Ethyltriphenylfosfonium bromide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from a study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

According to OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethyltriphenylfosfonium bromide

EC Number:

216-223-3

EC Name:

Ethyltriphenylfosfonium bromide

Cas Number:

1530-32-1

Molecular formula:

C20H20P.Br

IUPAC Name:

bromo(ethyl)triphenylphosphorane

Test material form:

solid: crystalline

Details on test material:

Name: Ethyltriphenylfosfonium bromide
IUPAC Name: Phosphonium, ethyltriphenyl-, bromide (1:1)
CAS No.: 1530-32-1
Molecular Weight: 371.25 g/mol
Molecular Formula: C20-H20-P.Br
SMILES: [Br-].CC[P+](c1ccccc1)(c2ccccc2)c3ccccc3

Specific details on test material used for the study:

Name: Ethyltriphenylfosfonium bromide
IUPAC Name: Phosphonium, ethyltriphenyl-, bromide (1:1)
CAS No.: 1530-32-1
Molecular Weight: 371.25 g/mol
Molecular Formula: C20-H20-P.Br
SMILES: [Br-].CC[P+](c1ccccc1)(c2ccccc2)c3ccccc3

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animals were procured from animal ethics committee approved vendor.
- Age at study initiation: 12-13 weeks
- Weight at study initiation: 228.17 g to 232.55 g
- Fasting period before study: No data available
- Housing: One to three rats were housed in each polycarbonate cage (length 37 cm X breadth 21 cm X height 20 cm). During mating, one male and two female rats were housed in a single cage. Pregnant females were housed individually. Sterilized corn-cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean.
- Diet (e.g. ad libitum): A conventional laboratory pellet diet from approved vendor was offered ad libitum.
- Water (e.g. ad libitum): Aquaguard™ filtered drinking water was offered ad libitum in regularly cleaned bottles.
- Acclimation period: Animals were acclimatised to the test conditions for at least 5 days prior to estrous cycle evaluation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 to 24.5 °C
- Humidity (%): 43.8 to 64.7 %.
- Air changes (per hr): At least 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark.

IN-LIFE DATES:
From: June 5, 2020
To: July 6, 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Distilled Water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The vehicle used for dose administration was distilled water. The test chemical was weighed and dissolved in distilled water to achieve the desired concentration of the test chemical at each dose level. Formulations were prepared on the same day or one day prior to dose administration and stored at room temperature until usage as the test chemical stability in solution was proved up to 24 hours.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Distilled water was used as a vehicle.
- Concentration in vehicle: 0 mg/ml, 1 mg/ml, 3 mg/ml and 9 mg/ml for 0, mg/kg bw/day, 10 mg/kg bw/day, 30 mg/kg bw/day and 90 mg/kg bw/day, respectively
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Mobile Phase
A - Acetonitrile:
B – Water
Ratio: (A:B = 45:55, v/v)
Column: Zorbax Extend C18 (150 mm x 4.6 mm x 5 µm)
Oven Temperature: 25 °C
Detector: VWD
Wavelength: 210 nm
Flow rate: 1.0 mL/min
Injection Volume: 10 µL
The active ingredient content in dose formulation of test item was determined using a validated analytical method (High Performance Liquid Chromatography – HPLC with VWD). The analytical method was validated in terms of specificity, linearity, precision (% RSD), and accuracy (% Recovery), homogeneity and stability in dose formulation samples using Distilled water as Vehicle. It was confirmed from the results of the validation that the method was specific, linear, precise, accurate, homogeneous and stable for the analysis of the test chemical in dose formulation samples. The results of validation criteria were well within the acceptable limits of the guideline specifications.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: Yes
- M/F ratio per cage: For mating, two females were kept with a single male (2:1 pairing)
- Length of cohabitation: Until the evidence of copulation was observed.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: No Data
- 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 of pregnancy
- Any other deviations from standard protocol: No data available

Duration of treatment / exposure:

Gestation Day 5 to Gestation 19

Frequency of treatment:

Once Daily from GD 5 to 19.

Duration of test:

120-130 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control Group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Low Dose Group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Mid Dose Group

Doses / concentrations 4

Dose / conc.:

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

Remarks:

High Dose Group

No. of animals per sex per dose:

25 pregnant females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected on the basis of previously done dose range finding study wherein the test chemical was administered orally to rats in graduated doses of 20, 60 and 180 mg/kg body weight for 14 consecutive days. Mortality was observed in only one animal of 180 mg/kg body weight group, while no mortalities were observed at 20 or 60 mg/kg body weight dose groups. Body weight was affected for both male and female rats of 180 mg/kg body weight dose group, while no change was observed for animals of mid and low dose groups. Further, test chemical related and dose-dependent lesions in stomach and intestine were observed at all the dose groups, which were also confirmed by histopathological analysis. Based on the above stated results, the doses 0, 10, 30 and 90 mg/kg body weight were selected for control, low, mid and high dose groups of the test chemical in the current study.

- Rationale for animal assignment (if not random): Randomization was done based on body weight of the pregnant females on GD 0.The animals were assigned in an unbiased manner to the control and treatment groups manually. As mating was carried out in batches, females inseminated with same male were evenly distributed across the groups. Individual body weights were within ± 20% of the respective groups mean after randomization.The mean body weights on the day of randomization were analysed by One-way ANOVA and it was found that the group means were statistically comparable to each group.

- Other: No Data Available

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for any morbidity and/or mortality, throughout the acclimatization and study period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for clinical signs and symptoms after dose administration. These observations were regularly performed approximately 1 hour after dose administration, since onset of clinical signs after oral gavage is anticipated in that time frame.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed at the time of receipt, on GD 0, on the first day of dosing (GD 5), and on gestation days 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, The weight of feed provided and that leftover was recorded for each cage on the same day as animal body weight. This data was used to calculate mean feed consumption (g/day/rat) on gestation days 5, 8, 11, 14, 17 and 20.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: The total weight of the gravid or non-gravid uteri including cervix were recorded for all animals. The ovarian and placental weights were determined for all pregnant animals. Also, thyroid and parathyroid examinations were also performed. Also, changes in the visceral organs were also examined.

OTHER: No data available

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: Placental weights were determined for all pregnant animals.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

Raw data were processed using statistical software “Sigma Plot 14.0”. The mean and standard deviation were calculated using the software and all data were summarized in tabular form. All continuous data (body weight, feed consumption, hormone estimation, absolute and relative organ weights, maternal and pup parameters) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using Kruskal-Wallis, ANOVA on ranks. Gross, skeletal and visceral abnormalities were represented as both the total no. of foetuses and litters affected and the percentage of foetuses and litters affected. Further, the data of percent incidences of abnormalities were analysed for dose dependency by Pearson’s correlation. P values of < 0.05 were deemed to be statistically significant.

Indices:

Maternal Fertility Index, Pregnancy Index, Gestation Index, Implantation Index, Resorption Index, Litter Index, Fetal Viability Index,

Historical control data:

No data available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs or symptoms were observed in any of the animals through the study period.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No mortality or morbidity was recorded in animals of any of the experimental groups throughout the duration of the experiment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight of pregnant and non-pregnant females remained comparable among all the groups for every time point (GD 0, 5, 8, 11, 14, 17 and 20) recorded during the study period. The percent change in body weights with respect to the first day of gestation was also statistically comparable among all the groups of Pregnant and non-pregnant females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean feed consumed per day for pregnant females remained statistically equivalent in all groups of pregnant and non-pregnant females.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Triiodothryronine (T3) and thyroid stimulating hormone (TSH) levels in sera were found to be statistically comparable among the control and treatment groups in both pregnant and non-pregnant females. However, significant decrease in thyroxine (T4) levels were observed in pregnant females of G4 (90 mg/kg) group as compared to G1 (Control) group (P<0.01).

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute weights and weights relative to body weight of gravid uterus and ovary remained comparable for pregnant females of control and treatment groups. A statistically significant increase was observed in absolute weight of thyroid-parathyroid in pregnant females of G4 group (90 mg/kg) as compared to G1 (control) group (P<0.05). However, when thyroid-parathyroid weights relative to body weights were compared, no significant difference was observed between control and treatment groups. Absolute and relative weights of uterus, ovary, thyroid and parathyroid of non-pregnant females between control and treatment groups remained comparable. The mean feed consumed per day for pregnant females remained statistically equivalent in all groups of pregnant and non-pregnant females.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Dams were euthanized and subjected to gross pathology examination. No gross pathological alterations were observed for pregnant and non-pregnant females of control (G1) or treated groups (G2, G3, G4).

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid glands from G1 (control) and G4 (90 mg/kg body weight) were subjected to microscopic examination. Congenital cyst (ultimobranchial origin) of thyroid gland was found in 3 animals of each G1 (control) and G4 (90 mg/kg body weight) group. Although congenital cysts were observed for thyroid and parathyroid glands of G4 (90 mg/kg body weight) animals, they were concurrently observed in G1 (Control) group. Furthermore, these cysts are spontaneous in nature and not attributable to the test chemical.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

No treatment-related clinical signs or symptoms were observed in any of the animals through the study period. No mortality or morbidity was recorded in animals of any of the experimental groups throughout the duration of the experiment. The mean body weight of pregnant and non-pregnant females remained comparable among all the groups for every time point (GD 0, 5, 8, 11, 14, 17 and 20) recorded during the study period. The percent change in body weights with respect to the first day of gestation was also statistically comparable among all the groups of Pregnant and non-pregnant females. Triiodothryronine (T3) and thyroid stimulating hormone (TSH) levels in sera were found to be statistically comparable among the control and treatment groups in both pregnant and non-pregnant females. However, significant decrease in thyroxine (T4) levels were observed in pregnant females of G4 (90 mg/kg) group as compared to G1 (Control) group (P<0.01). Absolute weights and weights relative to body weight of gravid uterus and ovary remained comparable for pregnant females of control and treatment groups. A statistically significant increase was observed in absolute weight of thyroid-parathyroid in pregnant females of G4 group (90 mg/kg) as compared to G1 (control) group (P<0.05). However, when thyroid-parathyroid weights relative to body weights were compared, no significant difference was observed between control and treatment groups. Absolute and relative weights of uterus, ovary, thyroid and parathyroid of non-pregnant females between control and treatment groups remained comparable. The mean feed consumed per day for pregnant females remained statistically equivalent in all groups of pregnant and non-pregnant females. Dams were euthanized and subjected to gross pathology examination. No gross pathological alterations were observed for pregnant and non-pregnant females of control (G1) or treated groups (G2, G3, G4). Thyroid glands from G1 (control) and G4 (90 mg/kg body weight) were subjected to microscopic examination. Congenital cyst (ultimobranchial origin) of thyroid gland was found in 3 animals of each G1 (control) and G4 (90 mg/kg body weight) group. Although congenital cysts were observed for thyroid and parathyroid glands of G4 (90 mg/kg body weight) animals, they were concurrently observed in G1 (Control) group. Furthermore, these cysts are spontaneous in nature and not attributable to the test chemical.

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

At termination 24/25, 24/25, 24/25 and 21/25 females were found to be pregnant from G1 (control), G2 (10 mg/kg body weight), G3 (30 mg/kg body weight) and G4 (90 mg/kg body weight) group respectively. This effect was not considered to be related to the test chemical administration.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No test chemical administration related adverse effect on the pre- and post-implantation losses was observed and remained comparable among all the treatment groups and control group.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No significant difference was observed for the mean litter size as there were no significant litter losses by any resorptions across all dose groups.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

One female from each G1, G2 and G4 had very early resorption which was identified by staining the uterus with ammonium sulphide solution. This effect was no considered as treatment related since it did not have any dose dependent increase and was also observed in the control group.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses were observed across all dose groups due to the test chemical administration.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No changes in pregnancy duration was observed in all females across all the dose groups due to the test chemical administration.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

At termination 24/25, 24/25, 24/25 and 21/25 females were found to be pregnant from G1 (control), G2 (10 mg/kg body weight), G3 (30 mg/kg body weight) and G4 (90 mg/kg body weight) group respectively. This effect was not considered to be related to the test chemical administration.

Other effects:

not specified

Details on maternal toxic effects:

No test chemical administration related adverse effect on the pre- and post-implantation losses was observed and remained comparable among all the treatment groups and control group. No significant difference was observed for the mean litter size as there were no significant litter losses by any resorptions across all dose groups. One female from each G1, G2 and G4 had very early resorption which was identified by staining the uterus with ammonium sulphide solution. This effect was no considered as treatment related since it did not have any dose dependent increase and was also observed in the control group. No dead fetuses were observed across all dose groups due to the test chemical administration. No changes in pregnancy duration was observed in all females across all the dose groups due to the test chemical administration. At termination 24/25, 24/25, 24/25 and 21/25 females were found to be pregnant from G1 (control), G2 (10 mg/kg body weight), G3 (30 mg/kg body weight) and G4 (90 mg/kg body weight) group respectively. This effect was not considered to be related to the test chemical administration.

Effect levels (maternal animals)

1

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

clinical biochemistry

Remarks on result:

other: Please see 'remarks'

Remarks:

Significant decrease in thyroxine (T4) levels were observed in pregnant females of G4 (90 mg/kg) group as compared to G1 (Control) group. This decrease in T4 level is deemed to be test chemical administration related.

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

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 consumption and compound intake

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

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The weights of placenta remained comparable for control (G1) and treatment groups (G2, G3 and G4). The mean weight of male foetuses significantly increased in G2 (10 mg/kg body weight) (P<0.01) as compared to G1 (control). Such change in body weight was not observed in other dose groups and therefore it was considered as an incidental finding.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no dead fetuses and reduction in number of live offsprings across all the dose groups due to test chemical administration.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant changes in the sex ratio of fetuses were observed across all the dose groups due to test chemical administration.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No significant changes in the litter sizes and weights were observed across all the dose groups due to test chemical administration.

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Gross external observation of foetuses revealed that 5.26, 2.08, 2.76 and 7.11 percentage of foetuses from G1 (control), G2 (10 mg/kg), G3 (30 mg/kg) and G4 (90 mg/kg) respectively showed malformations/variations. The variation included haemorrhage in 0.79 percent of foetuses of G4 (90 mg/kg body weight) group and the malformations included: retarded growth (Runt): 3.86 (G1), 2.08 (G2), 2.76 (G3) and 5.93 (G4) percent of foetuses; dome-shaped head (Meningo-encephalocele): 1.4 (G1) and 1.19 (G4) percent of foetuses. No correlation was observed for the doses of the test chemical and the total percent of foetuses with malformations/variations. Similarly, no significant correlation was observed when litter was used as the unit for comparing the malformations/ variations or when data of individual malformations/variations were compared. These observations were found to be independent of the treatment and rather seem to be spontaneous in nature as observed by other studies

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations observed in skull included: hole in skull (mainly in interparietal and occipital region) observed in 5 (1) foetuses (litter) from G1 (Control group) and 3 (1) foetuses (litter) from G4 (90 mg/kg body weight); Bipartite ossification of inter-parietal found in 4 (1) foetuses (litter) from G1 (control) and 3 (1) foetuses (litter) from G4 (90 mg/kg body weight); Bipartite ossification and incomplete ossification of occipital found in 1 foetus from G1 (control) and G4 (90 mg/kg body weight) respectively. Although the number of foetuses with malformations of skull seems to be high, these observations were only present in foetuses of 1 litter each from G1 and G4 group. One rib was absent in 1 foetus from G3 (30 mg/kg body weight) and the same foetus had branched rib; 2 (2), 4 (3), 2 (2) and 2 (1) foetuses (litters) from G1, G2, G3 and G4 respectively showed misaligned ribs. Unossified/incompletely ossified sternebrae were observed in 3 (1), and 2 (1) foetuses (litters) of G1 and G3 respectively; misaligned sternebrae were seen in 1 foetus of G2. Fused cervical vertebrae were observed in 1 foetus of G1. The total percentage of foetuses with skeletal malformations/variations were 6.80, 2.65, 3.36 and 3.76 for G1 (control), G2 (10 mg/kg), G3 (30 mg/kg) and G4 (90 mg/kg) respectively while the percentage of litters affected were found to be 17.39 (G1), 13.04 (G2), 16.67 (G3) and 10.00 (G4). No significant correlation was observed between treatment and individual skeletal malformations/variations or total percentages of foetuses and litters affected. The skeletal malformations/variations noted in treatment group were concurrently seen in control group. Moreover, the occurrence and incidences of these malformations/variations are common in developing foetuses. Therefore, these skeletal developmental malformations/variations were not considered to be test chemical related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In visceral observations, hematoma (ankle of left hind limb) was found in 1 foetus from G2 (10 mg/kg body weight) and enlarged adrenal with reduced thymus size was found in 1 foetus from G3 (30 mg/kg body weight) group. No malformations/variations were observed in any other foetuses of control or treated groups. Head razor examination of foetuses did not show any malformations/variations in control or treated groups.

Other effects:

no effects observed

Description (incidence and severity):

No significant effects were observed for the AGD and normalised AGD among all the groups. Similarly, the crown to rump length (CRL) of foetuses were comparable for control (G1) and treatment groups (G2, G3, G4)

Details on embryotoxic / teratogenic effects:

The weights of placenta remained comparable for control (G1) and treatment groups (G2, G3 and G4). The mean weight of male foetuses significantly increased in G2 (10 mg/kg body weight) (P<0.01) as compared to G1 (control). Such change in body weight was not observed in other dose groups and therefore it was considered as an incidental finding. There were no dead fetuses and reduction in number of live offsprings across all the dose groups due to test chemical administration. No significant changes in the sex ratio of fetuses were observed across all the dose groups due to test chemical administration. No significant changes in the litter sizes and weights were observed across all the dose groups due to test chemical administration. Gross external observation of foetuses revealed that 5.26, 2.08, 2.76 and 7.11 percentage of foetuses from G1 (control), G2 (10 mg/kg), G3 (30 mg/kg) and G4 (90 mg/kg) respectively showed malformations/variations. The variation included haemorrhage in 0.79 percent of foetuses of G4 (90 mg/kg body weight) group and the malformations included: retarded growth (Runt): 3.86 (G1), 2.08 (G2), 2.76 (G3) and 5.93 (G4) percent of foetuses; dome-shaped head (Meningo-encephalocele): 1.4 (G1) and 1.19 (G4) percent of foetuses. No correlation was observed for the doses of the test chemical and the total percent of foetuses with malformations/variations. Similarly, no significant correlation was observed when litter was used as the unit for comparing the malformations/ variations or when data of individual malformations/variations were compared. These observations were found to be independent of the treatment and rather seem to be spontaneous in nature as observed by other studies. Skeletal malformations observed in skull included: hole in skull (mainly in interparietal and occipital region) observed in 5 (1) foetuses (litter) from G1 (Control group) and 3 (1) foetuses (litter) from G4 (90 mg/kg body weight); Bipartite ossification of inter-parietal found in 4 (1) foetuses (litter) from G1 (control) and 3 (1) foetuses (litter) from G4 (90 mg/kg body weight); Bipartite ossification and incomplete ossification of occipital found in 1 foetus from G1 (control) and G4 (90 mg/kg body weight) respectively. Although the number of foetuses with malformations of skull seems to be high, these observations were only present in foetuses of 1 litter each from G1 and G4 group. One rib was absent in 1 foetus from G3 (30 mg/kg body weight) and the same foetus had branched rib; 2 (2), 4 (3), 2 (2) and 2 (1) foetuses (litters) from G1, G2, G3 and G4 respectively showed misaligned ribs. Unossified/incompletely ossified sternebrae were observed in 3 (1), and 2 (1) foetuses (litters) of G1 and G3 respectively; misaligned sternebrae were seen in 1 foetus of G2. Fused cervical vertebrae were observed in 1 foetus of G1. The total percentage of foetuses with skeletal malformations/variations were 6.80, 2.65, 3.36 and 3.76 for G1 (control), G2 (10 mg/kg), G3 (30 mg/kg) and G4 (90 mg/kg) respectively while the percentage of litters affected were found to be 17.39 (G1), 13.04 (G2), 16.67 (G3) and 10.00 (G4). No significant correlation was observed between treatment and individual skeletal malformations/variations or total percentages of foetuses and litters affected. The skeletal malformations/variations noted in treatment group were concurrently seen in control group. Moreover, the occurrence and incidences of these malformations/variations are common in developing foetuses. Therefore, these skeletal developmental malformations/variations were not considered to be test chemical related. In visceral observations, hematoma (ankle of left hind limb) was found in 1 foetus from G2 (10 mg/kg body weight) and enlarged adrenal with reduced thymus size was found in 1 foetus from G3 (30 mg/kg body weight) group. No malformations/variations were observed in any other foetuses of control or treated groups. Head razor examination of foetuses did not show any malformations/variations in control or treated groups.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Treatment related:

no

Any other information on results incl. tables

Table for Mortality and Morbidity

Group	Dose (mg/kg)	No. of Animals	Gestation Day	Observation
				Morning	Evening
G1	0	25	0-20	No mortality/ morbidity	No mortality/ morbidity
G2	10	25	0-20	No mortality/ morbidity	No mortality/ morbidity
G3	30	25	0-20	No mortality/ morbidity	No mortality/ morbidity
G4	90	25	0-20	No mortality/ morbidity	No mortality/ morbidity

Table for General Clinical Signs Observation

Group	Dose (mg/kg)	No. Of Animals	Animal Number	Gestation Day	Clinical Signs
G1	0	25	1-25	0-20	Normal
G2	10	25	26-50	0-20	Normal
G3	30	25	51-75	0-20	Normal
G4	90	25	76-100	0-20	Normal

Table for Body Weight

			Pregnant Females
Group	Dose (mg/kg)		Day of gestation and body weight in grams
			0	5	8	11	14	17	20
G1	0	Mean	228.17	250.57	260.83	274.70	290.26	318.48	355.43
		SD	14.31	14.59	15.97	17.15	19.11	18.02	23.23
		N	23	23	23	23	23	23	23
G2	10	Mean	230.26	254.22	264.09	278.22	294.39	320.57	358.96
		SD	14.08	15.03	16.09	16.21	18.10	20.63	24.42
		N	23	23	23	23	23	23	23
G3	30	Mean	231.04	253.33	263.38	277.83	293.33	318.71	357.42
		SD	12.61	12.74	14.56	15.62	15.56	16.06	17.55
		N	24	24	24	24	24	24	24
G4	90	Mean	232.55	254.75	262.85	278.00	293.15	318.15	358.00
		SD	14.52	17.01	17.92	19.43	20.58	22.12	26.77
		N	20	20	20	20	20	20	20

 

			Non-Pregnant Female
Group	Dose (mg/kg)		Day of gestation and body weight in grams
			0	5	8	11	14	17	20
G1	0	Mean	230.50	252.50	255.50	270.00	260.00	251.50	259.00
		SD	33.23	33.23	34.65	33.94	33.94	31.82	32.53
		N	2	2	2	2	2	2	2
G2	10	Mean	217.50	227.00	217.50	205.00	215.50	217.50	222.00
		SD	9.19	19.8	6.36	28.28	9.19	12.02	4.24
		N	2	2	2	2	2	2	2
G3	30	Mean	235.00	255.00	263.00	272.00	263.00	259.00	268.00
		SD	./.	./.	./.	./.	./.	./.	./.
		N	1	1	1	1	1	1	1
G4	90	Mean	232.20	258.20	265.20	274.80	266.60	262.60	263.40
		SD	9.73	13.65	16.48	14.52	17.43	15.42	14.45
		N	5	5	5	5	5	5	5

Key: SD: Standard deviation; N: No. of animals; ./.: Not applicable

Table for Body weight Change:

			Pregnant Females
Group	Dose (mg/kg)		Gestation Day and body weight change (%)
			5	8	11	14	17	20
G1	0	Mean	9.86	14.35	20.44	27.25	39.68	55.84
		SD	2.27	2.83	3.80	4.21	3.93	5.76
		N	23	23	23	23	23	23
G2	10	Mean	10.44	14.73	20.91	27.91	39.29	55.99
		SD	2.32	3.02	3.84	3.83	5.13	7.25
		N	23	23	23	23	23	23
G3	30	Mean	9.69	14.03	20.28	27.03	38.04	54.87
		SD	1.96	2.87	3.13	3.93	4.31	6.39
		N	24	24	24	24	24	24
G4	90	Mean	9.56	13.04	19.58	26.09	36.90	54.08
		SD	2.99	3.44	4.90	5.18	6.83	9.57
		N	20	20	20	20	20	20

 

			Non-Pregnant Females
Group	Dose   (mg/kg)		Gestation Day and body weight change (%)
			5	8	11	14	17	20
G1	0	Mean	9.65	10.92	17.30	12.91	9.25	12.52
		SD	1.39	0.95	2.18	1.56	1.95	2.11
		N	2	2	2	2	2	2
G2	10	Mean	4.27	0.15	-5.39	-0.75	0.21	2.21
		SD	4.7	7.16	17.01	8.42	9.77	6.27
		N	2	2	2	2	2	2
G3	30	Mean	8.51	11.91	15.74	11.91	10.21	14.04
		SD	./.	./.	./.	./.	./.	./.
		N	1	1	1	1	1	1
G4	90	Mean	11.19	14.19	18.33	14.87	13.15	13.46
		SD	3.06	4.25	2.69	7.05	6.16	4.59
		N	5	5	5	5	5	5

Key: SD: Standard deviation; N: No. of animals; ./.: Not applicable

 

Table for Maternal Evaluation

Groups	G1	G2	G3	G4
Dose (mg/kg body weight)	0	10	30	90
Initial animals per group	25	25	25	25
Confirmed Pregnancy at necropsy	24	24	24	21
Litters with live foetuses	23	23	24	20
Litters with early resorptions	1	1	0	1
Pregnancy rate (%)	92	92	96	80
No. of Corpora lutea	13.43 ± 1.41	13.65 ± 1.61	13.67 ±1.40	13.60 ± 2.09
No. of implantation sites	12.87 ± 1.74	13.22 ± 1.70	12.71 ± 1.65	13.35 ± 1.84
No. of resorptions	0.48 ± 0.67	0.61 ± 0.84	0.63 ± 0.82	0.70 ± 1.13
Pre-implantation loss (%)	4.30 ± 7.27	3.07 ± 6.96	6.78 ± 9.84	1.55 ± 4.06
Post-implantation loss (%)	3.63 ± 5.05	4.58 ± 5.74	4.94 ± 6.38	5.20 ± 7.84
Litter size	12.39 ± 1.73	12.57 ± 1.44	12.13 ± 1.75	12.65 ± 1.95
No. of live foetuses	12.39 ± 1.73	12.57 ± 1.44	12.08 ± 1.79	12.65 ± 1.95
No. of dead foetuses	0.00 ± 0.00	0.00 ± 0.00	0.04 ± 0.20	0.00 ± 0.00
Live Foetuses (%)	100.00 ± 0.00	100.00 ± 0.00	99.62 ± 1.86	100.00 ± 0.00

Values are represented as Mean ± SD.

Pregnancy rate(%) = (No. of females with live foetuses/ No. of mated females) x 100;

Pre-implantations loss = [(no. of corpora lutea − no. of implantations)/ no. of corpora lutea] x 100;

Post-implantation loss = [(no. of implantations - no. of live foetuses)/ no. of total implantations] x 100

 

Table for Hormone levels (T3, T4 & TSH)

Pregnant Females
Group	Dose (mg/kg)		T3 (ng/mL)	T4 (ng/mL)	TSH (µIU/mL)
G1	0	Mean	0.37	56.89	0.44
		SD	0.11	9.19	0.12
		N	23	23	23
G2	10	Mean	0.35	57.45	0.43
		SD	0.12	8.54	0.15
		N	23	23	23
G3	30	Mean	0.34	51.03	0.43
		SD	0.11	10.37	0.13
		N	24	24	24
G4	90	Mean	0.37	48.35 ↓↓	0.41
		SD	0.09	10.24	0.10
		N	20	20	20

 

Non- Pregnant Females
Group	Dose (mg/kg)		T3 (ng/mL)	T4 (ng/mL)	TSH (µIU/mL)
G1	0	Mean	0.26	48.34	0.44
		SD	0.04	2.35	0.12
		N	2	2	2
G2	10	Mean	0.22	37.50	0.37
		SD	0.06	1.17	0.16
		N	2	2	2
G3	30	Mean	0.25	56.67	0.31
		SD	./.	./.	./.
		N	1	1	1
G4	90	Mean	0.33	34.00	0.54
		SD	0.07	5.35	0.11
		N	5	5	5

Key: SD: Standard deviation; N: No. of animals; ↓↓:Decreased as compared to control (P<0.01)

 

Table for External Gross Evaluation of Foetuses

Group	G1	G2	G3	G4
Dose level (mg/kg body weight)	0	10	30	90
Total No. of Litters with live foetuses	23	23	24	20
Total No. of live Foetuses	285	283	290	253
Total No. of Male Foetuses	136	151	137	109
Total No. of Female Foetuses	149	138	153	144
Weight of foetuses (g)	3.46 ± 0.44	3.53 ± 0.33	3.54 ± 0.51	3.42 ± 0.33
Weight of male foetuses (g)	3.57 ± 0.46	3.65 ± 0.32 ↑↑	3.64 ± 0.57	3.56 ± 0.30
Weight of female foetuses (g)	3.37 ± 0.38	3.41 ± 0.28	3.45 ± 0.45	3.32 ± 0.30
Weight of Placenta (g)	0.46 ± 0.06	0.46 ± 0.06	0.47 ± 0.08	0.45 ± 0.06
Crown to rump length (cm)	3.45 ± 0.22	3.51 ± 0.16	3.51 ± 0.20	3.46 ± 0.27
AGD of Male foetuses (mm)	2.32 ± 0.17	2.35 ± 0.17	2.33 ± 0.08	2.31 ± 0.08
AGD of Female foetuses (mm)	1.16 ± 0.11	1.16 ± 0.10	1.15 ± 0.05	1.15 ± 0.06
Normalised AGD of Male foetuses	1.52 ± 0.13	1.53 ± 0.11	1.52 ± 0.08	1.52 ± 0.06
Normalised AGD of Female foetuses	0.77 ± 0.08	0.77 ± 0.07	0.76 ± 0.04	0.77 ± 0.04
Male/Female sex ratio (per dam)	1.20 ± 0.97	1.37 ± 0.96	1.03 ± 0.54	0.88 ± 0.51
External observations
Total no. of Foetuses with No Abnormality Detected	270	283	282	235
Total no. of Foetuses with abnormalities	15	6	8	18
Foetuses with abnormalities (%)	5.26	2.08	2.76	7.11
Total no. of Litters with No Abnormality Detected	12	17	17	9
Total no. of Litters with abnormalities	11	6	7	11
Litters with abnormalities (%)	47.83	26.09	29.17	50.00
Abnormalities
Haemorrhage (V)	0(0)	0(0)	0(0)	2(2)
Haemorrhage (%)	0(0)	0(0)	0(0)	0.79(10)
Runt (M)	11(11)	6(6)	8(7)	15(10)
Runt (%)	5.26(47.83)	2.08(26.09)	2.76(29.17)	5.93(50.00)
Dome-shaped Head (M)	4(1)	0(0)	0(0)	3(1)
Dome-shaped Head (%)	1.4(4.35)	0(0)	0(0)	1.19(5.00)

Values are represented as Mean ± SD; AGD: Anogenital distance; Normalised AGD: AGD/ Body weight ^(1/3); the incidence of the individual defect is presented as a number/ Percent of foetuses (number/ percent of litters); Abnormalities: includes malformations and variations; M: Malformations; V: Variations; ↑↑:Increased as compared to control (P<0.01).

 

Table for Skeletal Anomalies of the Foetuses

Groups	G1		G2		G3		G4
Dose (mg/kg body weight)	0		10		30		90
	No.	%	No.	%	No.	%	No.	%
Foetuses examined	147		151		149		133
No abnormality detected	137	93.20	147	97.35	144	96.64	128	96.24
Total and % Foetuses with abnormalities	10	6.80	4	2.65	5	3.36	5	3.76
Skull
Hole in Skull	5	3.40	0	0.00	0	0.00	3	2.26
Bipartite Ossification of interparietal	4	2.72	0	0.00	0	0.00	3	2.26
Bipartite Ossification of Occippital	1	0.68	0	0.00	0	0.00	1	0.75
Incomplete Ossification of Occipital	0	0.00	0	0.00	0	0.00	1	0.75
Ribs
Rib Misaligned	2	1.36	4	2.65	2	1.34	2	1.50
Rib Absent	0	0.00	0	0.00	1	0.67	0	0.00
Rib Branched	0	0.00	0	0.00	1	0.67	0	0.00
Sternebrae
Sternebra Unossified	3	2.04	0	0.00	2	1.34	0	0.00
Sternebra Misaligned	0	0.00	1	0.66	0	0.00	0	0.00
Vertebrae
Fused Cervical Vertebrae	1	0.68	0	0.00	0	0.00	0	0.00

The incidence of the individual defects presented as a number and percentages of foetuses; Abnormalities: includes malformations and variations; M: Malformations; V: Variations

 

Table for Skeletal Anomalies of the Foetuses (with respect to Litters) (continued)

Groups	G1		G2		G3		G4
Dose (mg/kg body weight)	0		10		30		90
	No.	%	No.	%	No.	%	No.	%
Litters examined	23		23		24		20
No abnormality detected	19	82.61	20	86.96	20	83.33	18	90.00
Total and % Litters with abnormalities	4	17.39	3	13.04	4	16.67	2	10.00
Skull
Hole in Skull	1	4.35	0	0.00	0	0.00	1	5.00
Bipartite Ossification of interparietal	1	4.35	0	0.00	0	0.00	1	5.00
Bipartite Ossification of Occippital	1	4.35	0	0.00	0	0.00	1	5.00
Incomplete Ossification of Occipital	0	0.00	0	0.00	0	0.00	1	5.00
Ribs
Misaligned	2	8.70	3	13.04	2	8.33	1	5.00
Absent	0	0.00	0	0.00	1	4.17	0	0.00
Branched	0	0.00	0	0.00	1	4.17	0	0.00
Sternebrae
Unossified	1	4.35	0	0.00	1	4.17	0	0.00
Misaligned	0	0.00	1	4.35	0	0.00	0	0.00
Vertebrae
Fused Cervical Vertebra	1	4.35	0	0.00	0	0.00	0	0.00

The incidence of the individual defect is presented as a number and percentages of litters

Abnormalities: includes malformations and variations; M: Malformations; V: Variations

Applicant's summary and conclusion

Conclusions:

Based on all the available data, it was concluded that there were no test chemical related adverse effects that were observed on the maternal reproductive/developmental toxicity and also fetal parameters. Although, significant decrease in T4 levels in thyroid estimations indicate towards maternal systemic toxicity. Therefore, the LOAEL for maternal systemic toxicity was observed to be 30 mg/kg bw/day, while the NOAEL for the maternal reproductive/developmental toxicity and fetal developmental toxicity was observed to be 90 mg/kg bw/day.

Executive summary:

A study according to Prenatal Developmental Toxicity Study, adopted: 25^thJune 2018 was performed using a total of 100 pregnant female Wistar rats. The test chemical was administered to these pregnant female Wistar rats by oral: gavage route and the vehicle used to dissolve the test chemical was distilled water. The test chemical was soluble in distilled water in the in-house solubility test and therefore, distilled water was chosen as the vehicle. The 100 pregnant females were randomized into 4 experimental groups containing 25 animals each. The groups, viz., G1, G2, G3 and G4 received 0, 10, 30 and 90 mg/kg bw/day of the test chemical, respectively. The test chemical was administered to these pregnant rats during Gestation Day 5 to 19 at the frequency of once daily. The doses were selected on the basis of previously done, dose range finding study (DRF study). In this study, the test chemical was administered orally to rats in graduated doses of 20, 60 and 180 mg/kg body weight for 14 consecutive days. Mortality was observed in only one animal of 180 mg/kg body weight group, while no mortalities were observed at 20 or 60 mg/kg body weight dose groups. Body weight was affected for both male and female rats of 180 mg/kg body weight dose group, while no change was observed for animals of mid and low dose groups. Further, test chemical related and dose-dependent lesions in stomach and intestine were observed at all the dose groups, which were also confirmed by histopathological analysis. Based on the above stated results, the doses 0, 10, 30 and 90 mg/kg body weight were selected for control, low, mid and high dose groups, respectively in the main study. In this study, all the animals were observed twice daily for any morbidity and/or mortality, throughout the acclimatization and study period. All animals were observed daily for clinical signs and symptoms after dose administration. These observations were regularly performed approximately 1 hour after dose administration, since onset of clinical signs after oral gavage is anticipated in that time frame. Animals were weighed at the time of receipt, on GD 0, on the first day of dosing (GD 5), and on gestation days 8, 11, 14, 17 and 20. The weight of feed provided and that leftover was recorded for each cage on the same day as animal body weight. This data was used to calculate mean feed consumption (g/day/rat) on gestation days 5, 8, 11, 14, 17 and 20. From all dams at termination, blood was collected in the morning; serum was separated and stored under appropriate conditions. All collected blood samples were assessed for serum levels of thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) using commercially available Rat ELISA kits. All pregnant females were weighed and euthanized on GD 20 (one day prior to expected day of delivery) by using CO2 asphyxiation. Fetuses were euthanized humanely as per the internal SOP. All animals were subjected to complete gross necropsy. From all dams at termination, thyroid gland were collected, weighed (after fixation) and preserved for histopathological examination. Immediately after the termination, uteri were removed, and the pregnancy status of the animals was evaluated. Uteri that appear non-gravid were further examined using ammonium sulphide staining to confirm non-pregnant status. The total weight of the gravid or non-gravid uteri including cervix were recorded for all animals. Further, the number of corpora lutea and the ovarian and placental weights were determined for all pregnant animals. The uterine contents were also examined for the number of implantation sites, number of live/viable fetuses, number of dead fetuses and number of resorptions. Using the above information, pre-implantation and post-implantation loss was calculated as follows: pre-implantations loss = [(no. of corpora lutea – no. of implantations)/ no. of corpora lutea] x 100 and post-implantation loss = [(no. of implantations – no. of viable fetuses/ no. of implantations] x 100. All fetuses were weighed, sexed and examined for external abnormalities and crown to rump length. For live fetuses, anogenital distances (AGD) were measured. The fetuses were examined for skeletal and soft tissue abnormalities with special emphasis to reproductive organs. Also, male fetuses were evaluated for incomplete testicular descent/cryptorchidism. The one half of each litter were prepared and examined for skeletal abnormalities (growth retardation, delayed ossification, etc.) using Alcian blue and Alizarin red double staining technique and the remainder were prepared for and examined for soft tissue alterations (visceral and head razor sectioning). During observations, no treatment-related clinical signs or symptoms were observed in any of the animals through the study period. No mortality or morbidity was recorded in animals of any of the experimental groups throughout the duration of the experiment. The mean body weight of pregnant and non-pregnant females remained comparable among all the groups for every time point (GD 0, 5, 8, 11, 14, 17 and 20) recorded during the study period. The percent change in body weights with respect to the first day of gestation was also statistically comparable among all the groups of Pregnant and non-pregnant females. Triiodothryronine (T3) and thyroid stimulating hormone (TSH) levels in sera were found to be statistically comparable among the control and treatment groups in both pregnant and non-pregnant females. However, significant decrease in thyroxine (T4) levels were observed in pregnant females of G4 (90 mg/kg) group as compared to G1 (Control) group (P<0.01). Absolute weights and weights relative to body weight of gravid uterus and ovary remained comparable for pregnant females of control and treatment groups. A statistically significant increase was observed in absolute weight of thyroid-parathyroid in pregnant females of G4 group (90 mg/kg) as compared to G1 (control) group (P<0.05). However, when thyroid-parathyroid weights relative to body weights were compared, no significant difference was observed between control and treatment groups. Absolute and relative weights of uterus, ovary, thyroid and parathyroid of non-pregnant females between control and treatment groups remained comparable. The mean feed consumed per day for pregnant females remained statistically equivalent in all groups of pregnant and non-pregnant females. Dams were euthanized and subjected to gross pathology examination. No gross pathological alterations were observed for pregnant and non-pregnant females of control (G1) or treated groups (G2, G3, G4). Thyroid glands from G1 (control) and G4 (90 mg/kg body weight) were subjected to microscopic examination. Congenital cyst (ultimo branchial origin) of thyroid gland was found in 3 animals of each G1 (control) and G4 (90 mg/kg body weight) group. Although congenital cysts were observed for thyroid and parathyroid glands of G4 (90 mg/kg body weight) animals, they were concurrently observed in G1 (Control) group. Furthermore, these cysts are spontaneous in nature and not attributable to the test chemical. No test chemical administration related adverse effect on the pre- and post-implantation losses was observed and remained comparable among all the treatment groups and control group. No significant difference was observed for the mean litter size as there were no significant litter losses by any resorptions across all dose groups. One female from each G1, G2 and G4 had very early resorption which was identified by staining the uterus with ammonium sulphide solution. This effect was no considered as treatment related since it did not have any dose dependent increase and was also observed in the control group. No dead fetuses were observed across all dose groups due to the test chemical administration. No changes in pregnancy duration was observed in all females across all the dose groups due to the test chemical administration. At termination 24/25, 24/25, 24/25 and 21/25 females were found to be pregnant from G1 (control), G2 (10 mg/kg body weight), G3 (30 mg/kg body weight) and G4 (90 mg/kg body weight) group respectively. This effect was not considered to be related to the test chemical administration. The weights of placenta remained comparable for control (G1) and treatment groups (G2, G3 and G4). The mean weight of male foetuses significantly increased in G2 (10 mg/kg body weight) (P<0.01) as compared to G1 (control). Such change in body weight was not observed in other dose groups and therefore it was considered as an incidental finding. There were no dead fetuses and reduction in number of live off springs across all the dose groups due to test chemical administration. No significant changes in the sex ratio of fetuses were observed across all the dose groups due to test chemical administration. No significant changes in the litter sizes and weights were observed across all the dose groups due to test chemical administration. Gross external observation of foetuses revealed that 5.26, 2.08, 2.76 and 7.11 percentage of foetuses from G1 (control), G2 (10 mg/kg), G3 (30 mg/kg) and G4 (90 mg/kg) respectively showed malformations/variations. The variation included haemorrhage in 0.79 percent of foetuses of G4 (90 mg/kg body weight) group and the malformations included: retarded growth (Runt): 3.86 (G1), 2.08 (G2), 2.76 (G3) and 5.93 (G4) percent of fetuses; dome-shaped head (Meningo-encephalocele): 1.4 (G1) and 1.19 (G4) percent of fetuses. No correlation was observed for the doses of the test chemical and the total percent of fetuses with malformations/variations. Similarly, no significant correlation was observed when litter was used as the unit for comparing the malformations/ variations or when data of individual malformations/variations were compared. These observations were found to be independent of the treatment and rather seem to be spontaneous in nature as observed by other studies. Skeletal malformations observed in skull included: hole in skull (mainly in interparietal and occipital region) observed in 5 (1) fetuses (litter) from G1 (Control group) and 3 (1) fetuses (litter) from G4 (90 mg/kg body weight); Bipartite ossification of inter-parietal found in 4 (1) fetuses (litter) from G1 (control) and 3 (1) fetuses (litter) from G4 (90 mg/kg body weight); Bipartite ossification and incomplete ossification of occipital found in 1 fetus from G1 (control) and G4 (90 mg/kg body weight) respectively. Although the number of fetuses with malformations of skull seems to be high, these observations were only present in fetuses of 1 litter each from G1 and G4 group. One rib was absent in 1 fetus from G3 (30 mg/kg body weight) and the same fetus had branched rib; 2 (2), 4 (3), 2 (2) and 2 (1) fetuses (litters) from G1, G2, G3 and G4 respectively showed misaligned ribs. Unossified/incompletely ossified sternebrae were observed in 3 (1), and 2 (1) fetuses (litters) of G1 and G3 respectively; misaligned sternebrae were seen in 1 fetus of G2. Fused cervical vertebrae were observed in 1 fetus of G1. The total percentage of fetuses with skeletal malformations/variations were 6.80, 2.65, 3.36 and 3.76 for G1 (control), G2 (10 mg/kg), G3 (30 mg/kg) and G4 (90 mg/kg) respectively while the percentage of litters affected were found to be 17.39 (G1), 13.04 (G2), 16.67 (G3) and 10.00 (G4). No significant correlation was observed between treatment and individual skeletal malformations/variations or total percentages of fetuses and litters affected. The skeletal malformations/variations noted in treatment group were concurrently seen in control group. Moreover, the occurrence and incidences of these malformations/variations are common in developing fetuses. Therefore, these skeletal developmental malformations/variations were not considered to be test chemical related. In visceral observations, hematoma (ankle of left hind limb) was found in 1 fetus from G2 (10 mg/kg body weight) and enlarged adrenal with reduced thymus size was found in 1 fetus from G3 (30 mg/kg body weight) group. No malformations/variations were observed in any other fetuses of control or treated groups. Head razor examination of fetuses did not show any malformations/variations in control or treated groups. Based on all the available data, it was concluded that there were no test chemical related adverse effects that were observed on the maternal reproductive/developmental toxicity and also fetal parameters. Although, significant decrease in T4 levels in thyroid estimations indicate towards maternal systemic toxicity. Therefore, the LOAEL for maternal systemic toxicity was observed to be 30 mg/kg bw/day, while the NOAEL for the maternal reproductive/developmental toxicity and fetal developmental toxicity was observed to be 90 mg/kg bw/day.