[No public or meaningful name is available]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 December 2021 to 03 February 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

OECD Guideline for Testing of Chemicals: Prenatal Developmental Toxicity Study, No. 414, Adopted: 25th June 2018.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty alcohols C13-15 (odd numbered, linear and branched), reaction products with ethylene oxide, sodium chloroacetate and ethanolamine

Molecular formula:

R-O-(CH2CH2O)nCH2CO-NH-CH2CH2OH

IUPAC Name:

Fatty alcohols C13-15 (odd numbered, linear and branched), reaction products with ethylene oxide, sodium chloroacetate and ethanolamine

Test material form:

liquid: viscous

Details on test material:

- Physical state: yellow viscous liquid

Specific details on test material used for the study:

Name of the Test Item: AMIDET A 15

Description: White to off-white powder


Batch No.: 30905893

Date of Production: 01.02.2021

Date of Expiry: 24.01.2022

Date of Receipt: 08.05.2021

Sponsored by: Kao Chemical Europe, S.L.
Puig dels Tudons 10 –
08210 BARBERÀ DEL VALLÈS (Barcelona)
SPAIN

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Species: Rat (Rattus norvegicus) Strain : Wistar (Crl:WI)

Details on test animals or test system and environmental conditions:

No. of animals
(Range finding study) : 28 animals (14 males +14 females)


No. of animals (Main study): 150 animals (50 males and 100 females)

Source: Hylasco Bio-Technology (India) Pvt. Ltd.
No. 4B MN Park, Turkapally Village,
Shameerpet Mandal, Medchal District,
Hyderabad, Telangana 500 078.
Location of the Study

The study was conducted in the experiment unit-I, room no. 4 at the animal house facility, IIBAT, Padappai - 601 301, Tamil Nadu, India.

Animal House Condition
Temperature was maintained between 21.1 and 22.8 °C, and the relative humidity between 48 and 60%. Temperature and relative humidity were recorded once daily. The test room was provided with 12-hour artificial light and 12-hour dark condition.

Housing
Animals were housed in clean, sterilized solid floored standard polypropylene rat cages covered with a stainless steel grill with provisions for feed and water. Cages were placed on stainless steel racks. Cage rotation was done at weekly intervals. Clean and sterilized corn cob was used as bedding material. During the acclimatization period, animals were housed in groups of two/three per cage. After the acclimatization period, one female was housed with one male until the confirmation of mating (for a maximum period of 14 days) in cages equipped with stainless steel bottom grills with blotting paper underneath. After the confirmation of mating, the females were housed individually. Clean and sterile nesting materials (Nestlets) was provided to all dams from gestation day (GD) 14 until scheduled sacrifice on GD 20. Male animals housed individually during the mating period, and females housed individually during the gestation period were provided with the enrichment devices (tunnels).

Sanitation
The floor of the experimental rooms was swept and mopped daily. Cages, enrichment devices, nesting materials and bedding material were changed twice weekly and water bottles were changed daily. All the experimental procedures were done in a clean environment.

 
Animal Welfare and Approval

IIBAT is committed to enhance animal welfare and registered with CPCSEA, Ministry of Environment and Forests, Government of India for Breeding and experiments of Animals (No.31/PO/RcBi - S/RcBi - L/1999/CPCSEA dated 11 May 2017). Use of live animals is inevitable to accomplish the purpose of this study. The recommendations regarding animal care and handling were followed and consistent with:

Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines for Laboratory Animal Facility, The Gazette of India, 2018.

The use of animals and the general procedures involved in the present study have been reviewed and approved by the Institutional Animal Ethics Committee of IIBAT (Approval Number: 25/155/IAEC/IIBAT/2021; dated 29.05.2021).

Feed and Water
Standard and conventional laboratory rodent pellet feed (manufactured by Special Diet Services - England, supplied by Vivo Biotech Ltd., Hyderabad, India), and potable well water (IIBAT) processed through reverse osmosis was provided ad libitum to all animals. Phytooestrogen levels in the feed and bedding material was tested and was ensured to be within the acceptable limits (6.94 mg/kg of Genistein and 0.50 mg/kg in the rodent laboratory feed and bedding material respectively). Analysis reports on phytoestrogen levels in feed and bedding material, microbial load and chemical contaminants in feed and water, and nutrient content of feed are retained with the facility records and an authorized photocopy was maintained with the raw data.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol (PEG) 300 was selected as vehicle for test item preparation. It was selected based on the available information on the test item (ECHA, 2010)). A solubility check was performed using the test item prior to initiation of dosing period.

Details on exposure:

Preparation of Test Item Formulation and Administration of Doses

Dose formulations were prepared by mixing the test item with Polyethylene glycol (PEG) 300 at the desired concentrations. The required test item formulations were prepared approximately 30 minutes prior to the start of dosing of animals. The test item was administered at approximately the same time (±1 h) each day as a single dose orally by gavage using a stainless steel oral intubation needle fitted to a graduated syringe. The dose volume was maintained at 0.5 ml/100 g body weight (b.w.). Animals in all the groups were administered the same volume of either the test item formulation or vehicle. Vehicle control group received the vehicle in the highest amount used as in the low dose group.

Dose Formulation Analysis

The dose formulation was subjected to homogeneity, stability and active ingredient analysis once prior to the initiation of dosing and once during the last week of the dosing period. The sampling was performed from the top, middle, and bottom of the container with dose formulations in duplicate preparations for three dose levels. The analysis was performed by validated analytical method (IIBAT study no. 21140) at the Analytical Chemistry Department of IIBAT.



Study Design

Range finding study
Attached as appendix - 1

Main Study

The main study was conducted using three treatment groups and a vehicle control group. Mating procedure was followed as described in section Mating of animals, 50 males and 100 females targeted to yield a minimum of 20 pregnant rats to maximum 25 pregnant rats per group at the end of mating period. Pregnancy-confirmed females were randomly assigned to the groups to obtain the best possible distribution of day 0 gestation weights between groups and were dosed from GD 0 until GD 19. The dose levels for the main study were determined based upon the results of the range finding study. The animals were dosed from GD 0 till GD 19 based on the most recent individual body weights. Dams were sacrificed on GD 20 and were subjected for gross pathological evaluations. Males used for mating were not dosed in the study and were sacrificed at the end of the mating period on day 15.

The high dose was selected with the aim to induce some developmental and/or maternal toxicity but not death or severe suffering, an intermediate dose with minimal observable toxic effects, and a low dose with no evidence of either maternal or developmental toxicity. The dose level used in Group 4 was the maximum dose level recommended in the OECD 414 test guideline. Animals in the control group was handled in an identical manner to the test group animals, except that they were administered with vehicle alone.


Groups Dose levels Number of pregnant females per group*
G1 (Vehicle Control) 0 mg/kg b.w. 24
G2 (Low dose) 100 mg/kg b.w. 24
G3 (Intermediate dose) 300 mg/kg b.w. 23
G4 (High dose) 1000 mg/kg b.w. 23
*Based on the pregnancy status of the animals ascertained during terminal sacrifice on GD 20 (Refer appendix – 1 & 2).

OBSERVATIONS

Animals were observed at least once daily throughout the study period. All observations related to this study were recorded manually in the respective formats for raw data collection.

Morbidity/Mortality

Animals were observed for morbidity/mortality once daily from the day of acclimatization until the experiment completion.

Clinical Observations

General cage-side clinical observations were performed once daily in the animals. Pertinent behavioral changes, and all signs of toxicity, including mortality, morbidity were recorded in terms of time of onset, degree and duration of toxicity signs if any were also recorded. During pregnancy, maternal animals were monitored for signs of abortion or premature delivery, if any.

Body Weight

During gestation, in the main study females were weighed on GD 0, 5, 8, 11, 14, 17, and on GD 20 prior to terminal sacrifice.

Feed Consumption

The feed consumption of the animals during the main study was calculated for gestation days 0 to 5, 6 to 8, 9 to 11, 12 to 14, 15 to 17, and 18 to 20. The feed consumption was calculated from the feed input and leftover feed data and reported as g/rat/day. The feed consumption was also recorded during the mating period.

The cage-wise feed consumption was estimated by following formula:

Food consumption (g/rat/day)=(Food consumption during interval per cage (g))/(No.of days in the interval)

Blood sample collection and analysis of thyroid hormones

Blood samples was collected individually from all dams on gestation day 20 and all non-pregnant females individually at scheduled termination day (GD 20). The blood samples were drawn from the animals under isoflurane anesthesia, from the retro - orbital plexus without anticoagulant. The serum was separated from the blood and stored at -20 °C until analysis. Blood samples were collected at approximately the same time (± 30 min) at each day.

Blood samples were analyzed for thyroid hormones triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) using an ELISA method.

Pathology

Gross pathology examination

In the study, all surviving dams were humanely euthanized on GD 20 by carbon dioxide asphyxiation and were subjected to detailed examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. Evaluation of the dams during caesarean section and subsequent fetal analyses and sample collection were done randomly across available groups to avoid sampling bias.

Examination of Uterine Contents

During termination or as soon as possible after death, the uteri of the animal were removed and the pregnancy status of the animals were ascertained. Uteri that appear non-gravid were examined further by ammonium sulphide staining to confirm the non-pregnant status. Gravid uteri including the cervix were weighed. Gravid uterine weights were not recorded from animals found dead during the study. The number of corpora lutea were determined in pregnant animals. The uterine contents were examined for number of embryonic or fetal deaths and viable fetuses. The degree of resorption was described (early, late) to estimate the relative time of death of the conceptus. The number of implantation sites were counted in females .

Based on the results, the following was calculated (Hong, 2014):


Preimplantation loss (%)= ((number of corpora lutea - number of implantations))/((number of corpora lutea) )×100

Postimplantation loss (%)= ((number of implantations - number of live fetuses))/((number of implantations) )×100


Fetal death =resorptions + dead fetuses

Examination of Fetuses

During termination live fetuses was euthanized by placing on a pre-chilled ice-cold plate covered with paper towel and marked with an appropriate identification number. Fetuses were examined externally for gross abnormalities. Particular attention was paid to the external genitals which were examined for signs of altered development of the fetuses. Each live fetus was measured for its crown-rump length, weighed, sexed and marked individually soon after termination of dam. Each fetus was examined at termination to establish the number and sex of fetus, stillbirths, live births, runts (fetuses that are significantly smaller than corresponding control fetus) and the presence of gross anomalies and/or other abnormalities.

The anogenital distance (AGD) of each live fetus was measured using a calibrated digital Vernier caliper with 0.01 mm accuracy. For male fetus AGD was measured from the anterior edge of the anus to the base of the anogenital aperture, and for female fetus, it was measured from the anterior edge of the anus to the base of the urinary aperture. AGD were normalized to a measure of fetus size (cube root of body weight) by calculating the AGD Index.

AGD Index = AGD of live fetus/Cube root of body weight
Fetuses were examined for skeletal and soft tissue alterations including variations and malformations or anomalies.

External fetal sex determined by gross examination was compared with internal (gonadal) sex in all fetuses examined for both skeletal and soft tissue malformations.

Approximately one-half of each litter was prepared for skeletal alterations. The remaining half of the litter was prepared and examined for soft tissue alterations. The alternate fetuses were selected for skeletal examination starting with the first fetus (closest to the right ovary) in even-numbered dams and the second fetus in the odd-numbered dams (Narotsky, 2003).

Visceral (Internal)

Fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. Fetal kidneys were examined and graded for renal papillae development. This examination includes the heart and major vessels. The sex of all fetuses was confirmed by internal examination.

The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for subsequent processing and soft-tissue examination using the Wilson sectioning technique. The heads from the remaining one-half of the fetuses in each litter were examined by a mid-coronal slice.

Skeletal Examinations

All carcasses, including the carcasses without heads, was eviscerated and fixed in alcohol and processed for staining of the skeletal and cartilaginous tissues using Alizarin Red S and Alcian Blue. These specimens were cleared in potassium hydroxide solution and, following staining, they were stored in glycerin for subsequent examination of the skeletons.

Organ weighing and Histopathological examination

Organ weighing was performed post fixation for thyroid gland in dams. Thyroid glands of all dams were preserved in 10% neutral buffered formalin for histopathological examination.

Detailed histological examination of the thyroid gland was performed on the dams of all treatment groups and the control group.

Disposal of waste generated during the study

Waste generated during the study was disposed as per SOP/GEN/003.

STATISTICAL ANALYSIS

Data from non-gravid females was excluded from calculation of means and from comparative statistics.
Continuous data variables such as maternal body weights, gravid uterine weights, weight changes, feed consumption data, thyroid hormone assessment data, fetal body weight (separately by sex, and combined), organ weights (absolute and relative to body weight), anogenital distance, and crown-to-rump length was initially analyzed for normality using Shapiro-Wilk test, and homogeneity of variance between groups was checked by Levene’s test. Where significant homogeneity was detected, a one-way analysis of variance (ANOVA) was carried out followed by parametric multiple comparison procedures using Dunnett's test for post hoc comparison. For non-normally distributed data, non-parametric ANOVA, Kruskal-Wallis test was used. If the results of the test were significant (p<0.05), the Mann-Whitney U test or Dunn’s test (pairwise comparison) was performed to identify the group which shows a significant difference from control.
The group mean numbers of corpora lutea, implantation sites, viable fetuses, the mean litter proportions of prenatal data (% per litter of viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss and the fetal sex distribution) was analysed using Kruskal-Wallis, non-parametric ANOVA test to determine the intergroup difference. If the results of the non-parametric ANOVA were significant (p<0.05), the Mann-Whitney-U test was performed, where appropriate, to identify the group which shows a significant difference from control.

The mean litter proportion (% per litter) of total fetal malformations and developmental variations (external, visceral, skeletal and combined) and of each particular external, visceral and skeletal malformation or variation was tabulated. The mean litter proportions of fetal malformations and developmental variations was subjected to the Kruskal-Wallis non-parametric ANOVA test followed by Mann-Whitney U test (if appropriate) as described above.

A p-value of <0.05 was considered statistically significant. The analysis was performed using IBM SPSS Statistics, version 28.

Analytical verification of doses or concentrations:

yes

Remarks:

The dose formulation was subjected to homogeneity, stability and active ingredient analysis once prior to the initiation of dosing and once during the last week of the dosing period.

Details on analytical verification of doses or concentrations:

Dose Formulation Analysis

The dose formulation was subjected to homogeneity, stability and active ingredient analysis once prior to the initiation of dosing and once during the last week of the dosing period. The sampling was performed from the top, middle, and bottom of the container with dose formulations in duplicate preparations for three dose levels. The analysis was performed by validated analytical method (IIBAT study no. 21140) at the Analytical Chemistry Department of IIBAT.

Details on mating procedure:

Mating of animals

Mating of animals was done in two batches. After 5 days of acclimatization, for the first batch of mating, 50 male and 50 female animals were randomly cohabited in 1:1 ratio (male:female). After confirming mating, male animals were separated and returned to their respective cage and the second batch of mating with 50 female animals was continued as mentioned above. The males were humanely euthanized by carbon dioxide asphyxiation after the last day of mating period.

Once daily until evidence of copulation, the female animals were examined for presence of vaginal plug, or sperm by vaginal smear examination. Day 0 (GD 0) of pregnancy is defined as the day on which mating evidence (vaginal plug, or sperm in the vaginal smear) was found. The mating of animals was continued for 7 days, if vaginal plug or sperm was not observed. If the mating was not successful even after 7 days, the females were remated with proven males / fresh males. The remated pairs were observed for another 7 days and were confirmed for gestation by following the above procedure. The females mated by same male were distributed among the groups. Females showing no-evidence of copulation were humanely euthanized by carbon dioxide asphyxiation after the last day of mating period (after a maximum of 14 days of mating period). Female Mating Index (measure of female’s ability to mate) and Female Fertility Index (measure of female’s ability to become pregnant) was calculated as follows:

Mating index of untreated females (%)= ((No.of untreated females mated))⁄((no.of untreated females placed with untreated males)) × 100
Female fertility index (%) = (No. of females pregnant/no. of females with confirmed mating) × 100.

Randomization
Females with confirmed mating (day 0 of gestation, GD 0) were randomly assigned to the groups using a stratified body weight distribution method (SOP/TOX/001) to equalize as best possible the gestation day 0 weights between groups, and were identified using ear-tags bearing a unique animal number. The body weight range among the animals used at each dose level did not exceed ± 20 % of the mean weight before start of the treatment.

Duration of treatment / exposure:

During the in-life phase of the study, from GD 0 till GD 19, all the dams were observed for morbidity/mortality, clinical signs of toxicity, body weight, feed consumption and for signs of premature delivery. Dams were sacrificed on GD 20 and were subjected for gross pathological evaluations, and examination of uterine contents, including the external, visceral and skeletal examinations of fetuses. Thyroid glands collected from the dams were weighed and subjected to histopathological examination. Blood samples collected from the dams during the terminal sacrifice (GD 20) were analyzed for thyroid hormones (T3, T4, TSH).

Frequency of treatment:

The test item, Amidet A 15, was formulated as a suspension using polyethylene glycol 300 as the vehicle. Dose formulations were administered at dose levels of 100 (G2), 300 (G3), and 1000 (G4) mg/kg/day and the dose volume was maintained at 5 mL/kg body weight. The vehicle polyethylene glycol 300 was administered to the vehicle control (G1) group at a volume of 5 mL/kg body weight.

Duration of test:

This study was performed to investigate the effects of prenatal exposure on pregnant test animals and on the developing organism by the repeated oral exposure of the test item AMIDET A 15 from implantation to one day prior to expected parturition.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control (G1)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose (G2)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate Dose (G3)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose (G4)

No. of animals per sex per dose:

Ninety-four mating-confirmed female Wistar rats were assigned to four groups viz., Vehicle control (G1), Low dose (G2), Intermediate Dose (G3) and High dose (G4). Groups G1 and G2 consisted of 24 female rats, Groups andG3 and G4 consisted of 23 female rats

Control animals:

yes, concurrent vehicle

Details on study design:

Justification for Route of Exposure

The oral route was chosen as it is recommended route of exposure in the OECD test guideline 414, and it is one of the possible routes of exposure to humans.
Healthy young adult animals were selected for the study after a detailed clinical examination by a veterinarian. Only animals free of obvious health abnormalities were used for the study. Female animals were nulliparous and non-pregnant at the time of receipt for the study. The body weight range among the animals used for the experiment did not exceed ± 20 % of the mean weight of each sex at the start of the acclimatization.

Identification of Animals
In each cage, animals were identified with the numbers marked on their tail (during mating period) or by ear-tag bearing unique animal number (after confirmation of mating). The cages were identified by attaching a cage card containing information such as study number, cage number, animal number(s), species, sex, dose level, test item name, date of confirmation of mating along with the signature of study director or study personnel.

Acclimatization
All animals were acclimatized for a period of five days. Animals were weighed on the day of receipt and observed daily. Detailed records of acclimatization were maintained in the raw data.

Justification for the Choice of the Test System
The rat was selected because it has been historically proven to be a suitable test system for developmental toxicity studies, and rat is one of the preferred test systems as per the OECD test guideline 414.

Examinations

Maternal examinations:

During the in-life phase of the study, from GD 0 till GD 19, all the dams were observed for morbidity/mortality, clinical signs of toxicity, body weight, feed consumption and for signs of premature delivery

Ovaries and uterine content:

Dams were sacrificed on GD 20 and were subjected for gross pathological evaluations, and examination of uterine contents, including the external, visceral and skeletal examinations of fetuses. Thyroid glands collected from the dams were weighed and subjected to histopathological examination.

Blood sampling:

Blood samples collected from the dams during the terminal sacrifice (GD 20) were analyzed for thyroid hormones (T3, T4, TSH).

Fetal examinations:

visceral and skeletal examinations of fetuses, fetus weight, ano-genital distance (AGD), AGD index and crown crump length.

Statistics:

STATISTICAL ANALYSIS

Data from non-gravid females was excluded from calculation of means and from comparative statistics.
Continuous data variables such as maternal body weights, gravid uterine weights, weight changes, feed consumption data, thyroid hormone assessment data, fetal body weight (separately by sex, and combined), organ weights (absolute and relative to body weight), anogenital distance, and crown-to-rump length was initially analyzed for normality using Shapiro-Wilk test, and homogeneity of variance between groups was checked by Levene’s test. Where significant homogeneity was detected, a one-way analysis of variance (ANOVA) was carried out followed by parametric multiple comparison procedures using Dunnett's test for post hoc comparison. For non-normally distributed data, non-parametric ANOVA, Kruskal-Wallis test was used. If the results of the test were significant (p<0.05), the Mann-Whitney U test or Dunn’s test (pairwise comparison) was performed to identify the group which shows a significant difference from control.
The group mean numbers of corpora lutea, implantation sites, viable fetuses, the mean litter proportions of prenatal data (% per litter of viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss and the fetal sex distribution) was analysed using Kruskal-Wallis, non-parametric ANOVA test to determine the intergroup difference. If the results of the non-parametric ANOVA were significant (p<0.05), the Mann-Whitney-U test was performed, where appropriate, to identify the group which shows a significant difference from control.

The mean litter proportion (% per litter) of total fetal malformations and developmental variations (external, visceral, skeletal and combined) and of each particular external, visceral and skeletal malformation or variation was tabulated. The mean litter proportions of fetal malformations and developmental variations was subjected to the Kruskal-Wallis non-para

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related clinical findings like salivation and pressing and pushing the head through the bedding material were noted in 4 females in 300 mg/kg b.w. and all females in the 1000 mg/kg b.w. groups at the daily examinations post dosing. All these animals recovered from the clinical signs of toxicity after approximately 10-15 mins following dosing. All the animals of G1 and G2 dose groups were observed to be normal throughout the experimental period. No bleeding or abortion was observed on GD5 following dosing.

No premature deliveries or signs of abortion were observed in any of the dams of the treatment or control groups. All the remaining animals in the study were found to be normal throughout the experiment period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two female animals exhibited mortality prior to scheduled termination, one in G1 and one in G2. No Mortality/morbidity was observed in any of the remaining animals pertaining to the treatment or control group throughout the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of the dams measured at various time points during the gestation period (GD 0, 5, 8, 11, 14, 17 and 20) were comparable between the control and treatment groups. Body weight change during gestation (weight of the dams on GD20 minus the weight of the dams during GD0) and the gestational body weight change corrected for gravid uterine weight (body weight change during gestation minus the gravid uterine weight) were also comparable between the control and treatment groups (Table - 1).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Feed consumption measured at various intervals during the gestation period (GDs 0-5, 6-8, 9-11, 12-14, 15-17 and 18-20) was comparable between the control and treatment groups and no statistical significance was observed (Table-2).

Endocrine findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weight (Thyroid)

The absolute and relative thyroid organ weights were comparable between the control and treatment groups (Table – 6).

Gross pathological findings:

no effects observed

Description (incidence and severity):

All the surviving dams were euthanized on GD 20 for gross pathological observation. None of the dams showed any signs of abortion or premature delivery prior to the scheduled termination on GD 20, and there was no premature delivery in the dams. Detailed examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents had revealed no abnormalities. No gross lesions were observed in the uterus and endometrial area between each implant site. No premature delivery was observed in the dams.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item related histopathological findings were observed in the thyroid organs of the treatment groups of animals. The histopathological findings observed in the study, such as colloidal alteration and degeneration with mononuclear infiltration in the thyroid follicles, were considered to be spontaneous or incidental and not related to the test item treatment (Brändli-Baiocco et al., 2018) (Table – 7).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Examination of Uterine Contents

At the termination (GD20), the uteri of the animals were removed, and the pregnancy status of the animals was ascertained. Uteri that appear non-gravid were stained with 10% (v/v) ammonium sulphide solution to confirm the non pregnant status of the animals. Out of 94 animals which showed the evidence of copulation, 85 were found to be pregnant, and 9 were non-pregnant. No stained implantation sites were observed in the uteri of non-pregnant animals. No variations were observed in the colour or volume of the amniotic fluid in the uterus of any dams.

Gravid uterine weight

The mean gravid uterine weight of treatment and control groups were similar. No test item-related findings were noted in corpus luteum count, implantation sites, total resorptions and total live fetus (Table – 4 and 8).

Thyroid hormones

Blood samples were analyzed for thyroid hormones (T3, T4, TSH) on GD 20 and the results were comparable between control and treatment groups. No test item-related findings were noted in absolute and relative thyroid organ weights.
No test item-related findings were noted in T3 and T4 hormones. A slight increase in TSH in G3 group was noted. This change was considered as non-dose dependent and incidental. During the experimental phase clinical signs such as lethargy, salivation and pressing the head inside the bedding material (due to irritation caused by the test item) was observed in the high dose group. (Table – 3 and 7).

Details on results:

Mating of animals

Out of 100 females were cohabitated with males in 1:1 ratio (male: female), evidence of copulation (confirmation of mating) was observed in 94 females. Female mating Index (measure of female’s ability to mate) was calculated as 94.00%. Mating was confirmed by the presence of vaginal plug in 94 females. Out of 94 females with confirmed evidence of mating, 94 showed the evidence of copulation within 7 days. 6 females which did not show evidence of copulation were humanely sacrificed by carbon dioxide asphyxiation after a maximum of 14 days of mating period.

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean number of corpora lutea, implantations sites, and mean percent pre-and post-implantation losses were similar between control and treatment groups (Table – 4).

Early or late resorptions:

no effects observed

Description (incidence and severity):

In all dams the aminotic fluid was normal in colour, no gross lesions were observed in the endometrial areas between each implants. No late resorptions or dead fetuses were observed in any of the dams of treatment and control groups. The mean number of early resorptions, mean number of live fetuses and the percent viable fetus per litter were similar between treatment and control groups (Table – 4).

Dead fetuses:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

Hence it is concluded that the no observed adverse effect level (NOAEL) for maternal and developmental effects for the test item AMIDET A 15 is greater than 1000 mg/kg b.w. to the Wistar rats.

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical signs

Remarks on result:

other: Hence it is concluded that the no observed adverse effect level (NOAEL) for maternal and developmental effects for the test item AMIDET A 15 is greater than 1000 mg/kg b.w. to the Wistar rats.

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean values of fetal weight (either separated by sex, or combined), anogenital distance, anogenital index, and crown-to-rump length were similar between treatment and control groups. The fetal sex was found to be evenly distributed among the control and treatment groups (Table-5 and 6).

Reduction in number of live offspring:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The mean values of fetal weight (either separated by sex, or combined), anogenital distance, anogenital index, and crown-to-rump length were similar between treatment and control groups. The fetal sex was found to be evenly distributed among the control and treatment groups (Table-5 and 6).

External malformations:

no effects observed

Description (incidence and severity):

No remarkable soft tissue alterations were observed in the any of the fetus of the treated dams or control dams (Table – 5).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In terms of skeletal examination, there was no incidence of major malformations observed in the fetuses of the treated group dams. Variants were observed in a minimal number of fetuses of the control and the treated dose group. Variations such as rudimentary ribs, bent ribs, short ribs and variation in the caudal centrum (0-5) were observed in the control and the treated groups. No anomalies were observed in the centrum of the vertebrae and the number of cervical, thoracic, lumbar and sacral vertebrae in the control and treated group fetuses. These normal variants were spontaneous and considered to be not due to the test item treatment, and have no toxicological significance. The incidence of malformations (major/minor) and normal variants in the fetuses were statistically similar between treatment and control groups. Caudal centrum ranging from 0 to 5 in the fetus of the control and the treatment groups is within normal limits (Table – 5).

Visceral malformations:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

External examination of the fetuses showed that Test item administered at the dose rate of (100 mg, 300 mg, 1000 mg/ kg body weight) did not cause any major abnormalities in any fetus of the treated dams. The external examinations pertaining to the length, cranium, eyes, limbs, tail, genitals and sex did not reveal any major abnormalities. The only finding observed was absence of tail in one fetus (2632-L4) in G3 group and one fetus (2646- L11) in G4 group . There was no external malformation observed in the any of the fetus of the high dose group. These findings are considered to be of spontaneous incidence and not related to treatment. All these findings were incidental and is of no toxicological significance (Table – 5).

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

it is concluded that the no observed adverse effect level (NOAEL) for maternal and developmental effects for the test item AMIDET A 15 is greater than 1000 mg/kg b.w. to the Wistar rats.

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: External examination of the fetuses

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

External examination of the fetuses showed that Test item administered at the dose rate of (100 mg, 300 mg, 1000 mg/ kg body weight) did not cause any major abnormalities in any fetus of the treated dams. The external examinations pertaining to the length, cranium, eyes, limbs, tail, genitals and sex did not reveal any major abnormalities. The only finding observed was absence of tail in one fetus (2632-L4) in G3 group and one fetus (2646- L11) in G4 group . There was no external malformation observed in the any of the fetus of the high dose group. These findings are spontaneous incidence and not related to treatment. All these findings were incidental and is of no toxicological significance

The mean values of fetal weight (either separated by sex, or combined), anogenital distance, anogenital index, and crown-to-rump length were similar between treatment and control groups. The fetal sex was found to be evenly distributed among the control and treatment groups

No remarkable soft tissue alterations were observed in the any of the fetus of the treated dams or control dams

In terms of skeletal examination, there was no incidence of major malformations observed in the fetuses of the treated group dams

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

Based on the results obtained in the current study, it was evident that the exposure of the test item to pregnant Wistar rats, from impregnation (gestation day 0) until one day prior to their expected day of parturition (gestational day 20), at the dose levels of 100, 300, and 1000 mg/kg b.w. did not cause any maternal or developmental toxicity. Hence it was concluded that the no observed adverse effect level (NOAEL), for both maternal toxicity and developmental effects, for the test item AMIDET A 15 was greater than 1000 mg/kg b.w. in the Wistar rat.

Executive summary:

This study was performed to investigate the effects of prenatal exposure on pregnant test animals and on the developing organism by the repeated oral exposure of the test item AMIDET A 15 from impregnation to one day prior to expected parturition.

Ninety-four mating-confirmed female Wistar rats were assigned to four groups viz., Vehicle control (G1), Low dose (G2), Intermediate Dose (G3) and High dose (G4). Groups G1 and G2 consisted of 24 female rats,  Groups G3 and G4 consisted of 23 female rats on GD0. 22 females were found to be pregnant in G1 and G2 group out of 24, 23 in G3 group and 22 were pregnant in G4 group on GD20. The test item, Amidet A 15, was formulated as a suspension using polyethylene glycol 300 as the vehicle. Dose formulations were administered at dose levels of 100 (G2), 300 (G3), and 1000 (G4) mg/kg/day and the dose volume was maintained at 5 mL/kg body weight. The vehicle polyethylene glycol 300 was administered to the vehicle control (G1) group at a volume of 5 mL/kg body weight.

The identity of the test item was based on the Certificate of Analysis (CoA) provided by the Sponsor. The test item formulations were found to be stable for 7 days when stored at room temperature and homogeneous in polyethylene glycol 300. The results of dose formulation analyses carried out before initiation of treatment and towards the end of treatment period showed that the mean concentration of the test item was within acceptable limits (90-110% of recovery for the nominal concentration).

During the in-life phase of the study, from GD 0 till GD 19, all the dams were observed for morbidity/mortality, clinical signs of toxicity, body weight, feed consumption and for signs of premature delivery. Dams were sacrificed on GD 20 and were subjected for gross pathological evaluations, and examination of uterine contents, including the external, visceral and skeletal examinations of fetuses. Thyroid glands collected from the dams were weighed and subjected to histopathological examination. Blood samples collected from the dams during the terminal sacrifice (GD 20) were analyzed for thyroid hormones (T3, T4, TSH).

Test item-related clinical findings like salivation and pressing and pushing the head through the bedding material were noted in 4 females in 300 mg/kg b.w. and all females in the 1000 mg/kg b.w. groups at the daily examinations post dosing. All these animals recovered from the clinical signs of toxicity after approximately 10-15 mins following dosing. All the animals of G1 and G2 dose groups were observed to be normal throughout the experimental period. No bleeding or abortion was observed on GD5 following dosing.

No test item-related findings were noted in fetus weight, ano-genital distance (AGD), AGD index and crown crump length. A statistically significant increase in crown crump length in G2 group female fetuses was observed. Astatistically significant increase in fetus weight and decrease in AGD index were observed in G3 group male fetuses. A statistically significant increase in fetal weight, and decrease in crown crump length and increase in AGD index was observed in G3 group female fetuses. These changes were all considered as non-dose dependant and sporadic. Intrauterine growth and survival in the 100, 300, and 1000 mg/kg b.w. groups were unaffected by test item exposure. One fetus in the 300 mg/kg b.w group and one fetus in the 1000 mg/kg b.w had absence of tail, which were considered to be unrelated to exposure.

Body weights of the dams during the gestation period and the gestational body weight change corrected for gravid uterine weight was comparable between the control and treatment groups. No test item related changes were observed in feed consumption and in the thyroid hormone values of treatment group animals when compared with the control group animals. No gross pathological changes were observed in any of the dams of control or treatment groups. 

Uterine content examination of the dams revealed no abnormalities. The gravid uterine weight, number of corpora lutea and implantation sites, and pre-and post-implantation loses were similar between control and treatment groups. The number of early resorptions and live fetuses and the percent viable fetus per litter were comparable between the treatment and the control groups.

No soft tissue alterations were evident in the visceral examination of the fetuses. No test item related major or minor skeletal malformations were observed in the skeletal examination of fetuses. The normal skeletal variations that were observed in the fetuses of all groups were of no toxicological significance.

No test item related changes were observed in the thyroid weights and in the histopathological examination of thyroids and no changes in thyroid hormone levels were observed.