1,4-dimethylpiperazine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20222-06-16 to 2022-10-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2022-03-23 to 2022-05-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this preliminary study was to select the doses for definitive prenatal developmental toxicity study (OECD 414) in Wistar rats. This study evaluated the adverse effects on the pregnant female rats and development of the embryo and fetus consequent to exposure of the test substance administered to pregnant rats by oral route during gestation days (GD) 6 to 19.

GLP compliance:

no

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 1,4-dimethylpiperazine
- Lot/batch number of test material: PFW210769
- Expiry data of lot/batch: 2023-11-25 (retest date)
- Physical State/Appearance: liquid
- Purity: 99.91 A%. This substance is total titratable amine content. Amine content is 17.39 meq/g
- Purity test date: not indicated
- pH: 11.4
- density: 0.84 g/cm³
- specific gravity: 0.822 at 20°C

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (+15 to +22 °C), protect from light/humidity, at temperature no higher than +22 °C under inert gas blanket
- Stability under storage conditions: not indicated
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the vehicle: The stability of the test item in the vehicle was established at 1 and 100 mg/mL under Study No. G24817 in Milli-Q water. Based on the results, the test item was stable in the vehicle up to 48 hours when stored at room temperature.

Species:

rat

Strain:

Wistar

Remarks:

Crl:Wistar IGS

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology Pvt. Ltd., 4B, M.N. park, Shameerpet Mandal, Turkapally Village, Mechdal District, Telangana 500078, India
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at start of treatment: 13-14 weeks
- Weight at start of treatment: 224.60 to 294.95 g
- Fasting period before study: Not indicated
- Housing: Rats were housed in standard polysulfone rat cages (size: Length 425 mm x Breadth 266 mm x Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube. Additionally, polycarbonate rat huts were placed inside the cage as environmental enrichment object which were replaced at least once a week. Steam sterilized corn cob was used as bedding and was changed along with the cage at least twice a week. Following was the housing pattern during different periods of the experiment: i. Premating / Acclimatization: Two rats of the same sex per cage were housed. ii. Mating: Female rats were cohabited with males in a 1:1 ratio. iii. Post-mating / Treatment: After mating confirmation, females were housed individually. Nesting material (paper cuttings) were placed inside the cages from Gestation Day 18 onwards.
Steam sterilized corn cob was used as bedding and was changed along with the cage at least twice a week.
- Diet (e.g. ad libitum): ad libitum, Altromin Rat/Mice Maintenance diets
- Water (e.g. ad libitum): ad libitum, deep bore-well water
- Acclimation period: 5 days (2022-03-23 to 2022-03-27). After detailed clinical examination for good health and suitability for the study, 35 females and 35 male rats were acclimatized for five days before initiation of mating.

DETAILS OF FOOD AND WATER QUALITY:
Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, and deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India was provided in polycarbonate bottles with stainless steel sipper tubes.
The feed and water provided to the rats were tested for contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23 °C
- Humidity (%): 64 - 66 %
- Air changes (per hr): 12-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2022-03-23 To: 2022-04-20

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Mili-Q water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Pre-calibration of the beaker to desired volume: Milli-Q® water was measured in a graduated measuring cylinder to the final volume of the batch size (Example for initial dose formulation preparation, the batch size was 40 mL, Milli-Q® water was taken till 40 mL mark in the graduated measuring cylinder). The measured Milli-Q® water was transferred into the clean beaker (to be pre-calibrated) and upper and lower meniscus of Milli-Q® water was marked on the beaker using a glass marker. Once these lines were marked, the Milli-Q® water was discarded and the beaker dried. The upper meniscus mark was used to make up to the volume while dose formulation preparation.
- Required quantities of the test item were weighed in a beaker (previously calibrated to a desired volume) for each dose levels separately and a small volume of vehicle (Milli-Q® water) was added and stirred using a glass rod till a uniform solution was obtained. The volume was made up to the upper meniscus mark using the vehicle to get the final desired concentrations.
- Dose formulations were prepared in an interval of 1 to 3 days and used within the established stability period of 48 hours.
- Similar procedure was followed for dose formulation preparation throughout the experiment, however, the volume of dose formulation prepared varied depending on the requirement and/or body weights of the rats recorded.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The Formulation Analytical Method Validation, Homogeneity and Stability, established in Milli-Q water under Study No. G24817. Further, OECD 407 (G19072) and OECD 421 (G19073) studies were conducted using Milli-Q water as vehicle for dose formulation preparation. Hence, Milli-Q water was used as vehicle for dose formulation preparation.
- Amount of vehicle (if gavage): 10 mL/kg
- Dose levels: 0, 400, 800 and 1000 mg/kg bw/day
- Concentration in vehicle: 0, 40, 80, 100 mg/mL

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- The females were cohabited in batches of required numbers. This procedure was continued until there were sufficient numbers of Day 0 pregnant rats for the study.

Duration of treatment / exposure:

14 consecutive days; from Day 6 to Day 19 of gestation

Frequency of treatment:

Daily

Duration of test:

18 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

G2

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

G3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4

No. of animals per sex per dose:

7 female rats per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels for the dose range finding (DRF) study were selected in consultation with the Sponsor based on previous 14 day study conducted (Study No. N6321).
- Justification for selection of species: the rat was selected because it is a standard acceptable rodent species for use in prenatal developmental toxicity studies, this species and strain has been demonstrated to be sensitive to developmental toxicants, and historical data and experience exist at the testing facility.
- Route of administration justification: route of test item administration was through oral gavage because it is the preferred route of administration of test item in prenatal developmental toxicity studies.
- the test item/vehicle was administered orally by gavage using disposable plastic syringe attached with a metal feeding cannula to rats of specific groups daily from GD6 to GD19 of presumed gestation, at approximately the same time each day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each rat during the acclimatization period and the period before treatment was observed once daily for clinical signs and twice a day during treatment days pre-dose and post-dose. The post dose observation was carried out at least 0.5 h after dose administration and was completed within 3 h post dose for each animal.
- Each rat was observed twice daily during treatment period for morbidity and mortality i.e., once in the morning and once in the afternoon.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: All females included in the study were weighed on gestation days 0, 3, 6, 9, 12, 15, 18 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- About 200 g of food (food input) was provided on Day 0. The food output was recorded and replenished to about 200 g on Days 3, 6, 9, 12, 15 and 18 and food output on Day 20 of presumed gestation was recorded.
- The quantity of food consumed by each female was recorded over the following intervals: gestation days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 20. Further food consumption for GD 0 - 6, GD 6 - 20 and GD 0-20 was derived.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- All the surviving rats on Gestation Day 20 were sacrificed (caesarean section) under isoflurane anaesthesia, and those rats which were found dead/moribund sacrificed were subjected to post-mortem examination. Gross necropsy involved external observation and examination of thoracic and abdominal viscera including uterine contents which were performed on all rats in the study sacrificed at term (caesarean section).
- Organs with macroscopic findings were collected and preserved for possible histological evaluation. The corresponding organs from sufficient controls were also collected for comparison purpose.
- At necropsy, thyroid gland was collected, weighed from each rat (post fixation) and preserved in 10 % NBF for possible histopathological examination.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Pregnancy status
- Gravid uterine weight (only for females killed at termination, and not from
animals found dead or sacrificed moribund during the study)
- Number of corpora lutea
- Number of implantation sites
- Number of early resorptions/early deaths
- Number of late resorptions/late deaths
- Gross evaluation of placenta

The uterus from dams that appeared non-gravid were subjected to 10% ammonium sulphide staining to observe implantation sites if any (identified as pregnant rats) or to confirm the non-pregnant status.

Blood sampling:

- Hormone analysis: Before caesarean section, from each surviving dam, blood was collected under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture for the determination of total Triiodothyronine (T3), Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) hormones.
Blood samples (about 1 mL from each dam) were collected in plain labelled tubes and kept on bench top for approximately 90 minutes before
centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4° C. The serum samples were placed in labelled plastic tubes and stored at approximately -70 °C until they were analysed.
- Thyroid profile hormone analysis: The kits manufactured by Endocrine Technologies Inc., USA were used for the assay. The following thyroid hormones were estimated in serum by Enzyme-Linked ImmunoSorbent Assay (ELISA) method using BIO-RAD microplate washer and BIO-RAD model 680 readers: Rodent Thyroid Stimulating Hormone (TSH), Rodent Thyroxine (T4), Rodent Triiodothyronine (T3)

Fetal examinations:

- On GD 20, the individual fetuses were delivered by hysterectomy and removed sequentially. The fetuses were euthanised using intraperitoneal injection of sodium thiopentone. Fetuses were subjected to external examination.
The following litter data was recorded:
- Total number of fetuses
- Number of live fetuses
- Individual fetal body weight (g)
- Anogenital distance (mm) from all live fetuses
- Fetus sex (external determination based on anogenital distance and internal sex based on gonadal examination).

- External examinations: All the fetuses were examined for external alterations and discarded post examination.
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: No

Statistics:

Data captured using the ProvantisTM laboratory information management system (LIMS), such as Maternal body weight, body weight change, food consumption, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, Pre/post implantation loss , no. of implantations, sex ratio, Number of corpora lutea, early and late resorptions, hormone analyses (T4, T3, TSH) and the weight of thyroid gland data were evaluated using the Levene test for homogeneity of variances and the Shapiro-Wilks test for normality of distributions. When data was found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA). When data was found to be nonhomogeneous or of nonnormal, data was subjected to transformation and ANOVA was performed on transformed data. When ANOVA was significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group.

The incidence of dams with resorptions were tested by using Chi-square test followed by Fisher’s exact test for group association.

All hypothesis testing was carried out at the 5% (2-sided) significance level.

Significant differences were designated throughout the report as below:

*: Statistically significant difference from the control group at p < 0.05.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity at 400 or 800 mg/kg/day doses. At 1000 mg/kg/day, the main treatment-related clinical sign noted was that of hypoactivity (7/7 incidences). In addition, the clinical signs of chromodacryorrhea and clear eye discharge were observed. One rat which was sacrificed moribund in addition to being hypoactive had other clinical signs of swelling on both hindlimb paws, shallow breathing and bilateral partially closed eye lids.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Only 2/7 dams from 1000 mg/kg/day dose group and all the females from other lower dose groups (7/group) survived till termination and were sacrificed at term.
There was treatment related 5/7 mortalities between gestation days 9 to 17 at the highest dose of 1000 mg/kg/day. In the lower dose groups of 400 and 800 mg/kg/day, all animals survived to planned termination including the vehicle control.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, only 2 dams survived till termination, hence the body weight related parameters calculated were not considered for any discussion.

At 800 mg/kg/day, the mean maternal body weight during GD 12 to 20 was signficantly lower (-11 to -19%). The overall mean maternal body weight gains were lower during GD 6 to 20 (-36%) and GD 0 to 20 (-29%). The mean body weight corrected for the gravid uterine weight (corrected/adjusted body weight) and gains were lower by -16% and -104%, respectively.

At 400 mg/kg/day, the mean maternal body weight was comparable to the vehicle control group.

The observed effects on maternal body weight, body weight gain, and adjusted body weight gain at 800 mg/kg/day were considered treatment related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, only 2 dams survived till termination, hence food consumption data calculated were not considered for any discussion.

At 800 mg/kg/day, signficant decreases in food consumption were noted between GD 6 to 20 with reduction varying from -21 to -41%, which coincided with lower body weight gain during this period. The overall mean food consumption was significantly lower during GD 6 to 20 (-25%) and GD 0 to 20 (-17%).

At 400 mg/kg/day, the mean maternal food consumption was comparable to the vehicle control group.

The lower food consumption associated with lower body weight gain when compared to the vehicle control group at 800 mg/kg/day was considered treatment related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

The lower serum T4 levels at 800 mg/kg/day was considered as a treatment related however was without any consistent/associated findings in TSH and T3 levels.
The thyroid weight changes were not dose related and no treatment effect was suspected.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross pathology examination of unscheduled deaths and moribund sacrificed animal at 1000 mg/kg/day revealed test item related changes of non-glandular stomach mucosa; Focus; depressed (2/7) or stomach enlarged (1/7), thymus small (3/7) or adernals enlarged (2/7). The cause of death/moribundity in these animals cound not be determined based on the gross findings in these test item related decedents and moribund animal.

At 800 mg/kg/day, test item related changes of non-glandular stomach mucosa; Focus; depressed (1/7) and Adrenal-Focus, white, unilateral: raised (1/7) were observed.

There were no test item related gross findings at 400 mg/kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

At 1000 mg/kg/day, only 2 dams survived till termination, hence maternal data calculated was not considered for any discussion.

The maternal parameters comprising of number of implantations, early and late resorptions and post implantation loss were comparable to vehicle control group up to the dose level of 800 mg/kg/day, except for significantly lower uterine weights at 800 mg/kg/day due to lower litter size and fetal weights in
this dose group.

Gross evaluation of placenta revealed no remarkable findings and there were
no macroscopic findings at any dose level.

Total litter losses by resorption:

not examined

Early or late resorptions:

no effects observed

Description (incidence and severity):

At 1000 mg/kg/day, only 2 dams survived till termination, hence maternal data calculated was not considered for any discussion.

The maternal parameters comprising of number of implantations, early and late resorptions and post implantation loss were comparable to vehicle control group up to the dose level of 800 mg/kg/day, except for significantly lower uterine weights at 800 mg/kg/day due to lower litter size and fetal weights in
this dose group.

Gross evaluation of placenta revealed no remarkable findings and there were
no macroscopic findings at any dose level.

Dead fetuses:

no effects observed

Description (incidence and severity):

The total number of fetuses was 85, 95, 96 and 32 corresponding to a mean litter size of 17.0, 15.8, 13.7 and 16.0 respectively at 0, 400, 800 and 1000 mg/kg/day dose groups. No dead fetuses were observed at any dose level.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Key result

Dose descriptor:

dose level:

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

mortality

necropsy findings

Remarks on result:

other: proposed high dose in prenatal development toxicity study

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant changes in sex ratio were observed.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day, the mean litter size was significantly lower (-19%) and the mean fetal weights were lower (not statistically significant) by -7% in males, by -9% in females and by -8% in combined sex.
At 400 mg/kg/day, no statistically significant changes in litter size and fetal weight were observed.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

No statistically significant changes in anogenital distance were observed.

Changes in postnatal survival:

not examined

External malformations:

not examined

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

At 400 mg/kg/day, no statistically significant changes in litter size, fetal weight, sex ratio and anogenital distance were observed.

Key result

Dose descriptor:

dose level:

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in litter size and weights

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

In conclusion, oral administration of the test item at 0, 400, 800 and 1000 mg/kg/day in pregnant Wistar rats from GDs 6-19 resulted in treatment-related mortalities (5/7) at the highest dose of 1000 mg/kg/day with treatment related clinical sign mainly in form of reduced activity (hypoactivity). Lower body weight gains associated with lower food consumption during gestation at 800 mg/kg/day. The mean litter size was significantly lower (-19%) and mean fetal weights (not statistically significant) were lower by 7-9% associated with lower uterine weights at 800 mg/kg/day dose. Gross pathology examination of unscheduled deaths at 1000 mg/kg/day revealed test item related changes of stomach, thymus or adrenals, and in stomach and adrenals at 800 mg/kg/day. Lower T4 hormone levels at 800 mg/kg/day were considered treatment related without any consistent/associated findings in TSH and T3 levels. The thyroid weight changes were not dose related and hence no treatment effect was suspected.

Conclusions:

Based on the above findings, 1000 mg/kg/day is considered to be above the maximum tolerated dose as it caused mortality in 5/7 animals. The following dose levels are proposed for the definitive study:

Vehicle control (G1) - 0 mg/kg/day
Low dose (G2) - 200 mg/kg/day
Mid dose (G3) - 400 mg/kg/day
High dose (G4) - 800 mg/kg/day

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,4-dimethylpiperazine

EC Number:

203-412-0

EC Name:

1,4-dimethylpiperazine

Cas Number:

106-58-1

Molecular formula:

C6H14N2

IUPAC Name:

1,4-dimethylpiperazine

Details on test material:

- Name of test material (as cited in study report): Jeffcat DMP

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 1,4-dimethylpiperazine
- Physical state: liquid
- Source and lot/batch No. of test material: PFW210769
- Expiration date of the lot/batch: 2023-11-25
- Purity: ca. 99.91 A%. The substance is specified by total titratable amine content. Amine content is 17.39 meq/g.
- pH: 11.4
- density: 0.84 g/cm³
-specific gravity: 0.822 at 20°C

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (+15 to +22°C), protected from light/humidity. At temperature no higher than +22ºC. Recommend storage under a dry inert gas blanket, such as nitrogen, to minimize contamination resulting from contact with air and water.
- Stability of the test item in the vehicle: The stability of the test item in the vehicle was established at 1 and 100 mg/mL concentrations under Study No. G24817 in Milli-Q water. Based on the results, the test item was stable in the vehicle up to 48 hours when stored at room temperature.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology (India) Pvt. Ltd. 4B, M.N Park, Shameerpet Mandal, Turkapally Village, Medchal Dist, Telangana 500078, India
- Age at study initiation: 13-14 weeks
- Weight at study initiation: At the start of treatment, the females weighed between 240 to 309.68 grams.
- Fasting period before study: no data
- Housing: Rats were housed in standard polysulfone rat cages (size: Length 425 mm × Width 266 mm × Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube. Additionally, polycarbonate rat huts were placed inside the cage as environmental enrichment objects which were replaced once a week. Following was the housing pattern during different periods of the experiment: i. Pre-mating / Acclimatization: Two rats of the same sex per cage were housed. ii. Mating: Female rats were cohabited with males in a 1:1 ratio. iii. Post-mating / Treatment: After mating confirmation, females were housed individually. Nesting material (paper cuttings) were placed inside the cages from GD 18 onwards. Steam sterilized corn cob was used as bedding material and was changed along with the cage at least twice a week coinciding with body weight and food intake measurement.
- Diet (e.g. ad libitum): ad libitum; Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was used.
- Water (e.g. ad libitum): ad libitum; Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India was used
- Acclimation period: After detailed clinical examination for good health and suitability for the study, 120 females and 96 male rats were acclimatized for five days before initiation of mating. Only nulliparous females were used in the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23°C
- Humidity (%): 64-67%
- Air changes (per hr): 12-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light and 12-hours dark cycle

IN-LIFE DATES: From 2022-06-16 To 2022-07-21

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dose formulations were prepared in an interval of 1 to 3 days and used within the established stability period of 48 hours.
- Required quantities of the test item was weighed in a beaker (previously calibrated to a desired volume) for each dose levels separately and a small volume of vehicle (Milli-Q® water) was added and stirred using a glass rod till a uniform solution was obtained. The volume was made up to the upper meniscus mark using the vehicle to get the final desired concentration of 20, 40 and 80 mg/mL for the low (G2), mid (G3) and high (G4) dose groups, respectively.
- Pre-calibration of the beaker to desired volume: Milli-Q water was measured in a graduated measuring cylinder to the final volume of the batch size (i.e., when batch size was 50 mL, then 50 mL of Milli-Q water was poured in a graduated measuring cylinder). The measured Milli-Q water was then transferred into a clean beaker (to be pre-calibrated) and upper and lower meniscus of Milli-Q water were marked on the beaker using a marker. Once these lines were marked, the Milli-Q water was discarded, and the beaker was dried. The upper meniscus line was used to make up to the volume during the dose formulation suspension preparation.
- A similar procedure was followed for dose formulation preparation throughout the experiment; however, the volume of dose formulation prepared varied depending on the requirement and/or body weights of the rats recorded. The remaining dose formulations were stored in a container and sent for disposal at the end of the experiment.

VEHICLE
- Concentration in vehicle: 0, 20, 40 and 80 mg/mL
- Treatment volume 10 mL/kg
- Justification for the selection of vehicle: The Formulation Analytical Method Validation, Homogeneity and Stability, was established in Milli-Q water under Study No. G24817. Further, OECD 407 (Study No. G19072) and OECD 421 (Study No. G19073) studies with the same test item were conducted using Milli-Q water as vehicle for dose formulation preparation. Hence, Milli-Q water was used as vehicle for dose formulation preparation in this study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analysed for the test item concentration at the
initiation of treatment and 2 days before termination of treatment.
One set of samples was analyzed for concentration analysis and other set (second set) of samples were stored at ambient condition for reanalysis purpose as a backup and the second set of samples were discarded, as the analysis results of first set of samples were within the limits. For each set, composite sample were drawn in three replicates from each preparation and in case of control duplicate composite samples were drawn.
All the collected samples were sent under ambient conditions to Principal investigator at Test site, for formulation analysis to determine the concentration of the test item. The analysis was carried out within the established stability period. The analysis was carried out as per the method validated under Eurofins Advinus Study No.: G24817.
Formulations were considered acceptable as the mean results are within 80-120 % of the claimed concentration and the relative standard deviation (% RSD) is equal to or less than 20 %.

Details on mating procedure:

- During the mating period, female rats were cohabited with males in a 1:1 ratio and vaginal smears and (or) vaginal plug were examined in the morning of the subsequent day. When sperm was detected in a vaginal smear and/-or when vaginal plug was observed in the morning, the female was confirmed to be mated. This day was regarded as GD 0. The females were cohabited in batches of required numbers. This procedure was continued until there were sufficient numbers of GD 0 pregnant rats for the study.
- GD 0 pregnant rats obtained on each day were randomly distributed to different groups by body weight stratification method using ProvantisTM software. After grouping and ascertaining the group mean body weight, the rats were given accession number as applicable to the group on each day of assignment.
- Note: After confirming mating, females were housed individually, and the unselected females and males used for mating purpose were removed from the experiment.

Duration of treatment / exposure:

From GD 6 to GD 19 of presumed gestation, at approximately the same time each day (varying by ± 3 hours)

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

low dose group

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

medium dose group

Doses / concentrations 3

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

high dose group

No. of animals per sex per dose:

24 females/group; Total = 96

Control animals:

yes, concurrent vehicle

Details on study design:

- Justification for selection of species: The rat was selected because it is a standard acceptable rodent species for use in prenatal developmental toxicity studies, this species and strain has been demonstrated to be sensitive to developmental toxicants, and historical data and experience exist at the testing facility
- Dose selection justification: A preliminary dose range finding study (DRF) in pregnant female rats was carried out using 7 rats per group with the test substance dosed at 400, 800 and 1000 mg/kg/day along with the concurrent vehicle control group (Study No. N6327). The pregnant rats were treated with the dose formulations once daily by oral gavage at a dose volume of 10 mL/kg body weight during GD 6 to GD 19 and observed for clinical signs and mortality. On GD 20, animals were subjected to necropsy and uterine examinations were performed.
Oral administration of the test substance at 0, 400, 800 and 1000 mg/kg/day in pregnant Wistar rats from GDs 6-19 resulted in treatment-related mortalities (5/7) at the highest dose of 1000 mg/kg/day with treatment related clinical sign mainly in form of reduced activity (hypoactivity). There were lower body weight gains associated with lower food consumption during gestation at 800 mg/kg/day. The mean litter size was significantly lower (-19%) and mean fetal weights were lower by 7- 9% (not statistically significant). This was associated with lower uterine weights at 800 mg/kg/day dose. Gross pathology examination of unscheduled deaths at 1000 mg/kg/day revealed test item related changes of stomach, thymus or adrenals, and in stomach and adrenals at 800 mg/kg/day. Lower T4 hormone levels at 800 mg/kg/day were considered treatment related without any consistent/associated findings in TSH and T3 levels. The thyroid weight changes were not dose related and hence no treatment effect was suspected.
Based on the above findings, 1000 mg/kg/day was considered to be above the maximum tolerated dose as it caused mortality in 5/7 animals.
- Route of administration and justification of choice: The route of test item administration was through oral gavage. The oral route was chosen as it is the standard route as accepted by various regulatory authorities and the preferred route of administration of the test item in prenatal developmental toxicity studies.
- Dose administration: Vehicle alone and test item formulations were administered orally by gavage using a disposable plastic syringe attached with a metal feeding cannula to rats of specific groups daily from GD 6 to GD 19 of presumed gestation, at approximately the same time each day (varying by ± 3 hours). The test item was administered at a fixed dose volume of 10 mL/kg body weight. The actual volume was calculated for individual animals using the most recently recorded body weight. The animals in the vehicle control group were handled in an identical manner to the treatment group and were administered vehicle (Milli-Q water) only.

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS:
Each rat during the acclimatization period and the period before treatment were observed once daily for clinical signs and twice a day during treatment days pre-dose and post-dose. The post dose observation was carried out at least 0.5 h after dose administration and was completed within 3 h post dose for each animal.
Each rat was observed twice daily during the treatment period for morbidity and mortality i.e., once in the morning and once in the afternoon. Based on the assessment, as there were no clinical signs of concern, observation for morbidity and mortality was carried out once during weekends and public holidays.

BODY WEIGHT:
All females included in the study were weighed on gestation days 0, 3, 6, 9, 12, 15, 18 and 20.
Intermittent body weight gains were calculated from GD 0 - 3, 3 - 6, 6 - 9, 9 -12, 12 - 15, 15 - 18 and 18 - 20. Further body weight gains for GD 0 - 6, GD 6 – 20 and GD 0 - 20 were derived. Body weight data were statistically analyzed and presented only for rats found pregnant at caesarean section.
The corrected/adjusted body weight was obtained by subtracting terminal body weight (body weight on GD 20) from the unopened uterine weight. The corrected body weight gain was calculated by subtracting the corrected body weight from body weight on GD 6.

FOOD CONSUMPTION:
About 200 g of food (food input) was provided on GD 0. The food output was recorded and replenished to about 200 g on 3, 6, 9, 12, 15 and 18. Prior to terminal sacrifice, food left over was recorded on GD 20. The quantity of food consumed by each female was calculated for the following intervals: 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 20. Food consumption for GD 0 - 6, GD 6 - 20 and GD 0-20 were also calculated. Food consumption was statistically analyzed and presented only for rats found pregnant at caesarean section. No spillage of food was observed during the food measurement intervals.

POST-MORTEM EXAMINATIONS:
On gestation Day 20, all rats were sacrificed (caesarean section) under isoflurane anaesthesia and gross pathological changes in the placenta and in all visceral organs of rats were examined. Gross necropsy involved external observation and examination of thoracic and abdominal viscera including uterine contents which were performed on all rats in the study sacrificed at term (caesarean section). Organs/tissue with macroscopic findings were collected and subjected to histological evaluation, and findings reported. The corresponding organs from sufficient controls were also collected and examined for comparison.
At necropsy, the thyroid gland was collected and weighed from each rat (postfixation) and subjected to histopathological examination. The tissue was processed for routine paraffin embedding, and 4-5-micron sections were stained with Haematoxylin and Eosin stain. Liver was collected and weighed from each rat and stored for possible histopathology examination. Unused tissue will be archived.

Ovaries and uterine content:

Immediately after termination, the gravid uterus along with the cervix was excised, weighed, and examined.
The following maternal endpoints were investigated:
a. Pregnancy status
b. Gravid uterine weight
c. Number of corpora lutea
d. Number of implantation sites
e. Number of early resorptions/early deaths
f. Number of late resorptions/late deaths
g. Gross evaluation of placenta

Uteri from rats that appeared non-gravid were subjected to 10% ammonium sulphide staining to observe implantation sites if any (identified as pregnant rats) or to confirm the non-pregnant status.

Blood sampling:

Before caesarean section, from each surviving rat, blood was collected under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture for the determination of total Triiodothyronine (T3), Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) hormones.
Blood samples (about 1 mL from each rat) were collected in plain labelled tubes and kept on bench top for approximately 90 minutes before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4° C. The serum samples were placed in labelled plastic tubes and stored at approximately -70 °C until they were analyzed. The left-over samples from hormone analysis were discarded at the time of final
report preparation.
- thyroid hormone profiles: The kit manufactured by Endocrine Technologies Inc., USA was used for the
assay.
The thyroid hormones (TSH, T4, T3) were estimated in serum by Enzyme-Linked ImmunoSorbent Assay (ELISA) method using BIO-RAD microplate washer PW 40 and BIO-RAD model microplate readers 680

Fetal examinations:

On gestation Day 20, the individual fetuses were delivered by caesarean section and removed sequentially. The fetuses were euthanized using an intraperitoneal injection of sodium thiopentone. Fetuses were subjected to external and visceral or skeletal examination. The following litter endpoints were investigated:
a. Total number of fetuses
b. Number of live fetuses
c. Individual fetal body weight (g)
d. Anogenital distance (mm) from all live fetuses
e. Sex of the fetus - external determination based on anogenital distance that was confirmed based on gonadal examination (internal sex) during visceral examination.

Fetuses were examined for external and visceral or skeletal alterations and the observations were classified as variants, anomalies and malformations as defined below:
- Variants: An incidence distributed throughout a population in consistently large percentage, e.g. ossification variations.
- Anomaly: Alteration in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal.
- Malformation: Structural anomalies that alter general body conformity, disrupt or interfere with normal body function, may have a detrimental impact on postnatal life, or may be incompatible with postnatal survival.
- Fetus external examination: All fetuses were examined for external alterations/malformations.
- visceral (soft tissue) and skeletal examination: Approximately half the number of fetuses from each litter were subjected to visceral examination and the remaining half were subjected to skeletal examination.
Fetal Visceral Examination: About half of the fetuses from each litter were prepared for fresh tissue visceral organ examination using gross dissection technique. Indication of incomplete testicular descent/cryptorchidism in male
fetuses were also examined. The fetuses subjected to visceral examination were decapitated and heads were stored in 70% alcohol for sectioning using the modified Wilsons Razor blade sectioning technique. Individual bottles
containing fetal heads were labeled with study number, code number and fetal number among total number of fetuses. After head sectioning and evaluation, as no abnormalities were observed, fetal heads were discarded.
Fetal Skeletal Examination: The remaining half of the fetuses of each litter were prepared for skeletal examination by wet skinning followed by evisceration and staining. Fetuses were fixed in 70 % ethyl alcohol, eviscerated, and dehydrated in 100 % ethyl alcohol; macerated in 1.5 % solution of KOH and stained with saturated, aqueous Alizarin red S in Mall’s solution. The excess stain was removed in Mall’s solution and fetuses were cleared by passing through grades of glycerol with thymol crystals. Individual bottles containing fetuses for skeletal examination were labeled with study number, code number and fetal number among total number of fetuses.
After the skeletal examination, fetuses were pooled dam-wise in bottles containing 100 % glycerol with crystals of thymol to prevent fungal growth. The bottles were labeled with study number, code number and number of fetuses evaluated among total number of fetuses. All findings recorded are presented in the report as per the standard reporting format. As there were no malformations observed during fetus external, soft tissue and skeletal examinations, cross verification activity by another specialist in this prenatal developmental toxicology study was not carried out.

Statistics:

Data captured using the ProvantisTM laboratory information management system (LIMS), for parameters such as Maternal body weight, body weight change, food consumption, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, Pre/post implantation loss, Number of implantations, sex ratio, Number of corpora lutea, early and late resorptions, hormone analyses (T4, T3, TSH) and the weight of thyroid gland and liver were evaluated using the Levene test for homogeneity of variances and the Shapiro-Wilks test for normality of distributions. When data was found to be homogeneous and of normal distribution, data was analyzed by analysis of variance (ANOVA). When data was found to be nonhomogeneous or of nonnormal data, data was subjected to transformation and ANOVA was performed on the transformed data. When ANOVA was significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences. Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group.
The incidence of dams with resorptions were tested by using Chi-square test followed by Fisher’s exact test for group association.
The incidence of fetus and litter (incidence and percent) observations for skeletal observations were tested using Cochran-Armitage trend test and pairwise comparison was tested by Fisher’s exact test for group association.
All hypothesis testing were carried out at the 5% (2-sided) significance level. Significant differences are designated throughout the report as below:
*: Statistically significant difference from the vehicle control group at p < 0.05.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity at 200 and 400 mg/kg/day. At 800 mg/kg/day treatment related clinical signs of discolouration pink of tail (7/24) and ears (12/24) were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All the females from each group (24/group) survived till termination and were sacrificed at term. All animals survived to scheduled euthanasia up to the highest tested dose of 800 mg/kg/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day, the mean maternal body weight during GD 9 to 20 was significantly lower (-10 to -19%). The overall mean maternal body weight gains were lower during GD 6 to 20 (-59%) and GD 0 to 20 (-47%). The mean body weight corrected for the gravid uterine weight (corrected/adjusted body weight) and gains were lower by -18% and -143%, respectively.
At 400 and 200 mg/kg/day, the mean maternal body weight was comparable to the vehicle control group. The overall mean maternal body weight gains were lower during GD 6 to 20 by -9% and -11% at 200 and 400 mg/kg/day, respectively. These decreases were below 10% at 200 mg/kg/day and
marginally over 10% at 400 mg/kg/day and considered treatment related nonadverse effects.
The observed effects on maternal body weight, body weight gain, and adjusted body weight gain at 800 mg/kg/day were considered treatment related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 800 mg/kg/day, significant decreases in food consumption were noted between GD 6 to 20 with reduction varying from -24 to -49%, which coincided with lower body weight gain during this period. The overall mean food consumption was significantly lower during GD 6 to 20 (-35%) and GD 0 to 20 (-26%).
At 400 mg/kg/day, the mean maternal food consumption was comparable to the vehicle control group except during the initial part of treatment period GD6 to 9 and GD 9 to 12, wherein the food consumption was significantly lower and the reduction above 10% by -18% and -12%, respectively. The food consumption during GD 12 to 15 and overall mean food consumption GD 6 to 20 was significantly lower and the reduction being by -8% and -9%, respectively which was not above 10% to be considered of toxicological relevance. Thus, the food consumption reversed after the initial decrease and these changes are considered treatment related non-adverse effects.
At 200 mg/kg/day, the mean maternal food consumption was comparable to the vehicle control group.
The lower food consumption associated with lower body weight gains when compared to the vehicle control group at 800 mg/kg/day was considered treatment related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

The thyroid hormones TSH, T3 and T4 levels remained unaffected by the test item administration.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Thyroid and liver weights were significantly lower at 800 mg/kg/day and considered as secondary effects associated with the test item related decreased body weights.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related gross lesions. The incidences of tail discolouration at 400 and 800 mg/kg/day and ear discolouration at 800 mg/kg/day were considered as toxicologically insignificant findings as the tissues were normal on microscopic evaluation.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic evaluation of thyroid glands did not reveal test item-related alterations in any of the rats.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

The maternal parameters comprising of number of implantations, early and late resorptions and post implantation loss were comparable to vehicle control group up to the dose level of 800 mg/kg/day, except for significantly lower uterine weights by -21% at 800 mg/kg/day due to lower fetal weights in this dose group.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The maternal parameters comprising of number of implantations, early and late resorptions and post implantation loss were comparable to vehicle control group up to the dose level of 800 mg/kg/day, except for significantly lower uterine weights by -21% at 800 mg/kg/day due to lower fetal weights in this dose group.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The maternal parameters comprising of number of implantations, early and late resorptions and post implantation loss were comparable to vehicle control group up to the dose level of 800 mg/kg/day, except for significantly lower uterine weights by -21% at 800 mg/kg/day due to lower fetal weights in this dose group.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The maternal parameters comprising of number of implantations, early and late resorptions and post implantation loss were comparable to vehicle control group up to the dose level of 800 mg/kg/day, except for significantly lower uterine weights by -21% at 800 mg/kg/day due to lower fetal weights in this dose group.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses were observed at any dose level.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Details on maternal toxic effects:

The number of non-pregnant rats were 1, 2, 1 and 2 at 0, 200, 400 and 800 mg/kg/day, respectively. Hence, the number of litters available for external, visceral/skeletal evaluations were 23, 22, 23 and 22 at 0, 200, 400 and 800 mg/kg/day, respectively.
The total number of fetuses were 350, 324, 337 and 315 at 0, 200, 400 and 800 mg/kg/day dose groups, respectively. While all the fetuses were subjected to external examination, approximately half of the total fetuses were subjected to soft tissue examination and remaining half of the fetuses were subjected to skeletal examinations.
Number of fetuses subjected to soft tissue and skeletal examination:
- soft tissue examination: 171, 156, 161, 152 for vehicle, low, mid and high dose group
- skeletal examination: 179, 168, 176, 163 for vehicle, low, mid and high dose group

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean fetal weights at 800 mg/kg/day were significantly lower by -15% in males, by -16% in females and by -15% in combined sex which are considered treatment related secondary to lower maternal body weight gains and food consumption at this dose.
The mean male, female and combined sex fetal weights at 200 and 400 mg/kg/day dose groups were comparable to the mean fetal weights in the vehicle control group.

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No toxicologically significant changes in sex ratio was observed at any of the dose level.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The total number of live fetuses were 350, 324, 337 and 315 at 0, 200, 400 and 800 mg/kg/day dose groups, respectively. The corresponding mean litter size was 15.2, 14.7, 14.7 and 14.3 at 0, 200, 400 and 800 mg/kg/day dose groups, respectively.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

No toxicologically significant changes in AGD was observed at any of the dose level.

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed during external examinations of fetuses of rats treated up to the highest dose of 800 mg/kg/day.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of fetuses subjected to skeletal observations was 179, 168, 176 and 163 at 0, 200, 400 and 800 mg/kg/day dose groups, corresponding to 23, 22, 23 and 22 litters, respectively.
There were no skeletal malformations observed in any litter at any of the tested dose levels. The number of fetuses subjected to skeletal observations was 179, 168, 176 and 163 at 0, 200, 400 and 800 mg/kg/day dose groups, corresponding to 23, 22, 23 and 22 litters, respectively.
There were no skeletal malformations observed in any litter at any of the tested dose levels. There were significant increases in skeletal variations of not ossified or incomplete ossification of skull bones namely Zygoma, Supraoccipital, Squamosal, parietal, interparietal, hyoid or frontal at 800 and 400 mg/kg/day and a significant increase in incomplete ossification of pelvic girdle bones namely pubis and ischium at 800 and 400 mg/kg/day. Anomalies of ribs (slight wavy ribs) of 7th, 8th and 9th were observed at significantly higher incidences at 800 mg/kg/day.
Significantly higher incidences of not ossified or incomplete ossification of sternebral bones, 6th, 4th, 3rd and 1st at 800 mg/kg/day.
Ossification related observations are generally considered transient changes and these finding denotes generalized growth delays which normally ossify late in gestation or as the organism matures, and they do not have any general predictive value for teratogenicity [Carney and Kimmel (2007)].
Wavy ribs are manifestations of delayed ossification, are transient and reversible and are not of developmental importance since they are expected to resolve during maturation (Hayasaka et al., 1985; Kast, 1994; Mitchard &
Stewart, 2014). Thus, the observed variations and anomalies seen at 400 and 800 mg/kg/day doses were considered non-adverse, and they were likely secondary to maternal systemic toxicity of the test substance in the form of reduced body
weight and food consumption.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no treatment-related visceral observations during fresh fetal visceral examination at all the doses tested, compared to vehicle control group.
All fetal heads were normal upon the Wilsons-Razor blade sectioning.

Other effects:

not specified

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

2

Key result

Dose descriptor:

NOAEL

Remarks:

fetal development

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

800 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Oral administration of the test substance at 0, 200, 400 and 800 mg/kg/day in pregnant Wistar rats from GDs 6-19 did not cause any mortalities. Treatment related clinical signs in form pinkish discolouration of tail and ears were observed at 800 mg/kg/day without any microscopic correlates. Lower body weight gains associated with lower food consumption during gestation at 800 mg/kg/day. The number of corpora lutea and implantation sites, and thus the extent of pre-implantation loss, were similar between all groups. The resorptions were also comparable between the groups. The number of live fetuses was in a range 14.3 to 15.2 per female, with no treatment-related or statistically significant differences present between the groups. No dead fetuses were found in any group. The sex ratio in the different groups (45.4 to 53.7% male, 46.3 to 54.6% female) was within the normal range. The mean fetal weights were significantly lower by 15-16% associated with lower uterine weights at 800 mg/kg/day dose. Gross pathology examination revealed no test item related gross lesions. The thyroid hormones TSH, T3 and T4 levels remained unaffected by the test item administration. Thyroid and liver weights were significantly lower at 800 mg/kg/day and considered as secondary effects associated with the test item related decreased body weights. Lower thyroid weights were without any histological correlates. Fetal morphology (external, visceral and Skeletal) was unaffected by test item administration up to the exposure level of 800 mg/kg/day.
Based on the above findings, under the test conditions used in this study, the following NOAEL values were derived:
• NOAEL for maternal developmental toxicity was considered at 400 mg/kg/day as maternal body weight gains and food consumption were significantly affected at 800 mg/kg/day.
• NOAEL for fetal developmental toxicity was 400 mg/kg/day due to significant decreases in fetal weight at 800 mg/kg/day.
• NOAEL for teratogenicity was 800 mg/kg/day as the results from the fetal external, visceral, and skeletal examinations did not reveal any adverse effects of treatment.