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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10 weeks during pre-mating period (both P males and females), 2 weeks during mating period (both P males and P females), throughout gestation and lactation periods up to weaning of F1 animals (P females) as well as when administered to F1 generation animals from PND 21 to 95 (for C1A animals) and PND 21 to 102 (for C1B animals) under experimental conditions employed.
Therefore, the no-observed-adverse-effect-level (NOAEL) of the test item is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because an extended one-generation reproductive toxicity study is available

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Remarks:

In accordance with OECD Guideline for Testing of Chemicals, Number 443, “Extended One-Generation Reproductive Toxicity Study”, adopted on 25 June 2018.

Type of information:

experimental study

Remarks:

The Cohorts 1A and Cohort 1B (without extension to F2 generation) were selected for F1 generation assessments based on the available toxicity information of test item in consultation with the sponsor.

Adequacy of study:

key study

Study period:

14 May 2021 to 02 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The Cohorts 1A and Cohort 1B (without extension to F2 generation) were selected for F1 generation assessments based on the available toxicity information of test item in consultation with the sponsor

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

adopted on 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item. However, the estimated NOAEL of a structural analogue compound, Pigment Red 282 was 1000 mg/kg body weight/day obtained from a 28-Day Repeated Dose Oral Toxicity study in Wistar Rats performed according to OECD Test Guideline 407.
(Reference:https://echa.europa.eu/registration-dossier/-/registered-dossier/15097/7/6/2/? documentUUID=8787be8c-e05e-49c5-af69-63f79a57bcf6).
Based on the above information, a dose range finding study for prenatal developmental toxicity study [Bioneeds study no. BIO-DTX 053] with the test item by oral gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 was conducted with the doses of 0, 111, 333 and 1000 mg/kg body weight/day for vehicle control, low, mid and high dose groups, respectively in consultation with the sponsor. The study included the general and maternal end point assessments such as, maternal body weights, feed consumption, corrected body weight for maternal body weight gain, gravid uterus weight, uterine content observations, implantations, and gross pathological examinations. The dose range finding study also included the fetal/prenatal developmental assessments such as, fetal weights (sex wise) per litter, fetal external examinations, and fetal gross soft tissue examinations. The results of this range finding study did not produce any indication of maternal and fetal (prenatal developmental) toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Hence, the same dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid, and high dose groups, respectively in consultation with the sponsor for the present “Extended One-Generation Reproductive Toxicity Study with the test item by oral gavage in Sprague Dawley Rats”.

The test item was administered continuously in graduated doses to three groups of males and females. The parental (P) generation animals were dosed for a defined
pre-mating period (10 weeks) and a two-week mating period. Treatment of the
P females was continued during pregnancy and lactation until termination following weaning of respective litters. The F1 offspring received further treatment with the test item from weaning till adulthood.
The Cohorts 1A and Cohort 1B (without extension to F2 generation) were selected for F1 generation assessments based on the available toxicity information of test item in consultation with the sponsor.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability and homogeneity of the test material in the vehicle under test conditions (e.g. in the exposure medium) and during storage: The stability and homogeneity of the test item in dose formulations was established by the analytical investigation
The prepared test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxymethyl cellulose within the mean percent recovery range of 85 to 115.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Triturated well in a mortar (grinding)

- Final concentration of a dissolved solid: Low dose-11.1, Mid dose-33.3 and High dose-100 mg/mL

FORM AS APPLIED IN THE TEST: Suspension

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Species: Rat (Rattus norvegicus)
Strain: Sprague Dawley
Justification for Selection of Species : Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Source of Supply : Hylasco Biotechnology India Pvt. Ltd,
Charles River Technology Licensee, CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA

Animals were housed under standard laboratory conditions in an environmentally monitored, temperature-controlled room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.8 to 23.1oC, relative humidity
48 to 64%, with 12 hours light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.
Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
P Animals:
i. Pre-mating
Maximum of two animals of same sex and group per cage were housed.
ii. Mating
During mating, two animals (one male and one female) of same group were housed.
iii. Post mating
After confirmation on 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 during gestation and lactation periods.
Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
F1 Animals:
i. During Lactation period (postnatal period)
All F1 pups of both sexes were housed along with the dam.
ii. Post Weaning
Two animals of same sex and group per cage were housed.
Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum to the rats throughout the experimental period. The contaminant analysis test reports for the feed are presented as Annexure 2.
A sample of each batch of the diet used during the study was retained under appropriate conditions (-20°C) and discarded on the day of finalization of the report.
Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% w/v

Details on exposure:

The test item formulations/vehicle were administered to animals through oral gavage using stainless steel intubation cannula attached to a disposable syringe once daily.
All the doses were administered in an equi-volume of 10 mL/kg with the concentration of 11.1, 33.3 and 100 mg/mL for 111, 333 and 1000 mg/kg body weight dose groups respectively. Vehicle was administered to vehicle control group at an equi-volume of 10 mL/kg body weight. The actual dose volume for each animal was calculated based on the most recent body weight.
Parental (P) Generation Animals:
-The parental (P) animals (both males and females) were treated for a period of
10 weeks during pre-mating period.
The parental (P) males were treated during mating period and continued the treatment until sacrifice [a total of 88 to 95 days of test item administration].
The parental (P) females were treated during cohabitation period until confirmation of mating, during mating, throughout gestation and lactation periods and up to weaning of F1 animals [a total of 113 to 124 days of test item administration]. .
F1 Animals:Direct dosing by oral gavage
Cohort 1A - From day of weaning (PND 21) to PND 95
Cohort 1B - From day of weaning (PND 21) to PND 102

Details on mating procedure:

Each P female was placed with a randomly selected, single and unrelated male from the same dose group (1:1 pairing) until evidence of mating. Day 0 of pregnancy was defined as the day on which mating evidence was confirmed (presence of sperm in vaginal smear).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item in dose formulations was established by the Analytical Department of Bioneeds India Private Limited (Bioneeds study no.:
BIO-ANM 1717).
The prepared test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxymethyl cellulose within the mean percent recovery range of 85 to 115.

Duration of treatment / exposure:

Parental (P) Generation Animals
The parental (P) animals (both males and females) were treated for a period of
10 weeks during pre-mating period.
The parental (P) males were treated during mating period and continued the treatment until sacrifice [a total of 88 to 95 days of test item administration]
The parental (P) females were treated during cohabitation period until confirmation of mating, during mating, throughout gestation and lactation periods and up to weaning of F1 animals [a total of 113 to 124 days of test item administration
F1 Animals
Cohort 1A - From day of weaning (PND 21) to PND 95
Cohort 1B - From day of weaning (PND 21) to PND 102

Frequency of treatment:

once daily

Details on study schedule:

Study Initiation Date: 14 May 2021
Experimental Starting Date: 14 May 2021
Parental (P) Generation
Acclimatization Date : Start: 14 May 2021; End: 19 May 2021
Treatment Date :
Males: Start: 20 May 2021; End: 22 August 2021
Females: Start: 20 May 2021; End: 20 September 2021
Cohabitation period: Start: 28 July 2021; End: 09 August 2021
Necropsy Date :
Males: Start: 16 August 2021; End: 23 August 2021
Females: Start: 10 September 2021; End: 21 September 2021
F1 Generation
Weaning Date: Start: 09 September 2021; End: 20 September 2021
Treatment Date :
Cohort 1A: Start: 09 September 2021; End: 03 December 2021
Cohort 1B: Start: 09 September 2021; End: 10 December 2021
Necropsy Date :
Cohort 1A: Start: 23 November 2021; End: 04 December 2021
Cohort 1B: Start: 30 November 2021; End: 11 December 2021
In-life End Date :11 December 2021
Histopathology Completion Date: 01 May 2022
Experimental Completion Date : 01 May 2022
Draft Report Submission Date : 30 May 2022
Study Completion Date: 02 August 2022

Dose / conc.:

0 mg/kg bw/day

Remarks:

Dose concentration = 0 mg/mL

Dose / conc.:

111 mg/kg bw/day

Remarks:

Dose concentration = 11.1 mg/mL

Dose / conc.:

333 mg/kg bw/day

Remarks:

Dose concentration = 33.3 mg/mL

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Dose concentration = 100 mg/mL

No. of animals per sex per dose:

A total of 200 (100 males + 100 females) Sprague Dawley rats were selected for parental (P) generation and distributed to four P generation groups. Each P generation group (G1, G2, G3 and G4) consisted of 25 males and 25 females. A total of 160 males and 160 females were selected on the day of weaning (PND21) and randomly assigned to two cohorts [Cohort 1A (80 males + 80 females) and Cohort 1B (80 males + 80 females)]. Each cohort consisted of four groups and consisted of 20 males and 20 females per group.

Control animals:

yes, concurrent no treatment

Details on study design:

There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item. However, the estimated NOAEL of a structural analogue compound, Pigment Red 282 was 1000 mg/kg body weight/day obtained from a 28-Day Repeated Dose Oral Toxicity study in Wistar Rats performed according to OECD Test Guideline 407.
(Reference:https://echa.europa.eu/registration-dossier/-/registered-dossier/15097/7/6/2/? documentUUID=8787be8c-e05e-49c5-af69-63f79a57bcf6).
Based on the above information, a dose range finding study for prenatal developmental toxicity study [Bioneeds study no. BIO-DTX 053] with the test item by oral gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 was conducted with the doses of 0, 111, 333 and 1000 mg/kg body weight/day for vehicle control, low, mid and high dose groups, respectively in consultation with the sponsor. The study included the general and maternal end point assessments such as, maternal body weights, feed consumption, corrected body weight for maternal body weight gain, gravid uterus weight, uterine content observations, implantations, and gross pathological examinations. The dose range finding study also included the fetal/prenatal developmental assessments such as, fetal weights (sex wise) per litter, fetal external examinations, and fetal gross soft tissue examinations. The results of this range finding study did not produce any indication of maternal and fetal (prenatal developmental) toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Hence, the same dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid, and high dose groups, respectively in consultation with the sponsor for the present “Extended One-Generation Reproductive Toxicity Study with by oral gavage in Sprague Dawley Rats”.

Parental animals: Observations and examinations:

Clinical Signs of Toxicity and Mortality/Morbidity
Detailed Clinical Examination
Body Weight
Feed Consumption
Oestrus Cyclicity
Reproductive Performance Evaluation
Delivery and Litter Observations
Uteri Observations
Clinical Pathology and Thyroid hormonal estimations
Thyroid Hormone Levels Estimation
Sperm Parameters
Gross Pathology
Histopathology

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored daily for a period of 2-weeks during pre-mating period before initiation of cohabitation.
During cohabitation period until evidence of mating.
At termination (on the day of necropsy) to correlate with histopathology of reproductive organs.

Sperm parameters (parental animals):

Sperm motility was evaluated immediately after sacrifice. The percentage of progressively motile sperms was determined subjectively. Samples for evaluating sperm morphology was taken from the suspension used for the sperm motility. Sperm sample was examined as fixed and 200 spermatozoa per sample were classified as either normal (both head and mid-piece/tail appear normal) or abnormal. Another testis reserved for spermatid head counts was stored in -20ºC from each animal and subjected to evaluation for homogenization resistance spermatid head counts to calculate daily sperm production.

Litter observations:

The day of parturition was considered as lactation day (LD) 1 for the dam and postnatal day (PND) 1 for pups. The number of male/female pups born (live/dead/cannibalized) per litter were recorded and sex ratio (m/f) and live birth index was calculated.
The number of pups survived/dead per litter were recorded during lactation period and pup survival index (%) per litter between lactation day 1 to 4, 5 to 7 and 8 to 14 and 15 to 21 was calculated

Postmortem examinations (parental animals):

All the parental males were sacrificed after completion of mating procedure. Males were randomized throughout all dose groups and restricted to maximum of 20 animals per day for necropsy.
The females not confirmed with mating were sacrificed after 25 days from the day of termination of cohabitation process. The females confirmed with mating but not littered were sacrificed 25 days after confirmation of mating. All the littered females were sacrificed on lactation day 22. Organ Weight and Tissue Preservation
The organs such as Adrenals, Bone marrow smear, Brain Epididymis [males], Eye with optic nerve, Gastrointestinal tract, Heart, Kidneys, Liver, Lungs, Ovaries, Peripheral (sciatic) nerve, Pituitary gland, Prostate -dorsolateral and ventral [males], Seminal vesicles with coagulating glands and their fluids as a unit – [males], Skeletal muscle Skin with mammary gland [both males and females], Spleen, Spinal cord , Testes [males], Thymus, Thyroid along with parathyroid, Trachea, Urinary bladder, Uterus with cervix [females], Vagina [females] and Vas deferens [males] from all P animals were collected, weighed, and preserved.

Histopathology
Histopathological examination was conducted on all the tissues collected from the vehicle control (G1) and high dose (G4) group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) sacrificed at termination.
Additionally, reproductive organs of all animals suspected of reduced fertility (those that failed to conceive or sire from all the dose groups) were subjected to histopathological examination.

Postmortem examinations (offspring):

Necropsy and Gross Pathology

The animals were euthanized using deep Isoflurane anaesthesia followed by exsanguination and subjected to gross pathological examination. Gross pathological examination was performed on all the animals sacrificed.
Cohort 1A and 1B Males and Females: The cohort 1A animals were sacrificed on PND 96 and the cohort 1B animals were sacrificed on PND 103.

Cohort 1A: The organs and tissues such as Adrenals, Bone marrow smear, Brain Epididymis [males], Eye with optic nerve, Gastrointestinal tract, Heart, Kidneys, Liver, Lungs, Ovaries, Peripheral (sciatic) nerve, Pituitary gland, Prostate -dorsolateral and ventral [males], Seminal vesicles with coagulating glands and their fluids as a unit – [males], Skeletal muscle Skin with mammary gland [both males and females], Spleen, Spinal cord , Testes [males], Thymus, Thyroid along with parathyroid, Trachea, Urinary bladder, Uterus with cervix [females], Vagina [females] and Vas deferens [males] of all high dose and control animals were examined for histopathology.

Cohort 1A Females (Ovarian Follicular and Corpora luteal Assessments):
Histopathological examination of ovaries including quantitative evaluation of primordial and small growing follicles, as well as corpora lutea was conducted for all control and high dose animals.

Cohort 1B: The reproductive and endocrine tissues were processed to the block stage.

Organ-typic Development:
C1A Males: Caput, corpus and cauda of the epididymis and the vas deferens were examined for appropriate organ-typic development.
C1A Females: Ovary with oviduct, uterus and vagina were examined for appropriate organ-typic development.

Statistics:

The raw data was subjected to computer statistical processing. The data was subjected to various statistical analyses using SPSS software version 27.

All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05) indicated by the aforementioned tests.

The statistical analysis was followed to the parameters as mentioned below table.
Parameter
Type of Analysis - One-way ANOVA with Dunnett's post-test
• Bodyweight (weekly/gestation/lactation)
• Percent Change in body weight (weekly/gestation/lactation)
• Feed consumption (weekly/gestation/lactation)
• Copulatory interval
• Gestation length
• Mean oestrus cycle length
• Absolute/relative organ weights
• Mean pup weight per litter
• Mean pup anogenital distance ratio per litter
• Serum thyroid hormonal values
• Occurrence of postnatal developmental landmarks of F1 pups
• Responses for sensory reflexes of F1 pups
• Sexual maturation time points of F1 animals
• Splenic lymphocyte sub-populations
• Sperm motility/morphological changes/daily sperm production
• Ovarian follicular count (Independent sample T - test) Parametric -
Type of Analysis - Non-parametric - Kruskal-Wallis followed by the Mann-Whitney
• Implantations/litter
• No. of pups/litter
• Sex ratio/litter at birth and during the lactation period
• Litter size at birth and during the lactation period
• Post-implantation loss/litter
• Postnatal loss/litter
Type of Analysis - Cross Tabs - Frequency comparison Chi-square test/ Fischer's Exact Test
• Reproductive indices
• No. of dams with/without live pups
• No. of dams with/without dead pups
• No. of litters with/without resorptions

Reproductive indices:

The following reproductive performance indices were calculated for all P animals.

Mating and Fertility Index :
The male / female mating, and fertility indices were calculated as mentioned below:
Male mating index (%) = No. of males with confirmed mating/ Total no. of males cohabited X 100

Female mating index (%)= No. of sperm-positive females/Total no. of females cohabited X 100

Male fertility index (%)=No. of males impregnating a female/Total no. of males cohabited X 100

Female fertility index (%)= No. of females with evidence of implantation sites/No. of sperm-positive females X 100

Cohabitation Record and Copulatory Interval (Pre-coital Interval)

The day of initiation of mating and day of confirmation of mating were recorded for each female, and the pre-coital interval was calculated as mentioned below:

Pre-coital interval (days)=Date of confirmation of mating – Date of initiation of cohabitation

Gestation Length: The day of confirmation of mating and day of parturition were recorded for each female and the gestation length per litter was calculated as mentioned below:

Gestation length (days)=Date of parturition – Date of evidence of mating (GD 0)

Gestation Index
The gestation index (%) per group was calculated as mentioned below:
Gestation index (%)=No. of females with live born/No. of females with evidence of pregnancy X 100

Parturition Index
The parturition index (%) per group was calculated as mentioned below:
Parturition index (%)= No. of females littered/No. of females with evidence of pregnancy X 100

Female Fecundity or Pregnancy Index
The pregnancy index (%) per group was calculated as mentioned below:
Pregnancy index (%) = No. of females evident of presence of live/dead fetuses/pups /No. of females with evidence of matingX 100

Offspring viability indices:

The day of parturition was considered as lactation day (LD) 1 for the dam and postnatal day (PND) 1 for pups. The number of male/female pups born (live/dead/cannibalized) per litter were recorded and sex ratio (m/f) and live birth index was calculated (litter as a unit) as mentioned below:
Sex ratio (m/f)= No. of male offspring/Number of female offspring
Live birth index (%) per litter=No. of pups born alive/Total no. of pups bornX 100

The number of pups survived/dead per litter were recorded during lactation period and pup survival index (%) per litter between lactation day 1 to 4, 5 to 7 and 8 to 14 and 15 to 21 was calculated (litter as a unit) as mentioned below:

Pup survival index (%) on LD 4/7/14/21 =Total no. of live pups on LD 4/7/14/21/No. of pups born/4/7/14X 100

The sex ratio (m/f) on LD 4, 7, and 13 was calculated per litter, as mentioned below:
Sex ratio (m/f) on LD 4/7/14/21=No. of male offspring on LD 4/7/14/21/No. of female offspring on LD 4/7/14/21

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Dark pink coloured faeces were noted in all the tested dose group animals of both sexes. This coloration is due to coloured nature of the test item but not an adverse sign.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In group G3, statistically significant reduction in percent change in mean gestational body weight gain during GD 7 to 14 was noted when compared with vehicle control group. This noted change is considered as incidental and unrelated to treatment, as the change is a single occurrence and there were no such incidences noted during other phases of the gestation such as during GD 0 to 7 and 14 to 20 in the same dose level.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

In group G2 males, statistically significant reduction in mean feed consumption was noted during week 1 when compared with vehicle control group. This noted change is considered as incidental and unrelated to treatment, as the change is a single occurrence for mean feed consumption and there were no such incidences noted throughout the experimental period at the same dose level.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

statistically significant increase in mean haematocrit value in all the tested dose group males and statistically significant increase in mean absolute lymphocytes of group G2 males were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain and also similar changes were not occurred in other sex of same dose level.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

statistically significant decrease in mean total protein levels of group G4 females were noted when compared with vehicle control group.This difference is considered as incidental and unrelated to treatment, as the obtained mean value is within in-house historical control range of same species and strain and also similar changes did not occur in other sex of same dose level.

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The few microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

A mean oestrus cycle length of 4.76, 4.91, 4.95 and 4.92 days was noted from groups G1, G2, G3 and G4 respectively. The mean length of oestrus cycle per female during treatment period was unaffected by the test item administration in any of the tested dose groups.
However, statistically significant increase in mean oestrus cycle length was noted in all the tested dose groups when compared with vehicle control group. This noted change is not considered as test item related as the obtained mean values are well within the
in-house historical control range and no irregularities in oestrus cycle were noted in any of the tested dose groups.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

The test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10 weeks during pre-mating period (both P males and females), 2 weeks during mating period (both P males and P females), throughout gestation and lactation periods.

Therefore, the no-observed-adverse-effect-level (NOAEL) of the test item is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.

Key result

Dose descriptor:

NOAEL

Remarks:

1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points

Effect level:

ca. 1 000

Based on:

test mat.

Remarks:

test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food efficiency

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

other:

Remarks on result:

other: test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

The dark pink coloured faeces were noted in all the testing dose group animals of both sexes. This coloration is due to coloured nature of the test item but not any adverse signs.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (C1A)
The noted non-toxicological and statistically significant increase in mean body weight on PND 42 in group G2 females when compared with vehicle control group .

A statistically significant decrease in mean body weights on PND 63, 70, 77, 84 and 91 and statistically significant decrease in percent change in mean body weight gain with respect to postnatal day (PND) 21 on PND 63, 70, 77, 84 and 91 in groups G3 and G4 males were noted when compared with vehicle control group.
These changes are not considered as adverse as there were no other systemic toxicity effects noted in any of these tested dose groups but are considered as stress induced effects due to test item administration.
Cohort 1B (C1B)
The noted non-toxicological and statistically significant increase in mean body weights on PND 77 and 84 in group G2-C1B females; increase in mean body weights on
PND 77, 84, 91 and 98 in group G3-C1B females; increase in mean body weights on PND 84, 91 and 98 in group G4-C1B females; increase in percent change in mean
body weight gain with respect to postnatal day (PND) 21 on PND 84 in group
G3-C1B females when compared with vehicle control group are considered as incidental.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (C1A)
A statistically significant decrease in mean feed consumption during week 4 in group G3-C1A males when compared with vehicle control group is considered as incidental.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase in mean haemoglobin, haematocrit, and prothrombin time levels in group G3-C1A females were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain and similar changes were not occurred in other sex of same dose level.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease in mean glucose, creatinine, cholesterol, and albumin levels of group G4 males; statistically significant decrease in mean creatinine levels in all the tested dose group females; statistically significant decrease in mean albumin levels in group G3 females was noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean value is within in-house historical control range of same species and strain and similar changes were not occurred in other sex of same dose level.

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (C1A)

In group G2-C1A,
- decrease in mean absolute weight of liver and increase in relative weight of brain (males).
- increase in mean absolute and relative weight of liver and increase in absolute weight of iliac lymph nodes (females).

In group G3-C1A,
- decrease in mean terminal body weight (males).
- increase in mean relative weight of adrenals, kidneys and thyroid along with parathyroid (males).
- increase in mean relative weight of liver (females).

In group G4-C1A,
- decrease in mean terminal body weight (males).
- decrease in mean absolute weight of epididymides, heart, brain, liver (males); increase in mean relative weight of adrenals, testes, kidneys, prostate and thyroid along with parathyroid (males).
- increase in mean absolute and relative weight of thymus (females).

Cohort 1B (C1B)

In group G2-C1B,
- decrease in mean relative weight of mandibular lymph nodes (females).
In group G3-C1B,
- decrease in mean relative weight of adrenals and thyroid along with parathyroid (males).
- increase in mean absolute weight of spleen (females).
In group G4-C1B,
- increase in mean terminal body weight (females).
- decrease in mean absolute and relative weight of adrenals (males).
- decrease in mean relative weight of iliac and mandibular lymph nodes (females).

These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain and also neither gross pathological changes nor microscopic changes were noted in any of these organs in all the tested dose group animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In test-item administered group of animals of both the sexes, the contents of gastrointestinal tract were found to be pink in colour which could be attributed to physical appearance of the test item (pink in colour).
A single incidence of small sized testis was observed in G3-C1A group male rat and was microscopically correlated with moderate atrophy of tubules. This change was considered as an isolated spontaneous finding in rats.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Few of the microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.
Quantitative ovarian follicular assessment (primordial and primary follicles) in all parental and C1A females of control and high dose groups did not reveal any test item related variations. Similarly, quantitative evaluation of corpora luteal count in
C1A females of control and high dose group did not reveal any test item related variations.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Splenic Lymphocyte Sub-populations statistically significant increase in mean T cytotoxic (CD8+) cells populations in group G4-C1A males; increase in mean T cytotoxic (CD8+) cells populations in group G3-C1A females; decrease in T "helper" (CD4+) cells populations in group G3-C1A females were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment as the obtained mean value is within in-house historical control range of same species and strain.

The test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day)
when administered to F1 generation animals from PND 21 to 95 (for C1A animals) and PND 21 to
102 (for C1B animals) under experimental conditions employed.

Therefore, the no-observed-adverse-effect-level (NOAEL) of the test item is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.

Key result

Dose descriptor:

NOAEL

Remarks:

1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points

Generation:

other: cohort 1A and cohort 1B

Effect level:

>= 1 000

Based on:

test mat.

Remarks:

the test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period o

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

food efficiency

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

other: Splenic Lymphocyte Subpopulation Analysis

Remarks on result:

other: the test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period o

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

In conclusion, the test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10 weeks during pre-mating period (both P males and females), 2 weeks during mating period (both P males and P females), throughout gestation and lactation periods up to weaning of F1 animals (P females) as well as when administered to F1 generation animals from PND 21 to 95 (for C1A animals) and PND 21 to 102 (for C1B animals) under experimental conditions employed.
Therefore, the no-observed-adverse-effect-level (NOAEL) of the test item is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.

Executive summary:

The objective of this Extended One-Generation Reproductive Toxicity (EOGRT) Study in Sprague Dawley Rats was to evaluate the effects of test item as a result of pre and postnatal exposure on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females, young and adult offspring.
The study was conducted to serve as a test for reproductive endpoints that required the interaction of males with females, females with conceptus, females with offspring and the F1 generation before and after sexual maturity. This study was also conducted to evaluate the spermatogenesis for males and oestrous cycles, follicle counts/oocyte maturation and ovarian integrity (histopathology) for females.

This EOGRT study was also conducted to evaluate gonadal function, the oestrus cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition and lactation. Furthermore, the present EOGRT study was conducted to provide detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, and was expected to identify specific target organs in the offspring. In addition, this study provided and/or confirmed information about the effects of Hostaperm Pink E on the integrity and performance of the adult male and female reproductive systems.

A total of 200 (100 males + 100 females) Sprague Dawley rats were selected for parental (P) generation and distributed to four P generation groups. Each P generation group (G1, G2, G3 and G4) consisted of 25 males and 25 females. A total of 160 males and 160 females were selected on the day of weaning (PND21) and randomly assigned to two cohorts [Cohort 1A (80 males + 80 females) and Cohort 1B (80 males
+ 80 females)]. Each cohort consisted of four groups and consisted of 20 males and
20 females per group. The animals in G1 (P generation) and G1-C1A/G1-C1B
(F1 generation) groups were administered with vehicle [0.5% w/v Carboxymethyl Cellulose], the animals in G2 (P generation) and G2-C1A/G2-C1B (F1 generation),
G3 (P generation) and G3-C1A/G3-C1B (F1 generation), and G4 (P generation) and G4-C1A/G4-C1B (F1 generation) groups were administered with Hostaperm Pink E at the dose levels of 111, 333 and 1000 mg/kg body weight/day respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight.

The stability and homogeneity of test item in dose formulations were established before initiation of the treatment. The test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxymethyl cellulose within the mean percent recovery range of 85 to 115 at the concentrations of 10.0 mg/mL and
100.0 mg/mL. Homogeneity and dose formulation analysis for dose concentration verification was performed during week 1, 8, 16 and 26 of the treatment periods and the mean results were within the range of 85 to 115% recovery to the nominal concentration and the relative standard deviation (% RSD) was less than 10%.

All the P generation animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights (throughout the experimental period) and feed consumption (throughout the experimental period, except during cohabitation period) was recorded once weekly for all the P animals. The assessment for haematology, clinical chemistry, urinalysis, and thyroid hormone (Thyroxine Hormone and Thyroid Stimulating Hormone) levels was conducted for randomly selected males and females from each P generation group at termination. Sperm parameters (motility, morphology, and sperm concentration/daily sperm production) were estimated for all P males. Oestrus cyclicity evaluation was determined during pre-mating and cohabitation period for all P females.
The body weights and feed consumption were recorded during gestation (Gestation Day 0, 7, 14 and 20) and during lactation (Lactation Day 1, 4, 7, 14 and 21) for all the
P females. The gross pathology and organ weighing was performed on the day of termination for all the P animals. Histopathological examination was conducted on all the tissues collected from the P generation vehicle control and high dose group animals. All the P males and females were evaluated for reproductive performance or indices such as, mating and fertility index (for P males and females) and pre-coital interval, gestation length, fecundity index, gestation index, post-implantation loss and postnatal loss (for females). All P females were observed for birth parameters (number of live/dead pups born, litter size, sex ratio, and live birth index per litter) and for litter observations (number of live/dead pups during lactation period, sex ratio and pup survival index per litter).

All the P males and females did not reveal any test item related changes in systemic and reproductive endpoints in any of the tested dose groups. However, in test-item administered group animals of both the sexes, the faecal matter was noted with ‘pink’ in colour which could be attributed to physical appearance of the test item (pink in colour). There were no test item-related histopathological findings noted in high dose parental animals of both sexes. Some of the noted microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats of this strain and age.

The F1 generation pups were observed once daily for external examinations and
twice daily for mortalities till termination, weighed individually on postnatal day (PND) 1, 4, 7, 14 and 21, observed for occurrences of postnatal developmental landmarks, observed for responses towards to sensory reflexes during postnatal period, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13, observed for gross pathological observations at termination. The assessment for serum Thyroxine (T4) levels was conducted for PND 4 pups and assessment for serum Thyroxine (T4) and Thyroid Stimulating Hormone levels was conducted for PND 21 pups.

There were no test item related changes in growth parameters, postnatal developmental landmarks, sensory reflexes, thyroid hormone levels in F1 generation pups. No gross pathological changes noted in of the any of the F1 pups of both sexes from all the tested dose group litters.

All the Cohort 1A (F1 generation) animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights and feed consumption were recorded once weekly. All the
C1A animals were evaluated for occurrence of sexual maturation (for balanopreputial separation in C1A males and for vaginal patency in C1A females). All C1A females were evaluated for mean occurrence of first cornified cells and time interval between vaginal patency and occurrence of first cornified cells. All C1A females were evaluated for oestrus cyclicity from PND 75 to until sacrifice. The assessment for haematology, clinical chemistry, urinalysis and thyroid hormone (Thyroxine Hormone and Thyroid Stimulating Hormone) levels was conducted for randomly selected males and females from each C1A group at termination. Sperm parameters (motility, morphology and daily sperm production) were estimated for all C1A males. The gross pathology and organ weighing were performed on the day of termination for all the C1A animals. Histopathological examination was conducted on all the tissues collected from the
C1A vehicle control and high dose group animals. The flow cytometric analysis of splenic samples for assessment of splenic lymphocyte sub-populations of T "helper" (CD4+) cells, T cytotoxic (CD8+) cells, natural killer (NK) cells and B lymphocytes was conducted for randomly selected males and females from each C1A group.

All the C1A males and females did not reveal any test item related changes in systemic and reproductive endpoints in any of the tested dose groups when compared with vehicle control group, except slight reduction in mean body weight and percent change in mean body weight gain in all the tested dose group males. There were no changes noted in sub-populations of splenic lymphocytes such as, T "helper" (CD4+) cells,
T cytotoxic (CD8+) cells, natural killer (NK) cells and B lymphocytes at any of the tested dose groups when compared with vehicle control group. There were no test item-related histopathological findings noted in high dose C1A animals of both sexes. Some of the noted microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

All the Cohort 1B (F1 generation) animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights and feed consumption were recorded once weekly. All the
C1B animals were evaluated for occurrence of sexual maturation (for balano-preputial separation in C1B males and for vaginal patency in C1B females). The gross pathology and organ weighing were performed on the day of termination for all the C1B animals.

All the C1B males and females did not reveal any test item related changes in systemic and reproductive endpoints, except reduction in mean body weight and percent change in mean body weight gain in all the tested dose group of both sexes.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

reliable without restriction

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available (further information necessary)

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 April 2021 to 08 November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

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

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

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

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

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

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Details on mating procedure:

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

Duration of treatment / exposure:

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

Frequency of treatment:

Once Daily

Duration of test:

15 days for each presumed pregnant female.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle Control

Dose / conc.:

111 mg/kg bw/day

Remarks:

Low Dose

Dose / conc.:

333 mg/kg bw/day

Remarks:

Mid Dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High Dose

No. of animals per sex per dose:

25 presumed pregnant females

Control animals:

yes, concurrent vehicle

Details on study design:

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

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

Maternal examinations:

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

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

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

Ovaries and uterine content:

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

Fetal examinations:

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

Statistics:

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

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

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

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

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

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

Indices:

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

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality:

no mortality observed

Description (incidence):

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

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

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

Food efficiency:

no effects observed

Description (incidence and severity):

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

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

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

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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

Other effects:

no effects observed

Description (incidence and severity):

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

Details on results:

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

Number of abortions:

no effects observed

Description (incidence and severity):

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

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

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

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

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

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

Early or late resorptions:

no effects observed

Description (incidence and severity):

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


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

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

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

Dead fetuses:

no effects observed

Description (incidence and severity):

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

Description (incidence and severity):

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

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

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

Details on maternal toxic effects:

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

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food efficiency

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

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

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

uterus

Description (incidence and severity):

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

Fetal body weight changes:

no effects observed

Description (incidence and severity):

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

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

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

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

Changes in sex ratio:

no effects observed

Description (incidence and severity):

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

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

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

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

External malformations:

no effects observed

Description (incidence and severity):

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

Skeletal malformations:

no effects observed

Description (incidence and severity):

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

Visceral malformations:

no effects observed

Description (incidence and severity):

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

Other effects:

no effects observed

Description (incidence and severity):

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

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

Details on embryotoxic / teratogenic effects:

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

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

other: anogenital distance and fetal crown rump length

Key result

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

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

Key result

Developmental effects observed:

no

TABLE 1. SUMMARY OF PREGNANCY STATUS RECORD

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

%: Percentage.

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

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

N: Normal.

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

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

SD: Standard Deviation; n: Number of Dams.             

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

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

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

SD: Standard Deviation; n: Number of Dams.             

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

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

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

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

Corrected Body Weight (g)

=

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

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

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

SD: Standard Deviation; n: Number of Dams.

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

TABLE 7. SUMMARY OF UTERINE OBSERVATIONS PER LITTER RECORD

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

SD: Standard Deviation; n: Number of Dams.

TABLE 8. SUMMARY OF MATERNAL RECORD PER LITTER

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

SD: Standard Deviation; n: Number of Dams.

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

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

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

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

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

SD: Standard Deviation; n: Number of Dams.

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

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

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

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

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

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

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

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

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

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

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

SD: Standard Deviation; n: Number of Dams.

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

TABLE 15. SUMMARY RECORD OF FETAL EXTERNAL EXAMINATION PER LITTER

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

No.: Number; %: Percentage.

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

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

No.: Number; %: Percentage.

TABLE 16. SUMMARY OF FETAL VISCERAL EXAMINATION PER LITTER RECORD

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

No.: Number; %: Percentage.

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

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

No.: Number; %: Percentage.

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

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

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

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

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

No.: Number; %: Percentage.

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

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

No.: Number; %: Percentage.

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

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

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

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

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

No.: Number; %: Percentage.

Conclusions:

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

Executive summary:

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

 

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

 

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

 

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

 

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

 

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

 

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

 

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

Reference 2

Endpoint:

developmental toxicity

Data waiving:

other justification

Justification for data waiving:

other:

Species:

other: second species not required

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

reliable without restriction

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification

There are no indicators of reproductive toxicity indentified in available studies.