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Diss Factsheets

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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

Constituent 1
Chemical structure
Reference substance name:
2,2,2-trichloro-1-phenylethyl acetate
EC Number:
201-972-0
EC Name:
2,2,2-trichloro-1-phenylethyl acetate
Cas Number:
90-17-5
Molecular formula:
C10H9Cl3O2
IUPAC Name:
2,2,2-trichloro-1-phenylethyl acetate
Test material form:
solid: crystalline
Remarks:
White in color

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
The health status of each animal used in this study was examined on receipt (initial examination) by a veterinarian. All received animals were found to be in good health and were acclimatised to the laboratory conditions. Before mating, a veterinary examination was carried out by the study veterinarian and it was ascertained that all animals were still in good condition. Prior to receipt of animals for the study, the study room, racks and cages were cleaned and disinfected. During the study, the floor of the experimental room and work tops were swept and mopped with disinfectant solution at least once every day and more frequently when required. Cages were cleaned regularly. 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 an approved supplier, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean. Bedding material of batch no. 720 (Krishna Corncob Industries, Aurangabad) was used and a copy of the analyses for microbial and chemical contaminants of bedding material provided by the manufacturer has been incorporated in the raw data.
Environmental conditions: The room temperature was maintained between 21.00 to 23.30 °C and the relative humidity achieved was in the range of 50.10 to 63.80%. Artificial light was set to give a cycle of 12 hours light and 12 hours dark. Adequately filtered air with at least 12 changes per hour was provided. A conventional laboratory pellet diet (Batch no. 040820) from approved supplier (Nutrivet Life Sciences, Pune) was available ad libitum. Copies of the composition, microbial and chemical contaminant reports analysed by the manufacturer have been incorporated in the raw data.Aquaguard™ filtered drinking water was available ad libitum in regularly cleaned bottles. Samples of the drinking water at sa-FORD are periodically subjected to tests for bacteriological and chemical contaminants. The latest test results are included in the raw data.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methyl cellulose
Details on exposure:
The dose volume for each animal was calculated based on the most recently recorded body weight. Each formulation was administered in a single dose through oral gavage at a constant volume of 10 ml per kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulation samples [of doses viz; G1 (Control), G2 (Low dose), G3 (Mid dose), and G4 (High dose)] were analysed by HPLC method. Two replicates of approximately 2 ml samples from each upper, middle, and lower layer were sent to Chemistry Department for homogeneity and active ingredient analysis. The concentrations were calculated and reported. The dose formulation analysis was carried out during the first and the last week of treatment using a validated analytical method
Details on mating procedure:
For mating, two females were kept with a single male (2:1 pairing) until evidence of copulation was observed. The presence of sperm in vaginal smear was considered as day 0 of pregnancy/gestation. Females showing sperm positive smear were separated and housed individually.
Duration of treatment / exposure:
15 days (i.e. on GD 5 up to and including 19).
Frequency of treatment:
Once daily.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
G2
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
G3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
G4
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
All animals were observed twice daily (once before and once after the day’s activities) for any morbidity and/or mortality during acclimatization and treatment. All animals were observed daily for clinical signs and symptoms after dose administration. These observations were regularly performed approximately 1 hour after dose administration sincethe onset of clinical signs after oral gavage can be 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. Blood samples were collected from all dams on the day of termination through retro-orbital plexus. Serum from all blood samples were separated, stored under appropriate conditions, and measured for serum levels of thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) using commercially available Rat ELISA kits (KinesisDx USA).
Ovaries and uterine content:
Immediately after the termination, uteri were removed, and the pregnancy status of each animal 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 the cervix, was recorded for each animal. The number of corpora lutea and the ovarian and placental weights were recorded for each pregnant animal. Uterine contents were examined for the number of implantation sites, number of live/viable foetuses, number of dead foetuses and number of resorptions (early and late resorptions). Using the above information, percent pre-implantation and post-implantation losses were calculated as follows: pre-implantation loss = [(no. of corpora lutea – no. of implantations)/ no. of corpora lutea] x 100 and post-implantation loss = [(no. of implantations – no. of viable foetuses/ no. of implantations] x 100. All animals were subjected to complete gross necropsy. From all dams at termination, thyroid glands were collected, weighed (after fixation) and preserved for histopathological examination.
Fetal examinations:
From every individual litter, each foetus was weighed, sexed and examined for external abnormalities and crown to rump length. For each live foetus, anogenital distances (AGD) was measured. One-half of the foetuses from each litter were prepared and examined for skeletal abnormalities (growth retardation, delayed ossification, etc.) using Alcian blue and Alizarin red double-staining technique. The remaining foetuses from each litter were prepared for and examined for soft tissue alterations (visceral and head razor sectioning) with special emphasis on thereproductive tract. Each male foetus was also evaluated for incomplete testicular descent/cryptorchidism.
Statistics:
Raw data were processed using statistical software “Sigma Plot 14.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviations 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 were analysed using ANOVA and data showing significance in their variance 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 number 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.
Historical control data:
Inhouse.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs or symptoms were observed in G1, G2 or G3 during the study period. Clinical signs in the G4 animals included hypothermia and incoordination (all animals on the first day of treatment) and numerous cases of chromodacryorrhea from the 2nd to 10th day of treatment
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All animals survived to planned death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant decreases in body weight in treated pregnant dams vs. controls were observed at 250 mg/kg (on GD 20, by 4.4%), at 500 mg/kg (on GD 17, by 5.5%; on GD 20, by 6.3%) and at 1000 mg/kg (on GD 8, by 5.3%; on GD 14, by 5.5%; on GD 17, by 6.1%; and on GD 20, by 7.3%).
Significant decreases in body weight gain in treated pregnant dams vs controls were observed at 250 mg/kg (on GD 0-20, by 11.2%), at 500 mg/kg (on GD 0-8, by 25.9%; on GD 0-14, by 14.5%; on GD 0-17, by 16.5%; and on GD 0-20, by 15.3%) and at 1000 mg/kg (on GD 0-8, by 53.5%; on GD 0-11, by 32.4%; on GD 0-14, by 28.6%; on GD 0-17, by 24.0%; and on GD 0-20, by 22.0%).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No significant decreases in food intake were observed in pregnant dams treated at 250 mg/kg. At 500 mg/kg, significant decreaes in food intake were observed on GD 8 (mean, 13.92 g. vs mean 17.89 g. at 0 mg/kg), GD 11 (mean, 17.41 g. vs. mean, 19.46 g at 0 mg/kg) and GD 14 (mean, 20.95 g. vs. mean, 22.74 g. at 0 mg/kg). At 1000 mg/kg, significant decreaes in food intake were observed on GD 8 (mean, 9.53 g. vs. mean 17.89 g. at 0 mg/kg) and GD 14 (mean, 20.05 g. vs. mean, 22.74 g. at 0 mg/kg).
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:
effects observed, non-treatment-related
Description (incidence and severity):
No significant differences were observed in Triiodothryronine (T3) and Thyroxin (T4) levels in pregnant females of any dose group as compared to the G1 control group. Thyroid stimulating hormone (TSH) level was slighly but statistically decreased in the G4 group (mean, 0.16 microlitre IU/ml) as compared to control group (mean 0.21 microlitre IU/ml)). Since no associated changes were observed in Triiodothryronine (T3) or Thyroxin (T4) levels and no associated gross or histopathological alterations of the thyroid were noted; the observed small decrease in TSH in G4 animals was considered incidental/non-adverse.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The absolute and organ weight relative weight to body weight of ovary and thyroid-parathyroid remained statistically comparable between all the groups. A significant reduction in absolute uterus weight was observed in G4 pregnant dams (mean 55.68 g.) as compared to the control group (mean, 67.15 g). No other significant changes in uterus weight (either absolute or relative to body weight) were observed in the study. Since also no gross findings were made in the uteri from G4, the significant decrease in absolute uterus weight was considered incidental/non-adverse. A significant increase in placenta weight was observed in G3 (mean, 0.47 g) and G4 (mean, 0.48 g) as compared to G1 (mean, 0.44g) group. The observed placenta weights in G3 and G4 group were well within the historical control range of the facility (0.40-0.52 g); hence the significant changes in placenta weights were considered incidental/non-adverse.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological alteration was observed in any of the dams.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Thyroid and parathyroid gland from G1 and G4 did not exhibit any microscopic lesion of toxicologic significance. Focal ultimobranchial cysts were found in 2 animals of G1 and in one animal from G4 group. The cyst formations were considered congenital as they were noticed in both control and treated groups, and therefore it was considered a spontaneous and non-test item-related effect. Dilatations of follicles of the thyroid gland were also noticed in G1 and G4 groups (viz., Dam No. 1, 5, 10, 13, for G1 and Dam No.76, 82, 89, 93 for G4). These dilated follicles were considered to be incidental changes as they appeared in both control and treated groups at comparable incidences.
Histopathological findings: neoplastic:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No cases of abortion were observed in any of the groups.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No significant changes were observed.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
The incidences of total litter resorption were 1 of 22 dams with confirmed pregnancy at necropsy at 500 mg/kg and 2 of 24 dams with confirmed pregnancy at necropsy at 1000 mg/kg. All three cases were attributed to early resorptions. This effect was considered to be incidental/non-adverse since no significant changes in pregnancy rate with live foetuses were observed. The pregnancy rate with live foetuses were 96, 92, 84, and 88% at G1, G2, G3, and G4, respectively, and all of these values were within the historical control range of the test facility (i.e. of 72-96%).
Early or late resorptions:
no effects observed
Description (incidence and severity):
No significant changes were observed.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetus was observed in any of the groups.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy rates were 92, 84, 88, and 96% in G1, G2, G3, and G4, respectively.
Other effects:
no effects observed
Description (incidence and severity):
No significant differences in the number of corpora lutea were observed.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
maternal general toxicity
Based on:
test mat.
Remarks on result:
not determinable because of methodological limitations
Remarks:
Mild but statistically significant changes in body weight were observed the lowest dose level
Dose descriptor:
LOAEL
Remarks:
maternal general toxicity
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
changes in number of pregnant
dead fetuses
early or late resorptions
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
other: Number of corpora lutea

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant decreases in the mean body weight of foetuses (both sex-combined) were observed in G3 (mean, 3.18 g, i.e. 5.9% decrease vs. controls) and G4 group (mean, 3.01 g, i.e. 11.0% decrease vs controls) as compared to G1 (mean, 3.38 g). Significant decreases in mean body weight of male foetuses were observed in G3 (mean, 3.29 g, i.e. 5.7% decrease vs controls) and G4 groups (mean, 3.14 g, i.e. 10.5% decrease vs controls) as compared to control group (mean, 3.49 g). Furthermore, significant decreases in the mean body weight of female foetuses were observed in G3 (mean, 3.03 g, i.e. 7.6% decrease vs controls) and G4 groups (mean, 2.92 g, i.e. 11.0% decrease vs controls) as compared to G1 (mean, 3.28 g).
The mean body weights of foetuses from the G3 group were well within the historical control range (male & female combined weight: 3.02-3.9 g; male: 3.11-4.03 g, female: 2.99-3.75 g). The significant decreases in foetal weight in G4 for sex-combined and females were minimally/slightly below the historical control range and were therefore considered treatment-related and adverse.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Mean numbers of live foetuses per dam were 12.44, 10.24, 9.56, and 10.00 in G1, G2, G3, and G4, respectively. No significant differences were observed.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
M/F sex ratios were 1.02, 1.26, 1.48, and 0.83 in G1, G2, G3 and G4, respectively. No significant differences were observed.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean litter sizes were 12.96, 11.13, 11.38, and 11.36 in G1, G2, G3, and G4, respectively. No significant differences were observed.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Gross external observation of foetuses revealed that 3.22 (29.17), 1.56 (17.39), 3.35 (28.57) and 3.20 (36.36) percentages of foetuses (litters) from G1, G2, G3 and G4, respectively demonstrated variations whereas 0.96 (12.50), 0.78 (4.35), 0.42 (4.76) and 2.80 (31.82) percentages of foetuses (litters) from G1, G2, G3 and G4, respectively showed malformations. The variation included haemorrhage observed in 3.22 (29.17), 1.56 (17.39), 3.35 (28.57) and 3.20 (36.36) percent of foetuses (litters) from G1, G2, G3 and G4, respectively. The malformations included: retarded growth (Runt): 0.64 (8.33) in G1, 0.78 (4.35) in G2, 0.42 (4.76) in G3 and 2.80 (31.82) in G4 percent of foetuses (litters); dome-shaped head: 0.32 (4.17) (G1) and 0.40 (4.55) (G4) percent of foetuses (litters); Short neck and opened eye each observed in 0.40 (4.55) (G4) percent of foetuses (litters). No significant difference were observed when individual or total malformations or variations were compared between the groups.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Skeletal malformation included unossified supraoccipital observed in skull of 0.76 (4.55) percentage of foetuses (litters) from G4. No skeletal malformation was found in foetuses of G1, G2 and G3 groups. The total percentage of foetuses (litters) with skeletal variations were 6.25 (25.00), 3.01 (17.39), 4.03 (14.29) and 12.88 (40.91) in G1, G2, G3 and G4 group, respectively. Skeletal variations observed in rib included: rudimentary rib in 0.76 (4.55) percentage of foetuses (litters) from G4; misaligned ribs found in 5.00 (20.83), 2.26 (13.04), 3.23 (9.52) and 4.55 (18.18) percentage of foetuses (litters) from G1, G2, G3 and G4, respectively. Skeletal variations observed in sternebrae included: unossified/incompletely ossified sternebrae observed in 4.38 (4.17), 0.75 (4.35), 3.23 (14.29) and 9.85 (31.82) percentage of foetuses (litters) of G1, G2, G3 and G4, respectively; misaligned sternebrae were seen in 3.75 (20.83), 2.26 (13.04), 3.23 (9.52) and 4.55 (18.18) percentage of foetuses (litters) from G1, G2, G3 and G4.
Most skeletal abnormalities noted in treatment groups were concurrently seen in the control group. Moreover, the occurrence and incidences of these malformations/variations are common in developing foetuses. However, a positive correlation was observed in the doses of the test item and % litters with the incidences of unossified sternebrae (P≤0.05 and r = 0.970). The increased incidence of this skeletal variation, which was notable at 500 and 1000 mg/kg bw/day, was considered treatment-related but non-adverse
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral variations observed included: hematoma found in 1 foetus each from G2 and G3 group; and congestion on left kidney and liver found in 2 foetuses of 1 dam (dam no. 65) from G3 group. These observations were considered spontaneous in nature and not treatment-related due to lack of dose dependency. No other malformations or variations were observed in any other foetuses of control or treated groups. The head razor examination did not show any malformation or variation in any of the foetuses in any of the groups.
Other effects:
no effects observed
Description (incidence and severity):
A significant decrease in crown to rump length (CRL) was observed in foetuses of G4 (mean, 3.29 cm) as compared to control group (mean, 3.42 cm). The observed decrease in CRL of G4 foetuses was within the historical control range of test facility (3.23-3.91 cm) and this effect was therefore considered to be non-adverse. A significant increase in normalized AGD was observed in male foetuses of G4 group (mean, 2.10) as compared to G1 (mean, 2.04). The mean normalized AGD was within the historical control range of facility (1.83-2.29) and thus the observed increase in normalized AGD in G4 males was not considered to be an adverse effect of treatment. No significant changes in normalized AGD were observed for the female foetuses between the doses.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
other: crown to rump length, AGD
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Applicant's summary and conclusion

Conclusions:
In this OECD 414 conducted with Wistar rats, the NOAELs for maternal developmental toxicity and foetal developmental toxicity were established at 1000 and 500 mg/kg bw/day, respectively. NOAEL for maternal general toxicity could not be established due to minor but statistically significant decreases in body weight and body weight gain at 250 mg/kg bw/day. The foetal effects observed at 1000 mg/kg bw/day included significant decreases in body weight (sex-combined [i.e. 11.0% decrease vs controls] and of females [i.e. 11.0% decrease vs controls]) that were minimally or slightly below the historical control range of the test facillity. These foetal effects were considered secondary to maternal general toxicity, which at 1000 mg/kg bw/day included significant decreases in body weight (on GD 8, by 5.3%; on GD 14, by 5.5%; on GD 17, by 6.1%; and on GD 20, by 7.3%) and significant decreases in body weight gain (on GD 0-8, by 53.5%; on GD 0-11, by 32.4%; on GD 0-14, by 28.6%; on GD 0-17, by 24.0%; and on GD 0-20, by 22.0%). A significant positive correlation was found between test item dose and the incidence of unossified sternebrae (skeletal variation) at 500 and 1000 mg/kg bw/day. The increased incidence of this skeletal variation was considered treatment-related but non-adverse.
Executive summary:

The present study aimed to provide information on systemic, maternal, and developmental toxicological endpoints associated with the repeated oral administration of graduated doses of Rose crystal ex benzaldehyde (CAS No. 90-17-5) to Wistar rats. For this purpose, the OECD Test Guideline 414 (Adopted 25 June 2018) was followed. A total of 100 pregnant female rats were randomised into 4 experimental groups each containing 25 animals. The groups, viz., G1, G2, G3 and G4, received 0, 250, 500 and 1000 mg/kg bw/day of Rose crystal ex benzaldehyde (CAS No. 90-17-5), respectively. The test item was administrated via oral gavage and the vehicle used in this study was 0.5% w/v methyl cellulose. All groups were administered with vehicle or with dose formulation once daily from gestation day (GD) 5 to 19 (i.e. to one day prior to scheduled sacrifice). To ensure the accurate administration of the test item, the concentrations and homogeneity of the dose formulation were verified through formulation analyses in the first and last week of treatment. The results of these analyses were within the acceptable limits on both occasions. Observations on the animals included mortality/morbidity, onset of clinical signs/symptoms, body weight, body weight changes, feed consumption, pregnancy observations, foetal developmental parameters, serum hormone levels, gross pathology and histopathology. All animals survived to planned death. No clinical signs or symptoms were observed in G1, G2 or G3 during the study period. Clinical signs in the G4 animals included hypothermia and incoordination (on the first day of treatment) and numerous cases of chromodacryorrhea between the 2nd and 10th day of treatment. Significant decreases in the mean body weight and % change in body weight with respect to GD 0 were observed in G2, G3, and G4 as compared to the control group. The decreases were dose-dependent and attributed to the test item. Significant decreases in feed consumption were observed in G3 and G4 as compared to the control group. No significant differences were observed in triiodothyronine (T3) and Thyroxin (T4) levels in pregnant females of any group as compared to the G1 control group. Thyroid stimulating hormone (TSH) level was significantly decreased in the G4 group (mean, 0.16 microlitre IU/ml) as compared to control group (mean 0.21 microlitre IU/ml)). Since no associated changes were observed in triiodothyronine (T3) or Thyroxin (T4) levels and no associated gross or histopathological alterations of the thyroid were noted; the observed small decrease in TSH in G4 animals was considered incidental/non-adverse. The absolute and organ weight relative weight to body weight of ovary and thyroid-parathyroid remained statistically comparable between all the groups. A significant reduction in absolute uterus weight was observed in G4 pregnant dams (mean 55.68 g.) as compared to the control group (mean, 67.15 g). No other significant changes in uterus weight (either absolute or relative to body weight) were observed in the study. Since also no gross findings were made in the uteri from G4, the significant decrease in absolute uterus weight was considered incidental/non-adverse. A significant increase in placenta weight was observed in G3 (mean, 0.47 g) and G4 (mean, 0.48 g) as compared to G1 (mean, 0.44g) group. The observed placenta weights in G3 and G4 group were well within the historical control range of the facility (0.40-0.52 g); hence the significant changes in placenta weights were not considered to be adverse effects of treatment. No treatment-related changes were observed during the gross or histopathologic examination. The incidences of total litter resorption were 1 of 22 dams with confirmed pregnancy at necropsy at 500 mg/kg and 2 of 24 dams with confirmed pregnancy at necropsy at 1000 mg/kg. All three cases were attributed to early resorptions. This effect was considered to be incidental/non-adverse since no significant changes in pregnancy rate with live foetuses were observed. The pregnancy rate with live foetuses were 96, 92, 84, and 88% at G1, G2, G3, and G4, respectively, and all of these values were within the historical control range of the test facility (i.e. of 72-96%). Maternal parameters including pregnancy ratio, number of corpora lutea, implantation sites, resorptions and percentage of pre- and post-implantation loss remained comparable between all groups. Significant decreases in the mean body weight of foetuses (both sex-combined) were observed in G3 (mean, 3.18 g, i.e. 5.9% decrease vs. controls) and G4 group (mean, 3.01 g, i.e. 11.0% decrease vs controls) as compared to G1 (mean, 3.38 g). Significant decreases in mean body weight of male foetuses were observed in G3 (mean, 3.29 g, i.e. 5.7% decrease vs controls) and G4 groups (mean, 3.14 g, i.e. 10.5% decrease vs controls) as compared to control group (mean, 3.49 g). Furthermore, significant decreases in the mean body weight of female foetuses were observed in G3 (mean, 3.03 g, i.e. 7.6% decrease vs controls) and G4 groups (mean, 2.92 g, i.e. 11.0% decrease vs controls) as compared to G1 (mean, 3.28 g). The mean body weights of foetuses from the G3 group were well within the historical control range (male & female combined weight: 3.02-3.9 g; male: 3.11-4.03 g, female: 2.99-3.75 g). The significant decreases in foetal weight in G4 for sex-combined and females were minimally/slightly below the historical control range and were therefore considered treatment-related. Mean numbers of live foetuses per dam were 12.44, 10.24, 9.56, and 10.00 in G1, G2, G3, and G4, respectively with no significant differences were observed. M/F sex ratios were 1.02, 1.26, 1.48, and 0.83 in G1, G2, G3 and G4, respectively with no significant differences were observed. Mean litter sizes were 12.96, 11.13, 11.38, and 11.36 in G1, G2, G3, and G4, respectively with no significant differences were observed. A significant decrease in crown to rump length (CRL) was observed in foetuses of G4 (mean, 3.29 cm) as compared to control group (mean, 3.42 cm). The observed decrease in CRL of G4 foetuses was within the historical control range of test facility (i.e. of 3.23 - 3.91 cm) and this effect was therefore considered to be incidental/non-adverse. A significant increase in normalized AGD was observed in male foetuses of G4 group (mean, 2.10) as compared to G1 (mean, 2.04). The mean normalized AGD was within the historical control range of facility (i.e. of 1.83-2.29) and thus the observed increase in normalized AGD in G4 males was not considered to be an adverse effect of treatment. No significant changes in normalized AGD were observed for the female foetuses between the doses. No treatment-related effects were observed during the gross, visceral or skeletal examination except for a significant positive correlation found between the doses of test item and the incidence of unossified sternebrae (skeletal variation). The increased incidence of this skeletal variation, which was notable at 500 and 1000 mg/kg bw/day, was considered treatment-related but non-adverse.