Violet sodium polysulfide aluminosilicate with a SOD-type framework structure

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 August 2020 to 03 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

DRF study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Violet sodium polysulfide aluminosilicate with a SOD-type framework structure

EC Number:

701-186-2

Molecular formula:

|Na+6-x+y+z (S2•-)y (S3•-)z (S4)t|[Al6-x Si6+x O24] – SOD Where: 6 ≤ 6-x+ y+z ≤ 8 0 ≤ x ≤ 1.2 0 < y+z +t ≤ 2 t > 0 SOD = Sodalite framework structure

IUPAC Name:

Violet sodium polysulfide aluminosilicate with a SOD-type framework structure

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: Minimum of 10 weeks
- Weight at study initiation: Males: 251.03 g to 285.09 g & Females: 200.23 g to 238.40 g
- Fasting period before study: No
- Housing: 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 was used as bedding material and was changed along with the cage at least twice a week
- Housing: Mating - During mating, three rats (one male and two females) were housed in standard polypropylene cages.
- Housing: 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 (e.g. ad libitum): ad libitum - Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): ad libitum - Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: for a minimum period of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1ºC to 23.2 ºC
- Humidity (%): relative humidity 50 to 61%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark

IN-LIFE DATES: From: To: 20 August 2020 to 13 October 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated many times to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% w/v Carboxy Methyl Cellulose, test item was insoluble in distilled water
- Concentration in vehicle: 100 mg/mL (maximum)
- Amount of vehicle (if gavage): 10mL/kg
- Lot/batch no. (if required): BCBN1690V
- Purity: n/a

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Verification of concentration and homogonetiy of the test item was conduced by BIONEEDS INDIA PRIVATE LIMITED DEVARAHOSAHALLY, SOMPURA HOBLI, NELAMANGALA TALUK, BANGALORE RURAL DISTRICT, PIN - 562 111, KARNATAKA, INDIA which hold the Good Laboratory Practice Certificate (GLP). 0.1 mL of sample was pipetted out into a digestion tube, a volume of 6 mL of concentrated nitric acid was added and placed in microwave sample preparation system and digested by the selected method. After digestion, the digestion tubes were cooled to room temperature and the contents were transferred to suitable volumetric flasks and diluted with Milli-Q water.


TECHNIQUE AND TEST METHOD: ICP-MS


TEST PARAMETERS: Timings Sweeps : 30 Seconds, Reading : 1, Replicates : 3, Analyte : Aluminium, Mass : 26.9815, Sample Flush : 35 seconds, Read Delay : 15 Seconds, Wash : 45 seconds, Mode : KED, Flow (Helium) : 4.0 mL, Vehicle: 0.5% Carboxymethyl Cellulose


ACCEPTANCE CRITERIA: Homogeneity and dose formulation analysis of prepared test formulations for the dose concentration verification were performed during first and last week of the treatment and the obtained mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:2 ratio (one male and two females)
- Length of cohabitation: until evidence of copulation was observed or for two weeks.
- 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: In-house bred animals
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

Gestation days 5 to 19

Frequency of treatment:

once daily

Duration of test:

Acclimatization: 20 August 2020 to 25 August 2020
Cohabitation: 26 August 2020 to 23 September 2020
Treatment: 01 September 2020 to 12 October 2020
Necropsy: 16 September 2020 to 13 October 2020

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose range finding study, conducted with the doses of 100, 300 and 1000 mg/kg body weight based on the available literature. Oral administration of test item to pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and prenatal-developmental toxicity at all these tested dose levels in the dose range finding study.
- Rationale for animal assignment (if not random): evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weight for all groups
- Fasting period before blood sampling for (rat) dam thyroid hormones: 0
- Time of day for (rat) dam blood sampling: within 2 hours before necropsy.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

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).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not feeding study
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Recorded as g/animal/day during gestation days 0-3, 3-5, 5-8, 8-11, 11-14, 14-17, 17-19 and 19-20
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not drinking water study


POST-MORTEM EXAMINATIONS: Yes / No / No data
- 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
- Other: observation of uterine content.

Blood sampling:

- Plasma: No
- Serum: Yes
- Volume collected - n/a
- Other: blood samples were collected as follows:
- From all dams at termination for mandatory assessment of thyroid hormones T4, T3 and thyroid stimulating hormone (TSH) within 2 hours before necropsy.
- Blood samples from non-pregnant females were also collected but were not pooled with the pregnant dams.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
- Anogenital distance of all live rodent pups: Individual fetus was measured for its anogenital distance on the day of caesarean section

Statistics:

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: 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 per dam, Percent of early/late resorptions 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: No. of Pregnant / Non-pregnant females (Pregnancy status), 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 weigh
- Percent of Live Fetuses per dam = (Number of Live Fetuses / Litter Size) x 100
- Percent of Dead Fetuses per dam = (Number of Dead Fetuses/ Litter Size) x 100
- Percent of Early Resorptions (%) per dam = (Number of Early Resorptions/Number of Implantation sites) x 100
- Percent of Late Resorptions (%) per dam = (Number of Late Resorptions / Number of Implantation sites)x 100
- Pre-implantation Loss (%) per dam = [(Number of Corpora lutea - Number of Implantation sites)/ Number of Corpora lutea]x 100
- Post-implantation Loss (%) per Dam = [(Number of Implantation sites - Number of Viable fetuses )/ Number of Implantation sites]x 100
- Sex Ratio (m/f) = Number of live male fetuses / Number of live female fetuses
- Male/Female Fetuses (%) = (Number of live male/female fetuses/ Total number of live fetuses)x 100
- Ano-genital Distance Ratio = Ano-genital distance (mm) / Cube root of Fetal weight (g)
- Fetal incidence (%) = (Number of Fetuses with particular observation per group/Total number of Fetuses examined per group)x 100
- Litter incidence (%) = (Number of Litters/dams with particular observation per group/Total number of Litters per group) x 100

Historical control data:

Historical control data not included in report. Historical control data from in-house of the same species and strain.

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 at all the tested dose groups and vehicle control group animals during the experimental period.

Mortality:

no mortality observed

Description (incidence):

There were no morbidity/mortality noted at all the tested dose groups and vehicle control group animals during the experimental period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase in mean gestation body weight on day 5 and statistically significant decrease in percent change in mean gestation body weight gain during day 5 to 8 was noted at group G4 when compared with the vehicle control group. These changes are considered as incidental and toxicologically insignificant as there were no changes noted in daily observations of animals and also no effects were noted in mean feed consumption during this period at this dose level.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase in mean gestation (maternal) feed consumption during day 19 to 20 was noted at group G4 when compared with the vehicle control group. This change is considered as incidental and toxicologically insignificant as there were no changes noted in daily observations of animals and also no effects were noted in mean percent change in gestation body weight gain during this period at this dose level.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone T3: There were no test item-related changes noted for this parameter across the dose groups when compared to the vehicle control group. The noted statistically significant decrease in mean serum T3 levels (ng/mL) at group G4 is considered as incidental and un-related to treatment with test item due to lack of a dose-response relationship and also the obtained values were within lab historical control range of same species and strain.

Thyroid hormone T4: There were no test item-related changes noted for this parameter across the dose groups when compared to the vehicle control group. The noted statistically significant decrease in serum T4 levels (ng/mL) at group G4 is considered as incidental and unrelated to treatment with test item due to lack of a dose-response relationship and also the obtained values were within the lab historical control range of same species and strain.

Thyroid stimulating hormone THS: There were no test item-related changes noted for this parameter across the dose groups when compared to the vehicle control group. The noted statistically significant decrease in serum TSH levels (μIU/mL) at group G3 is considered as incidental and unrelated to treatment with test item due to lack of a dose-response relationship and also the obtained values were within the lab historical control range of same species and strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

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 dose group. There were no changes noted for mean absolute and relative weight for this organ 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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in thyroid along with the parathyroid subjected to histopathological examination at all the tested dose group animals. Single incidence of minimal, diffuse hypertrophy of follicular cells in groups G2 and G4, single incidence of minimal infiltration of inflammatory cells in group G3 and single incidence of ultimobranchial cyst/s in thyroid gland in each group (G1, G2, G3 and G4) were noted during microspic examination. However, all these changes were considered as incidental findings and/or were expected for laboratory rats of this age (Elizabeth McInnes, 2012).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no statistically significant differences noted for percentage of pre- or post-implantation loss per litter at all the tested dose groups when compared to the vehicle control group.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

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

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no statistically significant differences noted for number of dead fetuses at all the tested dose groups when compared to the vehicle control group.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Pregnancy with rates of 96%, 92%, 92% and 96% in groups G1, G2, G3 and G4 respectively.

Other effects:

no effects observed

Description (incidence and severity):

The mean number of corpora lutea per litter was 11.63, 12.26, 11.78 and 11.96 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted at all the tested dose groups when compared with vehicle control group for number of corpora lutea.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: Highest concentration tested, no effects observed

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no changes 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):

There were no statistically significant differences noted for number of live fetuses at all the tested dose groups when compared to the vehicle control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no statistically significant differences noted for 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):

The mean litter sizes, assessed as the total number of fetuses in utero (live plus dead) per dam, was 10.33, 11.00, 10.26 and 10.67 for groups G1, G2, G3 and G4, respectively. There were no changes or no statistically significant differences noted at all the tested dose groups when compared with vehicle control group for litter size.

Anogenital distance of all rodent fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The mean male fetal anogenital distance per litter was 4.44 mm, 4.47 mm, 4.47 mm and 4.37 mm and the mean female anogenital distance per litter was 2.43 mm, 2.50 mm, 2.50 mm and 2.43 mm for groups G1, G2, G3 and G4 respectively.
The mean male fetal anogenital distance ratio per litter was 2.69, 2.75, 2.76 and 2.69 and the mean female anogenital distance ratio per litter was 1.52, 1.56, 1.56 and 1.52 for groups G1, G2, G3 and G4 respectively.

Statistically significant increase in mean female fetal anogenital distance ratio per litter was noted at groups G2 and G3 when compared with the vehicle control group. These changes are considered as incidental and unrelated to treatment as the obtained range is within in-house historical control range.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The general / developmental variations such as subcutaneous hemorrhagic spot/s beneath the skin on different regions of the body, pale skin colored fetuses, kinked tail and noted external malformations such as hyperextension of hindlimb – unilateral or short hindlimb - unilateral, noted across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain. Also, no remarkable differences or statistically significant changes were noted for these alterations in any of the tested dose groups when compared with vehicle control group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

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.
Occasional fetal incidences (litter incidences) of skeletal developmental variations were noted across the tested dose group litters: incomplete ossification of skull bones, incomplete ossification or unossification of sternabral bones, unilateral or bilateral wavy ribs, extra ossification site/s near to first lumbar vertebra i.e. immediately after 13th rib, dumbbell / semi-bipartite ossification of thoracic and lumbar verterbral centrum, incomplete ossification of thoracic verterbral centrum no. 10, unossification of metacarpal no. 5 of forelimb, incomplete ossification or unossification of proximal phalanges of forelimb - bilateral.
Occasional incidences of skeletal malformation were noted across the tested dose group litters:
-supplimentary rib - unilateral (14th rib) - two fetal incidences of different litters from group G3;
- supplimentary rib - bilateral (14th rib) - single fetal incidence from one litter from group G3;
- rudimentary rib - unilateral (14th rib) - two fetal incidences from one litter in group G3;
- thoracic vertebra centrum no. 9 - split - 1, 1, 0 and 0 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- thoracic vertebra centrum no. 10 - split 1, 1, 0 and 0 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- thoracic vertebra centrum no. 11 - split 1, 0, 0 and 1 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- thoracic vertebra centrum no. 12 - split 2, 1, 0 and 3 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- thoracic vertebra centrum no. 13 - split 1, 1, 1 and 0 fetal and litter incidences from group G1, G2, G3 and G4 respectively.

These skeletal developmental variations and skeletal malformations are considered as incidental and un-related to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependant manner. Also, the occurred mean litter/fetal proportions were within the lab historical control range of this species and strain.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal visceral/soft tissue malformations or developmental variations for any of the fetuses examined from all the tested dose group litters. The developmental variations such as pale or discolored lung lobes, pale/discolored liver lobes, fused liver lobes or dilatation of renal pelvis noted across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental variations are common for this species and strain. There were no remarkable differences or statistically significant changes noted for these variations in any of the tested dose groups when compared with vehicle control group.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The mean male fetal crown rump length per litter was 37.38 mm, 38.10 mm, 38.10 mm and 38.17 mm and the female mean crown rump length per litter was 36.12 mm, 37.39 mm, 37.55 mm and 37.31 mm for groups G1, G2, G3 and G4 respectively. Statistically significant increase in mean male and female fetal crown rump length per litter was noted at all the tested dose groups when compared with the vehicle control group. These changes are considered as incidental and unrelated to treatment as the obtained range is within the lab historical control range.

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:

other: Highest dose tested, no effects observed

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

TABLE 1. SUMMARY OF PREGNANCY STATUS, CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD                                                           

Group & Dose (mg/kg body weight/day)	Pregnancy Status				Clinical Signs and Mortality Record
	No. of Presumed Pregnant Animals / Group	No. of Females confirmed with Pregnancy	Rate of Pregnancy (%)		Clinical Signs of Toxicity revealed (No. of Animals)	No. of Mortalities / Total No. of animals
G1 & 0	25	24	96.0		N (25)	0/25
G2 & 100	25	23	92.0		N (25)	0/25
G3 & 300	25	23	92.0		N (25)	0/25
G4 & 1000	25	24	96.0		N (25)	0/25

                                                       

TABLE 2. 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	227.66	235.37	241.69	252.47	266.32	281.74	302.70	322.31	333.97
	±SD	7.24	8.55	8.64	8.80	10.03	11.24	12.17	13.09	14.30
	n	24	24	24	24	24	24	24	24	24
G2 & 100	Mean	229.32	237.70	243.76	254.17	268.02	284.04	306.43	327.34	339.75
	±SD	7.43	8.57	8.41	9.83	9.59	10.45	12.93	14.14	15.10
	n	23	23	23	23	23	23	23	23	23
G3 & 300	Mean	228.93	237.43	244.12	253.84	266.36	281.79	303.07	324.11	336.45
	±SD	6.68	6.94	6.96	8.31	8.54	8.44	11.08	15.03	15.90
	n	23	23	23	23	23	23	23	23	23
G4 & 1000	Mean	232.84	240.58	248.10*	257.18	270.47	285.42	306.45	327.75	339.95
	±SD	10.50	9.99	10.09	10.07	9.32	9.38	10.98	10.51	11.44
	n	24	24	24	24	24	24	24	24	24

*: Statistically significant (P<0.05) change than the control group

 

TABLE 3. SUMMARY OF PERCENT CHANGE IN GESTATION BODY WEIGHT (%) 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	3.38	2.69	4.47	5.48	5.79	7.44	6.49	3.61
	±SD	1.17	0.81	1.13	1.15	1.14	0.82	1.83	1.00
	n	24	24	24	24	24	24	24	24
G2 & 100	Mean	3.66	2.57	4.26	5.47	5.98	7.87	6.83	3.79
	±SD	1.97	1.68	1.24	1.40	1.11	1.71	1.69	0.79
	n	23	23	23	23	23	23	23	23
G3 & 300	Mean	3.72	2.82	3.97	4.94	5.80	7.54	6.92	3.81
	±SD	1.01	0.86	1.03	0.89	1.12	1.44	2.07	0.85
	n	23	23	23	23	23	23	23	23
G4 & 1000	Mean	3.34	3.14	3.67*	5.19	5.54	7.37	6.97	3.72
	±SD	1.18	1.11	0.94	1.21	0.87	1.66	1.51	0.93
	n	24	24	24	24	24	24	24	24

*: Statistically significant (P<0.05) change than the control group

 

TABLE 4. 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 20	Gravid Uterus Weight (g)	Corrected Body Weight
				g	%
G1 & 0	Mean	92.29	64.90	27.38	11.37
	±SD	10.08	8.44	6.38	2.84
	n	24	24	24	24
G2 & 100	Mean	95.99	69.64	26.35	10.84
	±SD	12.05	11.39	5.02	2.19
	n	23	23	23	23
G3 & 300	Mean	92.33	65.24	27.09	11.13
	±SD	14.08	12.54	6.28	2.72
	n	23	23	23	23
G4 & 1000	Mean	91.85	66.79	25.06	10.15
	±SD	10.71	8.17	5.81	2.52
	n	24	24	24	24

 

TABLE 5. SUMMARY OF AVERAGE GESTATION FEED CONSUMPTION (g/animal/day) RECORD

Group & Dose (mg/kg body weight/day)		Feed Consumption (g/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	16.52	17.59	17.45	18.80	20.35	21.65	25.41	24.34
	±SD	1.74	1.55	1.48	1.47	1.36	1.29	2.44	1.86
	n	24	24	24	24	24	24	24	24
G2 & 100	Mean	16.81	18.47	17.55	19.15	20.56	21.98	26.49	25.47
	±SD	1.51	1.29	1.49	1.40	1.28	1.25	1.80	1.74
	n	23	23	23	23	23	23	23	23
G3 & 300	Mean	16.77	18.12	17.36	18.68	20.17	21.86	25.88	25.23
	±SD	1.46	1.64	1.42	1.44	1.46	1.28	2.16	2.37
	n	23	23	23	23	23	23	23	23
G4 & 1000	Mean	16.11	18.39	17.09	18.72	20.46	22.20	26.52	26.19*
	±SD	1.59	2.07	1.28	1.27	1.20	0.92	1.88	1.96
	n	24	24	24	24	24	24	24	24

*: Statistically significant (P<0.05) change than the control group

 

TABLE 6. SUMMARY OF UTERI OBSERVATIONS PER LITTER RECORD

Group & Dose (mg/kg body weight/day)		No. of Corpora lutea	No. of Implantations	Litter Size	No. of Live Fetuses			No. of Dead Fetuses	No. of Early Resorptions	No. of Late Resorptions
					Total	Male	Female
G1 & 0	Mean	11.63	11.08	10.33	10.33	5.38	4.96	0.00	0.75	0.00
	±SD	2.04	2.00	1.83	1.83	1.74	1.78	0.00	0.79	0.00
	n	24	24	24	24	24	24	24	24	24
G2 & 100	Mean	12.26	11.61	11.00	11.00	6.17	4.83	0.00	0.61	0.00
	±SD	2.03	1.97	2.22	2.22	1.80	1.92	0.00	1.03	0.00
	n	23	23	23	23	23	23	23	23	23
G3 & 300	Mean	11.78	10.83	10.26	10.26	5.35	4.91	0.00	0.57	0.00
	±SD	2.07	2.08	1.96	1.96	1.99	1.35	0.00	0.73	0.00
	n	23	23	23	23	23	23	23	23	23
G4 & 1000	Mean	11.96	11.29	10.67	10.63	5.63	5.00	0.04	0.58	0.04
	±SD	1.88	1.57	1.71	1.69	1.56	1.32	0.20	0.72	0.20
	n	24	24	24	24	24	24	24	24	24

 

TABLE 7. SUMMARY OF MATERNAL DATA PER LITTER RECORD

Group & Dose (mg/kg body weight/day)		Pre-Implantation Loss (%)	Post-Implantation Loss (%)	Percent of Dead Fetus (%)	Percent of Early Resorptions (%)	Percent of Late Resorptions (%)	Male/Female Sex Ratio	Male Fetuses (%)	Female Fetuses (%)	Total Live Fetuses (%)
G1 & 0	Mean	4.49	6.61	0.00	6.61	0.00	1.38	52.26	47.74	100.00
	±SD	6.27	6.46	0.00	6.46	0.00	1.08	14.67	14.67	0.00
	n	24	24	24	24	24	24	24	24	24
G2 & 100	Mean	5.18	5.27	0.00	5.27	0.00	1.56	56.68	43.32	100.00
	±SD	7.16	9.08	0.00	9.08	0.00	0.88	13.54	13.54	0.00
	n	23	23	23	23	23	23	23	23	23
G3 & 300	Mean	8.04	4.99	0.00	4.99	0.00	1.21	51.22	48.78	100.00
	±SD	8.05	6.38	0.00	6.38	0.00	0.67	12.45	12.45	0.00
	n	23	23	23	23	23	23	23	23	23
G4 & 1000	Mean	5.21	5.98	0.35	5.36	0.30	1.26	52.71	47.29	99.65
	±SD	6.06	7.16	1.70	6.98	1.46	0.70	11.24	11.24	1.70
	n	24	24	24	24	24	24	24	24	24

 

TABLE 8. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group & Dose (mg/kg body weight/day)		Absolute Thyroid along with Parathyroid Weight (g)^#
G1 & 0	Mean	0.0194
	±SD	0.0011
	n	24
G2 & 100	Mean	0.0205
	±SD	0.0016
	n	23
G3 & 300	Mean	0.0191
	±SD	0.0025
	n	23
G4 & 1000	Mean	0.0200
	±SD	0.0012
	n	24

 

TABLE 9. SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TO TERMINAL BODY WEIGHT RECORD

Group & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Relative Thyroid along with Parathyroid weight (%)
G1 & 0	Mean	333.97	0.0058
	±SD	14.30	0.0004
	n	24	24
G2 & 100	Mean	339.75	0.0060
	±SD	15.10	0.0004
	n	23	23
G3 & 300	Mean	336.45	0.0057
	±SD	15.90	0.0007
	n	23	23
G4 & 1000	Mean	339.95	0.0059
	±SD	11.44	0.0004
	n	24	24

 

TABLE 10. SUMMARY OF SERUM TRIIODOTHYRONINE (T3) LEVELS (ng/mL) RECORD

Group & Dose (mg/kg body weight/day)		Serum T3 Levels (ng/mL)
G1 & 0	Mean	2.341
	±SD	0.347
	n	24
G2 & 100	Mean	2.219
	±SD	0.117
	n	23
G3 & 300	Mean	2.255
	±SD	0.155
	n	23
G4 & 1000	Mean	2.046*
	±SD	0.190
	n	24

* Statistically significant (P<0.05) change than the control group

 

TABLE 11. SUMMARY OF SERUM THYROXINE (T4) LEVELS (ng/mL) RECORD

Group & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1 & 0	Mean	65.090
	±SD	9.062
	n	24
G2 & 100	Mean	62.287
	±SD	5.247
	n	23
G3 & 300	Mean	63.891
	±SD	6.606
	n	23
G4 & 1000	Mean	58.756*
	±SD	6.290
	n	24

* Statistically significant (P<0.05) change than the control group

 

TABLE 12. 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	8.795
	±SD	7.224
	n	20
G2 & 100	Mean	7.262
	±SD	5.641
	n	19
G3 & 300	Mean	4.171*
	±SD	4.266
	n	21
G4 & 1000	Mean	9.573
	±SD	6.067
	n	17

* Statistically significant (P<0.05) change than the control group

 

TABLE 13. 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	4.32	4.10		37.38	36.12		4.45	2.43		2.70	1.52
	±SD	0.18	0.19		1.06	1.04		0.13	0.11		0.18	0.07
	n	24	24		24	24		24	24		24	24
G2 & 100	Mean	4.29	4.12		38.10*	37.39*		4.47	2.50		2.75	1.56
	±SD	0.12	0.11		0.66	0.65		0.13	0.10		0.06	0.05
	n	23	23		23	23		23	23		23	23
G3 & 300	Mean	4.26	4.10		38.09*	37.55*		4.47	2.50		2.76	1.56
	±SD	0.15	0.17		0.46	0.40		0.11	0.10		0.06	0.05
	n	23	23		23	23		23	23		23	23
G4 & 1000	Mean	4.27	4.09		38.17*	37.31*		4.37	2.41		2.69	1.51
	±SD	0.15	0.19		0.59	0.58		0.15	0.12		0.08	0.07
	n	24	24		24	24		24	24		24	24

* Statistically significant (P<0.05) change than the control group

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 14. SUMMARY RECORD OF FETAL EXTERNAL EXAMINATION PER LITTER

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		100		300		1000
Litters Evaluated for External Examination		No.	24		23		23		24
Fetuses Evaluated for External Examination		No.	248		253		236		255
Variations
Region/Tissue↓	Alteration↓
General	Subcutaneous hemorrhagic spot beneath the skin on different regions of the body
	Litter Incidences	No. (%)	6	(25.0)	8	(34.8)	6	(26.1)	9	(37.5)
	Fetal Incidences	No. (%)	9	(3.6)	10	(4.0)	8	(3.4)	10	(3.9)
General	Pale colored Fetus
	Litter Incidences	No. (%)	2	(8.3)	4	(17.4)	1	(4.3)	3	(12.5)
	Fetal Incidences	No. (%)	2	(0.8)	4	(1.6)	2	(0.8)	3	(1.2)
Tail	Kinked
	Litter Incidences	No. (%)	0	(0.0)	1	(4.3)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	1	(0.4)	0	(0.0)	0	(0.0)

 

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		100		300		1000
Litters Evaluated for External Examination		No.	24		23		23		24
Fetuses Evaluated for External Examination		No.	248		253		236		255
Malformations
Region/Tissue↓	Alteration↓
Hindlimb	Hyperextension (unilateral)
	Litter Incidences	No. (%)	1	(4.2)	0	(0.0)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	1	(0.4)	0	(0.0)	0	(0.0)	0	(0.0)
Hindlimb	Short (unilateral)
	Litter Incidences	No. (%)	0	(0.0)	1	(4.3)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	1	(0.4)	0	(0.0)	0	(0.0)

 

TABLE 15. SUMMARY OF FETAL VISCERAL EXAMINATION PER LITTER RECORD

Group			G1			G2			G3			G4
Dose (mg/kg body weight/day)			0			100			300			1000
Litters Evaluated for External Examination		No.	24			23			23			24
Fetuses Evaluated for External Examination		No.	118			121			111			120
Variations
Region/Tissue↓	Alteration↓
Lungs	Pale/discolored
	Litter Incidences	No. (%)	0	(0.0)	1		(4.3)	1		(4.3)	0		(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	1		(0.8)	1		(0.9)	0		(0.0)
Liver	Lobes Fused
	Litter Incidences	No. (%)	0	(0.0)	0		(0.0)	0		(0.0)	1		(4.2)
	Fetal Incidences	No. (%)	0	(0.0)	0		(0.0)	0		(0.0)	1		(0.8)
Liver	Pale/Discolored
	Litter Incidences	No. (%)	2	(8.3)	1		(4.3)	0		(0.0)	1		(4.2)
	Fetal Incidences	No. (%)	2	(1.7)	1		(0.8)	0		(0.0)	1		(0.8)
Kidneys	Renal Pelvis Dilatation (Bilateral)
	Litter Incidences	No. (%)	5	(20.8)	2		(8.7)	4		(17.4)	3		(12.5)
	Fetal Incidences	No. (%)	5	(4.2)	3		(2.5)	5		(4.5)	3		(2.5)
Ureters	Dilatation
	Litter Incidences	No. (%)	4	(16.7)	4		(17.4)	4		(17.4)	3		(12.5)
	Fetal Incidences	No. (%)	4	(3.4)	5		(4.1)	4		(3.6)	3		(2.5)

 

TABLE 16. SUMMARY OF SKELETAL EXAMINATION OF FETUSES PER LITTER RECORD

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		100		300		1000
Litters Evaluated for External Examination		No.	24		23		23		24
Fetuses Evaluated for External Examination		No.	130		132		125		135
Variations
Region↓	Alteration↓
Skull	Parietal bones - Incomplete ossification
	Litter Incidences	No. (%)	0	(0.0)	0	(0.0)	0	(0.0)	1	(4.2)
	Fetal Incidences	No. (%)	0	(0.0)	0	(0.0)	0	(0.0)	1	(0.7)
	Interparietal bones - Incomplete ossification
	Litter Incidences	No. (%)	0	(0.0)	1	(4.3)	0	(0.0)	1	(4.2)
	Fetal Incidences	No. (%)	0	(0.0	3	(2.3)	0	(0.0)	1	(0.7)
Sternum	Sternebra No. 2 - Unossified
	Litter Incidences	No. (%)	0	(0.0)	0	(0.0)	0	(0.0)	1	(4.2)
	Fetal Incidences	No. (%)	0	(0.0)	0	(0.0)	0	(0.0)	1	(0.7)
	Sternebra No. 3 - Unossified
	Litter Incidences	No. (%)	0	(0.0)	0	(0.0)	0	(0.0)	1	(4.2)
	Fetal Incidences	No. (%)	0	(0.0)	0	(0.0)	0	(0.0)	1	(0.7)
	Sternebra No. 5 - Unossified
	Litter Incidences	No. (%)	8	(33.3)	2	(8.7)	3	(13.0)	7	(29.2)
	Fetal Incidences	No. (%)	9	(6.9)	6	(4.5)	3	(2.4)	8	(5.9)
	Sternebra No. 6 - Unossified
	Litter Incidences	No. (%)	2	(8.3)	2	(8.7)	2	(8.7)	2	(8.3)
	Fetal Incidences	No. (%)	2	(1.5)	2	(1.5)	2	(1.6)	4	(3.0)
	Sternebra No. 4 - Incomplete ossification
	Litter Incidences	No. (%)	1	(4.2)	0	(0.0)	1	(4.3)	0	(0.0)
	Fetal Incidences	No. (%)	1	(0.8)	0	(0.0)	1	(0.8)	0	(0.0)
	Sternebra No. 5 - Incomplete ossification
	Litter Incidences	No. (%)	2	(8.3)	1	(4.3)	1	(4.3)	2	(8.3)
	Fetal Incidences	No. (%)	3	(2.3)	1	(0.8)	1	(0.8)	2	(1.5)
	Sternebra No. 6 - Incomplete ossification
	Litter Incidences	No. (%)	8	(33.3)	4	(17.4)	6	(26.1)	9	(37.5)
	Fetal Incidences	No. (%)	13	(10.0)	12	(9.1)	14	(11.2)	18	(13.3)

 

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		100		300		1000
Litters Evaluated for External Examination		No.	24		23		23		24
Fetuses Evaluated for External Examination		No.	130		132		125		135
Variations
Region↓	Alteration↓
Ribs	Rib No. 6 - Wavy (Unilateral)
	Litter Incidences	No. (%)	1	(4.2)	0	(0.0)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	1	(0.8)	0	(0.0)	0	(0.0)	0	(0.0)
	Rib No. 7 - Wavy (Unilateral)
	Litter Incidences	No. (%)	1	(4.2)	0	(0.0)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	1	(0.8)	0	(0.0)	0	(0.0)	0	(0.0)
	Rib No. 8 - Wavy (Unilateral)
	Litter Incidences	No. (%)	1	(4.2)	0	(0.0)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	1	(0.8)	0	(0.0)	0	(0.0)	0	(0.0)
	Rib No. 9 - Wavy (Unilateral)
	Litter Incidences	No. (%)	1	(4.2)	0	(0.0)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	1	(0.8)	0	(0.0)	0	(0.0)	0	(0.0)
	Rib No. 10 - Wavy (Unilateral)
	Litter Incidences	No. (%)	1	(4.2)	0	(0.0)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	1	(0.8)	0	(0.0)	0	(0.0)	0	(0.0)
	Rib No. 11 - Wavy (Unilateral)
	Litter Incidences	No. (%)	1	(4.2)	0	(0.0)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	1	(0.8)	0	(0.0)	0	(0.0)	0	(0.0)
	Rib No. 12 - Wavy (Unilateral)
	Litter Incidences	No. (%)	1	(4.2)	0	(0.0)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	1	(0.8)	0	(0.0)	0	(0.0)	0	(0.0)
	Rib No. 13 - Wavy (Unilateral)
	Litter Incidences	No. (%)	1	(4.2)	0	(0.0)	0	(0.0)	1	(4.2)
	Fetal Incidences	No. (%)	1	(0.8)	0	(0.0)	0	(0.0)	1	(0.7)
	Rib No. 4 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	(0.0)	2	(8.7)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	2	(1.5)	0	(0.0)	0	(0.0)
	Rib No. 5 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	(0.0)	2	(8.7)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	2	(1.5)	0	(0.0)	0	(0.0)
	Rib No. 6 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	(0.0)	2	(8.7)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	2	(1.5)	0	(0.0)	0	(0.0)
	Rib No. 7 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	(0.0)	2	(8.7)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	2	(1.5)	0	(0.0)	0	(0.0)
	Rib No. 8 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	(0.0)	2	(8.7)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	2	(1.5)	0	(0.0)	0	(0.0)
	Rib No. 9 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	(0.0)	2	(8.7)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	2	(1.5)	0	(0.0)	0	(0.0)
	Rib No. 10 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	(0.0)	2	(8.7)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	2	(1.5)	0	(0.0)	0	(0.0)
	Rib No. 11 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	(0.0)	2	(8.7)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	2	(1.5)	0	(0.0)	0	(0.0)
	Rib No. 12 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	(0.0)	2	(8.7)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	2	(1.5)	0	(0.0)	0	(0.0)
	Rib No. 13 - Wavy (Bilateral)
	Litter Incidences	No. (%)	0	(0.0)	2	(8.7)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	2	(1.5)	0	(0.0)	0	(0.0)
	Ossification site at first lumbar vertebra (Unilateral)
	Litter Incidences	No. (%)	4	(16.7)	1	(4.3)	4	(17.4)	1	(4.2)
	Fetal Incidences	No. (%)	5	(3.8)	1	(0.8)	5	(4.0)	2	(1.5)
	Ossification site at first lumbar vertebra (Bilateral)
	Litter Incidences	No. (%)	1	(4.2)	1	(4.3)	2	(8.7)	1	(4.2)
	Fetal Incidences	No. (%)	5	(3.8)	1	(0.8)	4	(3.2)	1	(0.7)

 

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		100		300		1000
Variations
Litters Evaluated for External Examination		No.	24		23		23		24
Fetuses Evaluated for External Examination		No.	130		132		125		135
Region↓	Alteration↓
Thoracic Vertebra	Centrum No. 5 - Dumbbell-shaped ossification
	Litter Incidences	No. (%)	0	(0.0)	1	(4.3)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	1	(0.8)	0	(0.0)	0	(0.0)
	Centrum No. 7 - Dumbbell-shaped ossification
	Litter Incidences	No. (%)	0	(0.0)	1	(4.3)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	1	(0.8)	0	(0.0)	0	(0.0)
	Centrum No. 8 - Dumbbell-shaped ossification
	Litter Incidences	No. (%)	0	(0.0)	0	(0.0)	1	(4.3)	1	(4.2)
	Fetal Incidences	No. (%)	0	(0.0)	0	(0.0)	1	(0.8)	1	(0.7)
	Centrum No. 9 - Dumbbell-shaped ossification
	Litter Incidences	No. (%)	1	(4.2)	4	(17.4)	2	(8.7)	1	(4.2)
	Fetal Incidences	No. (%)	1	(0.8)	6	(4.5)	3	(2.4)	2	(1.5)
	Centrum No. 10 - Dumbbell-shaped ossification
	Litter Incidences	No. (%)	9	(37.5	8	(34.8)	7	(30.4)	7	(29.2)
	Fetal Incidences	No. (%)	14	(10.8)	11	(8.3)	11	(8.8)	15	(11.1)
	Centrum No. 11 - Dumbbell-shaped ossification
	Litter Incidences	No. (%)	6	(25.0)	7	(30.4	7	(30.4)	7	(29.2)
	Fetal Incidences	No. (%)	16	(12.3)	17	(12.9	17	(13.6)	16	(11.9)
	Centrum No. 12 - Dumbbell-shaped ossification
	Litter Incidences	No. (%)	8	(33.3)	8	(34.8)	8	(34.8)	11	(45.8)
	Fetal Incidences	No. (%)	15	(11.5)	20	(15.2)	12	(9.6)	21	(15.6)
	Centrum No. 13 - Dumbbell-shaped ossification
	Litter Incidences	No. (%)	6	(25.0)	6	(26.1)	4	(17.4)	5	(20.8)
	Fetal Incidences	No. (%)	8	(6.2)	8	(6.1)	6	(4.8)	6	(4.4)
	Centrum No. 10 - Incomplete ossification
	Litter Incidences	No. (%)	0	(0.0)	1	(4.3)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	1	(0.8)	0	(0.0)	0	(0.0)

 

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		100		300		1000
Variations
Litters Evaluated for External Examination		No.	24		23		23		24
Fetuses Evaluated for External Examination		No.	130		132		125		135
Region↓	Alteration↓
Lumbar Vetebra	Centrum No. 1 - Dumbbell-shaped ossification
	Litter Incidences	No. (%)	2	(8.3)	3	(13.0)	0	(0.0)	2	(8.3)
	Fetal Incidences	No. (%)	2	(1.5)	3	(2.3)	0	(0.0)	2	(1.5)
	Centrum No. 2 - Dumbbell-shaped ossification
	Litter Incidences	No. (%)	1	(4.2)	0	(0.0)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	1	(0.8)	0	(0.0)	0	(0.0)	0	(0.0)
Forelimb	Proximal phalanges - Unossified (Bilateral)
	Litter Incidences	No. (%)	8	(33.3)	9	(39.1)	11	(47.8)	9	(37.5)
	Fetal Incidences	No. (%)	17	(13.1)	21	(15.9)	20	(16.0)	21	(15.6)
	Proximal phalanges - Incompletely ossified (Bilateral)
	Litter Incidences	No. (%)	1	(4.2)	1	(4.3)	2	(8.7)	2	(8.3)
	Fetal Incidences	No. (%)	1	(0.8)	2	(1.5)	3	(2.4)	3	(2.2)
	Metacarpel No. 5 - Unossified (Bilateral)
	Litter Incidences	No. (%)	0	(0.0)	1	(4.3)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	5	(3.8)	0	(0.0)	0	(0.0)

 

Group			G1		G2		G3		G4
Dose (mg/kg body weight/day)			0		100		300		1000
Litters Evaluated for External Examination		No.	24		23		23		24
Fetuses Evaluated for External Examination		No.	130		132		125		135
Malformations
Region↓	Alteration↓
Ribs	Rib No. 14 - Supplementary (Unilateral)
	Litter Incidences	No. (%)	0	(0.0)	0	(0.0)	2	(8.7)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	0	(0.0)	2	(1.6)	0	(0.0)
	Rib No. 14 - Supplementary (Bilateral)
	Litter Incidences	No. (%)	0	(0.0)	0	(0.0)	1	(4.3)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	0	(0.0)	1	(0.8)	0	(0.0)
	Rib No. 14 - Rudimentary (Unilateral)
	Litter Incidences	No. (%)	0	(0.0)	0	(0.0)	1	(4.3)	0	(0.0)
	Fetal Incidences	No. (%)	0	(0.0)	0	(0.0)	2	(1.6)	0	(0.0)
Thoracic Vertebra	Centrum No. 9 - Split
	Litter Incidences	No. (%)	1	(4.2)	1	(4.3)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	1	(0.8)	1	(0.8)	0	(0.0)	0	(0.0)
	Centrum No. 10 - Split
	Litter Incidences	No. (%)	1	(4.2)	1	(4.3)	0	(0.0)	0	(0.0)
	Fetal Incidences	No. (%)	1	(0.8)	1	(0.8)	0	(0.0)	0	(0.0)
	Centrum No. 11 - Split
	Litter Incidences	No. (%)	1	(4.2)	0	(0.0)	0	(0.0)	1	(4.2)
	Fetal Incidences	No. (%)	1	(0.8)	0	(0.0)	0	(0.0)	1	(0.7)
	Centrum No. 12 - Split
	Litter Incidences	No. (%)	2	(8.3)	1	(4.3)	0	(0.0)	3	(12.5)
	Fetal Incidences	No. (%)	2	(1.5)	1	(0.8)	0	(0.0)	4	(3.0)
	Centrum No. 13 - Split
	Litter Incidences	No. (%)	1	(4.2)	1	(4.3)	1	(4.3)	0	(0.0)
	Fetal Incidences	No. (%)	1	(0.8)	1	(0.8)	1	(0.8)	0	(0.0)

 

Applicant's summary and conclusion

Conclusions:

In a pre-natal developmental toxicity study conducted in Sprague Dawley Rats, the NOAEL of the test substance for both maternal and fetal toxicity was determined to be 1000 mg/kg body weight/day under the experimental conditions employed.

Executive summary:

The developmental toxicity of the test item was determined using OECD Guideline 414 under GLP conditions. The study used spraque dawley rats from an in-house line to test the daily oral administration via gavage of the test item using Carboxymethyl cellulose as vehicle. Based on a preliminary dose range study doses of 100, 300 and 1000 mg/kg bw/day were set with a concurrent vehicle control. On day 5 until day 19 of gestation, the test item was administered orally once daily to 25 pregnant females per group. During this time weight, food consumption, clinical signs of toxicity, mortality were observed. On day 20 all animals were euthanized, and tissue, organs and fetuses extracted and examined. There were no clinical signs of toxicity, mortality, morbidity, changes in body weight, food consumption changes, gross pathological or visceral changes in any group of maternal animals. Pregnancy rate, mean gravid uterus weight, live fetuses, mean litter size, mean sex ratio, mean number of implantation sites, mean number of resorptions, mean number of corpora lutae were similarly not affected by the treatment. Also, the mean pre-and post-implantation losses per dam and the mean absolute and relative thyroid along with the parathyroid weight were unaffected. No test item-related changes were noted in mean thyroid hormonal levels (T3, T4 and TSH). No test item-related microscopic changes were noted in thyroid along with the parathyroid of all the dams. There were no test item-related changes noted in mean fetal weight, mean fetal crown rump length and mean fetal anogenital distance measurement/ratio per litter. No test item related effects on developmental or structural alterations could be observed. Some external, visceral and skeletal alterations are considered incidental and not related to the test item as they occur in all groups, including the control and are not dose dependent. During the study no test item related effects in maternal or offspring animals could be observed. Therefore the "no observed adverse effect level (NOAEL) for reproduction and developmental toxicity is the highest dose used in this study of 1000mg/kg bw /day for maternal animals and offspring in rats.