group dams. All the dams were evaluated for Histopathology of thyroid along with the parathyroid was conducted for all the dams from each group.

 

All the fetuses collected from each litter were weighed and measured for its crown rump length and anogenital distance. The mean fetal weight, mean crown rump length, and mean anogenital distance measurement/ratio per litter was calculated. All the fetuses were subjected to external examination on the day of caesarean section. All the even numbered fetuses from each litter were subjected to fresh visceral (soft tissue) examination and fixed head sections examination. All the odd numbered fetuses from each litter were stained with Alizarin Red S stain and subjected to skeletal examination.

 

For maternal toxicity assessment, there were no clinical signs of toxicity and no mortality/morbidity noted in any of the dams in all the tested dose groups G2, G3 and G4. There were no changes noted in mean of maternal body weight, percent change in maternal body weight gain, maternal feed consumption and body weight corrected for maternal increase, number of live fetuses, litter size, sex ratio, number of corpora lutea, number of implantation sites, number of incidences of resorptions, pre-and post-implantation losses per dam in all the tested dose levels. Also, there were no changes noted in mean gravid uterus weight in all the tested dose levels. The mean absolute and or relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation and no test item-related changes were noted in mean thyroid hormonal levels (T3, T4 and TSH). There were no gross pathological changes noted in any of the dams during caesarean section and no test item-related microscopic changes were noted in thyroids and parathyroids of all dams during histopathological examination.

 

For fetal (pre-natal developmental) toxicity assessment, there were no changes noted in mean fetal weight, mean fetal crown rump length, and mean fetal anogenital distance measurement / ratio per litter in either sex in all the tested dose groups. There were no test item-related fetal developmental and or structural alterations noted from all the tested dose group litters when subjected to fetal external, visceral, and skeletal examinations. The observed fetal external/visceral/skeletal developmental variations and or malformations are considered as incidental and unrelated to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependent manner. Also, the occurred mean litter/fetal proportions were within the in-house historical control range of this species and strain.