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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

A reproduction/developmental toxicity study in rats was performed according to OECD guideline 421 and according to GLP requirements (Malleshappa, 2020). The NOEL for reproduction/developmental toxicity screening test was considered to be 500 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-03-13-to 2020-08-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016-07-29
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 39 days, up to and including the day before scheduled euthanasia. This included a two-week period prior to mating, during the mating period and post mating. Females were dosed throughout the treatment period. This included two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and 13 days after delivery, up to and including the day before scheduled euthanasia.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of the test item: 1,4-dimethylpiperazine
- Appearance: liquid
- Source and lot/batch number of test material: PFW190423
- Expiration date of the lot/batch: 2021-08-05
- Purity: 99.9 A%
- physicochemical properties: pH 11.42, specific gravity 0.84, density 0.84 g/cm³

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to +25°C); Temperature no higher than +22°C
- Stability under storage conditions: not indicated
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Stability of the test item in the vehicle was established at 1 and 100 mg/mL under study G19071. The test item was stable in the vehicle up to 24 hours when stored at room temperature.
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): not indicated
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities. The Wistar rat was selected due to the large amount of background data available for this strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hylasco Biotechnology Pvt. Ltd. Plot 4B, AKP, Turkapally Village, Shameerpet Mandal, RR Dist, Telangana 500078, India
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 12 weeks
- Weight at study initiation: 344.68 - 398.16 g (males); 222.47 - 272.37 g (females)
- Fasting period before study: No
- Housing:
* pre-mating: Two rats of the same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless-steel sipper tubes.
* Mating and post-mating: During mating, two rats (one male and one female) were housed in standard polysulfone cages with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plug (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term (Gestation Day 20).
* Enrichment: Polycarbonate rat huts were provided to the animals as environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall wellbeing. These objects were provided during the pre-mating and post-mating period for males and during the pre-mating period for females and changed along with cage twice a week.
* Bedding: Steam sterilized clean corn cob was used as bedding and changed along with the cage twice a week.
- Diet (e.g. ad libitum): Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum.
- Water (e.g. ad libitum): Deep bore-well water passed through an activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Limited., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: After detailed clinical examination for good health and the suitability for the study, the rats were acclimatized for five days before the pretreatment period. During the acclimatization period, animals were observed once daily for any abnormalities. Only the animals that were determined by the veterinarian to be suitable for use were assigned to this study. Female rats used in this study were nulliparous and non-pregnant.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24°C
- Humidity (%): 49-66%
- Air changes (per hr): 12-15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 2020-03-13 To: 2020-06-29
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Milli-Q
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- 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 obtaining a uniform solution. The volume was made up to the mark using the vehicle to get the final desired concentration of 12.5, 25 and 50 mg/mL for the G2, G3 and G4 groups, respectively. The solution was mixed well by stirring using a magnetic stirrer.
- Pre-calibration of the beaker to desired volume: Milli-Q water was measured in a graduated measuring cylinder to the final volume of 100 mL. The measured Milli-Q water was transferred into a clean beaker and the upper and lower meniscus of the Milli-Q water were marked on the beaker using a marker. The Milli-Q water was discarded and the beaker was dried. These lines were used to make up to the volume for the dose formulation suspension preparation.
- The dose formulations were prepared once daily before start of each day dosing and administered within 2 hours after preparation. Homogeneity of the test item solutions prior to sampling and during dosing was maintained by constant stirring using a magnetic stirrer.
- The volume of dose formulation to be prepared was varied depending on the requirement and/or body weights of the rats recorded during experimental period.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The Formulation Analytical Method Validation, Homogeneity and Stability was established in Milli-Q water under Eurofins Advinus study (G19071). Further, the same vehicle was used in the dose-range finding toxicity study (study no. N4793). Hence, Milli-Q water was used as vehicle for dose formulation preparation.
- Concentration in vehicle: 12.5, 25 and 50 mg/mL for the G2, G3 and G4 groups respectively.
- Amount of vehicle (if gavage): 10 mL/kg
Details on mating procedure:
One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there was evidence of sperms in the vaginal smear and /or vaginal plug. All the females were successfully copulated within five days from the day of cohabitation except for three females each in control, low and mid dose groups. These females were placed with a second proven male of the same group and when the presence of sperm in the vaginal smear and/or vaginal plug was not confirmed within 7 days, the female was euthanized 24-26 days after last day of mating period. Subsequently, pregnant females were housed individually until LD 14. All the mated females were maintained till they littered. Not-littered females were euthanised after 25 days from the day they were found sperm positive (by vaginal smear examination). The day of confirmed mating was designated as Gestation Day ‘0’ (GD ‘0’). The pre-coital time (days) was calculated for each female.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 of the treatment period and during the 2nd month (day 42) of treatment and was analysed inhouse. For each set, composite sample was drawn in six replicates from each preparation and in case of control duplicate composite samples were drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19071. One set of samples were analyzed and other set (second set) of samples were stored at room temperature for possible reanalysis as a backup and these samples were discarded, as the analysis results of first set of samples were within the acceptable limits.
Dose formulations were considered acceptable as the overall mean results were within ± 15 % of the theoretical concentration and the overall relative standard deviation (RSD) was less than 10%.
Duration of treatment / exposure:
Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily for 43 days at approximately the same time each day (varying by ± 3 hours) which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled euthanasia.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours). This includes 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 42-69 days), after which, pups were euthanised on LD 13 and parental females (dams) were euthanised on LD 14 after overnight fasting (water allowed).
The animals in the vehicle control group were handled in an identical manner to the treatment groups and were administered vehicle only.
The dose volume administered to each rat was 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of the individual rat.
Frequency of treatment:
Daily
Details on study schedule:
Route of administration and justification: The route of test item administration was through oral gavage. The oral route was chosen because it provides an exaggerated model of the normal exposure in humans.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
GI (Vehicle control)
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
G2 (low dose)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
G3 (Mid dose)
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
G4 (High dose)
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels of 125 (G2), 250 (G3) and 500 (G4) mg/kg/day were selected for this study in consultation with sponsor based on the outcome of 14 Day repeated dose oral toxicity study in Wistar Rats (study N4793). In addition to the test doses, vehicle control group was included. Animals in the vehicle control were handled in a manner similar to the treatment groups except for test item administration
- Rationale for animal assignment (if not random): Animals were randomly distributed to different groups by body weight stratification method using ProvantisTM software (Version 10.1.0.1, Instem LSS, Staffordshire ST15OSD, United Kingdom). Rats that are not selected were killed humanely without any further investigation. Grouping was done one day prior to start of treatment during pre-treatment period.
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs of toxicity, the observation for morbidity and mortality was carried out once in the morning during weekend and holidays. Each rat was observed for clinical signs twice daily during the treatment period i.e. once prior to dosing and 1-2 hour after the dosing. On the days of scheduled detailed clinical examination, clinical signs (after dosing) was included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examination was done prior to the treatment on Day 1 and 1 and 1-2 hour after dosing at weekly intervals (±1 days) thereafter during the treatment period.
- During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour like self-mutilation, walking backwards). On the days of detailed clinical examination, observations for general clinical signs were not performed except post-dose observations on treatment Day 1.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of males were recorded on Day 1 and at weekly (±1) intervals thereafter. Individual body weights of females were recorded on Day 1 (±1) and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
- All dams were weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The food consumption was measured at weekly intervals (± 1 day) during treatment period. The cage wise average food intake (g/rat/day) was calculated.
- Food consumption was not measured during the cohabitation period.
- Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Days 4 and 13 of the lactation period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER:
- Hormone analysis: Blood samples were collected and serum was separated as per the following schedule for the determination of total T4 and TSH:
- all adult males, prior to euthanasia and
- all dams, prior to euthanasia.
Blood samples were not collected from the non-littered females and the dams in which all pups were dead/cannibalized during lactation.
- Parturition: The duration of gestation was calculated from Day ‘0’ of pregnancy to the day of parturition (Gestation Length). Females were observed for signs of difficult or prolonged parturition.
Oestrous cyclicity (parental animals):
Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular 4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or precoital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal. Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle except for Rx6375 and Rx6380 in control. Rx6394 in low dose was killed moribund and one female rat in high dose (Rx 6440) was found dead. Hence, stage of oestrous was not recorded for these animals. A deviation was recorded for these animals.
Sperm parameters (parental animals):
Histopathology examination of the testes involved a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. Testes were sectioned at 3-4 µm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis.
Litter observations:
STANDARDISATION OF LITTERS
- At birth each day in the morning, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.
- The number of pups born (litter size), sex and individual pup body weight of male and female pups on LD 0 and 4 were recorded.
- On Day 4 after birth, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size drop below the culling target (8 pups/litter).


PARAMETERS EXAMINED
- Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.
- After standardization, the individual pup body weight was recorded on Day 13 of lactation.
- The ano-genital distance (AGD) of each pup was measured on PND 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from the cube root of body weight.
- The number of nipples/areolae in male pups was counted on PND 13.
- The litters were observed daily to note the number of alive, dead and cannibalized pups.
- In addition to daily clinical observations, any abnormal behaviour of the offspring was recorded.
- Fertility index for dams, sires as well as the pup survival index until lactation day 4 was calculated.

GROSS EXAMINATION OF DEAD PUPS: yes,
- all the dead and euthanised pups were examined for malformations and subjected to gross pathological examination

HORMONE ANALYSIS
Blood samples were collected and serum was separated as per the following schedule for the determination of total T4 and TSH:
- Two available surplus pups on Day 4 after birth.
- two available pups per litter on day 13
Pups were anesthetized with isoflurane and incised at the jugular vein in the neck region. The collected samples were pooled together for each litter. Blood samples were collected in plain labelled tubes and kept on the bench top for approximately 20 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 ca. -70°C until they were analyzed.

Postmortem examinations (parental animals):
SACRIFICE AND GROSS NECROPSY
All adult animals were examined macroscopically for any structural abnormalities or pathology changes. The adult animals killed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia.
The rats were subjected to detailed necropsy by a veterinary pathologist and findings were recorded. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.
The number of implantation sites were recorded for all the dams.

HISTOPATHOLOGY / ORGAN WEIGHTS
On completion of the gross pathology examination the tissues and organs noted above were collected and weighed from all terminally sacrificed adult animals. The organ weight ratios (organ to body weight) as percentage of fasting body weight were determined. The paired organs were weighed together and combined weight was presented. The tissues were preserved in 10% Neutral Buffered Formalin (NBF) unless noted otherwise.
- Organ weight determined for: adrenals, all gross lesions, epididymides, ovaries with oviducts, stomach, thymus, thyroid with parathyroids (weighed after formalin fixation), uterus with cervix, vagina, prostate (prostate + seminal vesicles with coagulating glands were weighed as a whole; subsequently prostate was separated and weighed. The derived weight was presented for the seminal vesicles and coagulating glands), seminal vesicles and coagulating glands, testes, levator ani bulbocavernosus muscle complex, Cowper’s glands, Glans penis
- Tissues collected from all terminally sacrificed adult animals were collected: adrenals, all gross lesions, epididymides, ovaries with oviducts, stomach, thymus, thyroid with parathyroids, uterus with cervix, vagina, prostate, seminal vesicles and coagulating glands, testes, levator anu bulbocavernosus muscle complex, cowper's glands, glans penis
- Histopathology: Tissues collected from all animals in the control and high dose groups and the found dead animal (Rx6440) were examined microscopically for histopathological changes including qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure in testes. Gross lesions were examined in all the animals. The reproductive organs were examined from Rx6405 (male failed to mate) , Rx6380 (non-pregnant)and Rx6375 (pregnant not littered), Rx6394 (unscheduled sacrifice on LD 7) and Rx6440 (found dead on LD 3 and all pups dead).The remaining tissues from the lower dose groups were not examined as no test item related changes were noted at high dose.
The available thyroid gland from a male and a female pup per litter (randomly selected) were also evaluated from all the groups.
The tissues were processed for routine paraffin embedding and 4-5 micron sections were stained with Mayer’s Haematoxylin Eosin stain. In addition, testes were sectioned at 3-4 µm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived.
List of tissues: adrenals, all gross lesions, epididymides, ovaries with oviducts, stomach, thymus, thyroid with parathyroids, uterus with cervix, vagina, prostate, seminal vesicles and coagulating glands, testes

Postmortem examinations (offspring):
SACRIFICE AND GROSS NECROPSY
- All pups were examined macroscopically for any structural abnormalities or pathology changes. All the surviving pups were necropsied on lactation Day 13 and findings were recorded. Dead pups were examined for defects and/or cause of death
- on lactation day 13, thyroid gland from available one male and female pup from each litter was collected and preserved in 10% NBF for the histopathological examination. The thyroid weight of pups were determined after fixation.

Statistics:
Data captured using ProvantisTM: Parameters of body weight, oestrous cycle, ano-genital distance, organ weights and terminal fasting body weight were analyzed using built-in statistical tests.
Derived data like net body weight change, food consumption, ano-genital ratio, oestrous cycle length, organ weight ratios, post implantation loss (%), number of nipples/areolae in male pups, no. of implantations, pre-coital interval, mean litter size, sex ratio and gestation length (days), mating, fertility and survival indices were also analyzed using above mentioned methods.
Hormone data was analyzed using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test was found to be significant. For two groups, the comparisons of mean between treatment and control group was done using Student’s ‘t’ test.
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated throughout the report as stated below:
*: Significantly different from the vehicle control group.
Reproductive indices:
Male mating index (%) = (Number of males with evidence of mating / Number of males cohabited) x 100
Male fertility index (%) = (Number of males siring a litter or impregnated a female / Number of males cohabited) x 100
Female mating index (%) = (Number of females mated / Number of females cohabited) x 100
Female fertility index (%) = (Number of pregnant females / Number of females used for mating ) x 100
Mean number of implantations or group = (Total number of implantations / Total number of pregnant animals) x 100
Post implantation loss (%) = ( (Number of implantations - Number of live pups) / Number of implantations ) x 100

Offspring viability indices:
Mean litter size per group = Total Number of pups born / Total Number of littered animals
Mean viable litter size = No. of viable pups / No. of females littered
Live birth index (%) = (No. of viable pups born (at first observation) / Total no. of pups born (at first observation) ) x 100
Day 4 survival index (%) = (Number of viable pups on lactation Day 4 / Number of viable pups born ) x 100
Sex Ratio (%) = (No. of male pups born / Total no. of pups born ) x 100
Ano-genital Distance Ratio (mm/g1/3 ) = Ano-genital distance / Cube root of body weight
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At 125 mg/kg/day, clinical sign of hypoactivity, piloerection, weakness and nasal discharge was observed in one female rat (Rx6394) Day 46 (LD7). The microscopic findings of congestion in adrenals, lymphoid depletion in thymus were noted in this animal. Lymphoid depletion in thymus was a secondary finding associated with stress. However, the cause of moribundity could not be determined based on the gross or microscopic findings.

At 500 mg/kg/day, transient clinical sign of slight salivation was observed soon after the dose administration in all animals (males and females) from treatment Day 4. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal. In addition hypoactivity, piloerection (slight) and vaginal discharge was observed in one female rat (Rx6440) on treatment Day 44 (LD 3). Subsequently, this female rat was found dead on Day 45 (LD4). Grossly, this animal showed nodules on the durface of heart, lung and thymus.
There were no clinical signs or mortalities observed during the treatment at 250 mg/kg bwt/day dose group in either sex.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female rat (Rx6394) dosed at 125 mg/kg was sacrified on moribund on lactation Day 46 (LD7). The cause of moribundity could not be determined based on the gross or microscopic findings.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weights and body weight gains were unaffected throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group.
Treatment had no effects on the maternal body weights measured during different intervals of the gestation period at all the tested doses when compared to vehicle control.
Treatment had no effects on the maternal body weights during measured different intervals of the lactation period at all the doses when compared to the vehicle control. Incidence of significantly lower body weight gain during days 4-13 was observed at 250 mg/kg/day. This statistically significant difference observed in absolute weight gain was toxicologically not significant as the total absolute weight gain from LD 0-13 was comparable to vehicle control.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The food consumption was unaffected throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group. Incidence of significantly lower food consumption during days 1-8 in females at 500 mg/kg/day was observed. This statistically significant difference observed in food consumption was toxicologically not significant as the mean body weights were not altered by the treatment.

Treatment had no effects on the maternal food consumption measured during different intervals of the gestation period at all the tested doses when compared to vehicle control.

Treatment had no effects on the maternal food consumption during measured different intervals of the lactation period at all the doses when compared to the vehicle control.
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):
There were no test item related changes in the T4 and TSH levels in Dams as well as in pups on Lactation Day 4 and Lactation Day 13.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item related microscopic changes. The observed findings were considered incidental/spontaneous and not related to test item administration.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks during the treatment period, prior to cohabitation with males.
The calculated mean oestrous cycle length was 4.7, 4.8, 4.5 and 4.6 days in vehicle control, low, mid and high dose groups, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the mean pre-coital time, gestation length (average days to litter), number of pregnancies and number of dams littered. No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested.
No test item-related changes were observed in the number of implantations and percentage of post implantation loww at all the tested doses when compared to vehicle control
Key result
Dose descriptor:
NOEL
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Test item had no treatment-related effects on the number of dead pups at first observation. There were no external abnormalities in live or dead pups in any of the groups. The mean total number of pups born and number of live pups on Day 0, 1, 4 and 13 were comparable to vehicle control at all the tested doses.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No treatment-related changes were observed in the survival data of pups up to LD 4 at all the tested doses.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The litter mean pup body weight of male, female and combined sex pups per litter were not affected by the treatment at all the tested doses.
Food consumption and compound intake (if feeding study):
not examined
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
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No changes attributable to the test item were detected in the Ano-genital distance and Ano-genital ratio in either sex.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The male pups did not exhibit areola/nipple retention on PND 13.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no effects on the thyroid hormone profile, terminal body weights/organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item related gross findings in pups.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no test item related histopathology findings in pups.
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Analytical verification


The results were considered acceptable, as the percent agreement of the analyzed concentrations were in the range, 85% to 115% of the claimed concentrations and the overall %RSD from six replicates at each dose level were <=10.0%.

Conclusions:
Parameters up to and including 500 mg/kg bw/day, the No Observed Effect Level (NOEL) for Reproduction/Developmental Toxicity Screening Test for the test substance is determined to be 500 mg/kg bw/day under the test conditions and doses employed.
Executive summary:

The daily oral (gavage) administration of the test item to Wistar rats at the dose levels of 125, 250 and 500 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, food consumption, pre-coital time, gestation length, mating and fertility parameters. Male and female reproductive organs did not reveal any changes. There were no treatment-related effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

At 500 mg/kg/day, transient clinical sign of slight salivation was observed soon after the dose administration in all animals from treatment Day 2-5. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal. The incidence of post-dosing salivation observed in 500 mg/kg/day, indicating that it was caused by the administration of test item. This finding may be attributed to oral mucosa irritation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity. One female rat (Rx6440) was found dead on LD 3. All the pups for this dam were found dead during LD 0-3. Grossly, this animal showed nodules on the surface of heart, lung and thymus. Microscopically, suppurative inflammation involving all these organs and uterus was noted. The systemic inflammation was related to the cause of death. One female rat (Rx6394) was sacrificed moribund on LD7 at 125 mg/kg/day. The microscopic findings of congestion in adrenals, lymphoid depletion in thymus were noted in this animal. Lymphoid depletion in thymus. was a secondary finding associated with stress. However, the cause of moribundity could not be determined based on the gross or microscopic findings

Treatment did not induce any test item-related adverse changes with respect to terminal fasting body weights, organ weights/ratios (including reproductive organs) and thyroid hormone profile. There were no test item related gross and microscopic changes findings at all the tested doses in either sex. 


 


Parameters up to and including 500 mg/kg bw/day, the NOEL for Reproduction/DEvelopmental Toxicity Screening Test for the test substance is determined to be 500 mg/kg bw/day under the test conditions and doses employed.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP-compliant study, according to OECD guideline
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Dose-Range finding study


A 14-day Repeated Dose toxicity study by oral gavage in Wistar rats (K1; Malleshappa, 2020) is conducted as Dose-Range Finding study (50/100, 150/300, 300/600 and 600/1000 mg/kg/day). At 1000 mg/kg/day, the clinical signs of toxicity included, salivation (slight to moderate) and reddish colour nasal discharge in males and slight salivation in females was observed in most of the animals from 9th day of treatment. At 600 mg/g/day, clinical sign of slight salivation was observed from 10th day of treatment period. At 1000 mg/kg/day, the body weight and body weight gains were lower, associated with decrease in the food consumption in males. Decrease in the total leukocyte count in males and lymphocyte count in both sexes at 1000 mg/kg/day. Increased neutrophil count at ≥ 600 mg/kg/day in females was observed. However, microscopic examination of hematopoietic organs did not reveal any associated morphological changes. The treatment did not induce any test item-related changes in coagulation parameters at all the tested doses in either sex. Clinical chemistry parameters analysis revealed, increase in blood glucose concentration at 1000 mg/kg/day in females. Organ weights showed decrease in absolute and relative weights of thymus associated with microscopic correlate of decreased cellularity of thymus in both sexes at 1000 mg/kg/day. Grossly, depressed foci in non-glandular tissue of stomach were observed in males at 1000 mg/kg/day and was microscopically confirmed as non-glandular epithelial hyperplasia. Microscopically, vacuolation of tubular epithelium in kidneys, duct epithelium of sub-mandibular and parotid salivary glands, acini of sublingual salivary glands, uterine glandular epithelium, glandular epithelium of stomach and ependymal cells of choroid plexus in brain were observed at 1000 mg/k/day. However, the stomach lesions observed in 1000 mg/kg/day were likely to be caused by the corrosivity/irritation of the test item. Based on these observations, the Maximum Tolerated Dose is considered to be 1000 mg/kg/day in the Reproduction / Developmental Toxicity Screening Study in Wistar rats. 


Screening for reproductive / developmental toxicity:


The purpose of this Reproduction / Developmental Toxicity Screening Study in Wistar rats by oral gavage was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provided initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The test item was dissolved Milli-Q and administered to rats at the graduated dose levels of 125, 250 and 500 mg/kg bwt/day to low dose (G2), mid dose (G3) and high dose (G4) group rats, respectively. The rats in the vehicle control group (G1) group received vehicle Milli-Q alone. The dose volume administered was 10 mL/kg body weight. Each group consisted of 10 male and 10 female rats. The dose formulations were administered once daily to a specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females. The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not done at the test facility. The stability and homogeneity of test item in the vehicle was carried out separately under Eurofins Advinus Study No. G19071 at 1 and 100 mg/mL concentrations. Based on the results, the test item was found to be stable for up to 24 hours when stored at room temperature. During the conduct of this study, the prepared dose formulations and vehicle were analyzed for concentration of the test item on Day 1 and during 2nd month (Day 42) of treatment. The results indicated that the overall mean concentration of test item in the tested formulation was found to be within ± 15% of the targeted concentration with the relative standard deviation (RSD) less than 10%. This indicates that the prepared dose formulation met the acceptance criteria for concentration and % RSD. All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly interval except during the cohabitation period. All dams were weighed on Gestation Days (GD) 0, 7, 14 and 20 and on Lactation Days (LD) 0, 4, 7 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams on LD 14 (at termination). The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected. Tissues collected from all animals in the control and high dose groups and the found dead animal (Rx6440) were examined microscopically for histopathological changes including qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure in testes. Gross lesions were examined in all the animals. The reproductive organs were examined from Rx6405 (male failed to mate), Rx6380 (non-pregnant)and Rx6375 (pregnant not littered), Rx6394(unscheduled sacrifice on LD 7) and Rx6440 (found dead on LD 3).The remaining tissues from the lower dose groups were not examined as no test item related changes were noted at high dose.


Under the experimental conditions employed, the following results were obtained:


Clinical signs and Mortality: There were no clinical signs observed at all the tested doses. The clinical sign of salivation (slight) was observed at high dose. The incidence of post-dosing salivation observed in 500 mg/kg/day, indicating that it was caused by the administration of test item. This finding may be attributed to oral mucosa irritation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity. One female rat (Rx6440) in 500 mg/kg/day was found dead on LD 3. Grossly, this animal showed nodules on the surface of heart, lung and thymus. Microscopically, suppurative inflammation involving all these organs and uterus was noted. The systemic inflammation was related to the cause of death. One female rat (Rx6394) was sacrificed moribund on LD7 at 125 mg/kg/day. The microscopic findings of congestion in adrenals, lymphoid depletion in thymus were noted in this animal. Lymphoid depletion in thymus was a secondary finding associated with stress. However, the cause of moribundity could not be determined based on the gross or microscopic findings.


Body weights: The mean body weights and total body weight gains were unaffected by the treatment at all the tested doses in both sexes.


Food consumption: Treatment did not affect the food consumption at all the tested doses in either sex.


Maternal body weights and food consumption: The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the tested doses.
Fertility parameters: Treatment had no effect on pre-coital time or gestation length, oestrous cycle length, mating and fertility parameters in both sexes.
Hormone analysis: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats remained unaffected by test item administration.
Terminal fasting body weights, organ weights and its ratios: The terminal body weights were not affected by test item administration. There were no test item related changes in the organ weights and their ratios to body weights. 


Gross and histopathology: There were no test item related gross and microscopic changes findings at all the tested doses in either sex.


No Observed Adverse Effect Level (NOAEL): As there were no treatment related effects on reproduction and fertility parameters up to and including 500 mg/kg bwt/day, the No Observed Effect Level (NOEL) for Reproduction Screening Test for test item is determined to be 500 mg/kg bwt/day under the test conditions and doses employed.


 


EOGRTS


The extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity. According to column 1 of annex IX of REACH, an EOGRTS should only be performed in the annex IX tonnage band if the available repeated dose toxicity studies (e.g. 28-day or 90-day studies, OECD 421 or 422 screening studies) indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity. No adverse effects on reproductive organs or tissues, and no other concerns in relation to reproductive toxicity were observed in the available, reliable studies. 


 


 


 

Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity study in rats was performed according to OECD guideline 414 and according to GLP requirements (Ramesh, 2022). The NOAEL for fetal developmental toxicity was considered to be 400 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

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
Reason / purpose for cross-reference:
reference to other study
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
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.
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
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
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
medium dose group
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.
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.
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
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
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
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
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
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
Key result
Abnormalities:
no effects observed
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
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.
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
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP-compliant study, performed according to OECD guideline
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Dose range finder study, prior to OECD414


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. A total of 28 Day 0 pregnant rats were randomly distributed into different groups: 0, 400, 800, 1000 mg/kg/day. The day of confirmed mating (sperm positive vaginal smear or presence of vaginal plug) was designated as GD 0. 


The characterisation of the test item was provided by the study Sponsor by a Certificate of Analysis (CoA) and TIDS. The stability and homogeneity of the test item in the vehicle was established under Study No. G24817 at concentrations of 1 and 100 mg/mL. Based on the results, the test item was stable in the vehicle for 48 hours when stored at room temperature at both the concentrations.
The following parameters and end points were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival, number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, observations]. In addition, from each dam the thyroids were weighed, and thyroid hormones were estimated from the blood collected at terminal sacrifice (GD20). Thyroid tissue was preserved including gross lesions which was not subjected to histopathological examination. 


Results of the study are summarized below:
• Mortality: There were 5/7 treatment related mortalities between gestation days 9 to 17 at the highest dose of 1000 mg/kg/day. In the lower dose groups, all animals survived to planned termination.


• Clinical signs: There were no clinical signs of toxicity at 400 or 800 mg/kg/day. 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.
• Maternal Parameters: At 1000 mg/kg/day, only 2 dams survived till termination, hence the maternal parameters calculated were not considered for any discussion. At 800 mg/kg/day, the mean maternal body weight during GD 12 to 20 was signfiacntly 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. The lower body weights were associated with significantly lower overall mean food consumption during GD 6 to 20 (-25%) and GD 0 to 20 (-17%). At 400 mg/kg/day, the mean body weight, and body weight gain and food consumption was comparable to the control. The other 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. However, there was a significantly lower uterine weight at 800 mg/kg/day due to lower litter size and fetal weight. Gross evaluation of placenta revealed no remarkable findings and there were no macroscopic findings at any dose level.


• Gross Pathology: Gross pathology examination of unscheduled deaths and moribund sacrificed animals at 1000 mg/kg/day revealed test item related changes such as non-glandular stomach mucosa; Focus; depressed (2/7) or stomach enlarged (1/7), thymus small (3/7) or adernals enlarged (2/7). 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).


• Litter Parameters: At 800 mg/kg/day, the mean litter size was significantly lower (-19%) and non-significant lower mean fetal weights (by -7% in males; by -9% in females and by -8% in combined sex). No significant changes in sex ratio or anogenital distance were observed at any dose level.
• Fetal examination: The fetal external examination revealed no signs of malformations at any of the tested doses.
• Thyroid Hormone Levels: Lower T4 hormone levels at 800 mg/kg/day were considered treatment related without any consistent/associated findings in TSH and T3 levels.
• Thyroid gland weights and Histopathology: The thyroid weight changes were not dose related and hence no treatment effect was suspected, hence histopathological evaluation of thyroid tissue was not performed.


In conclusion, 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 treatmentrelated 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 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


Prenatal development study in rats as first species (OECD414)


The objective of th study was to evaluate the developmental and maternal toxicity in pregnant female Wistar rats and developing embryos/fetuses after oral gavage treatment with the test substance from gestation day (GD) 6 to 19 (Ramesh, 2022).
This study was performed to provide a rational basis for risk assessment in humans and to establish a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rats.
The dose levels for this study were selected based on Dose Range Finding study (DRF). Dose levels of 400, 800 and 1000 mg/kg/day were evaluated in the DRF study, Study No. N6327. In the study, a total of 96 Day 0 pregnant rats were randomly divided into different groups : 0 (vehicle), 200 (low dose), 400 (mid dose) and 800 (high dose) mg/kg bw/day. The day of confirmed mating (sperm positive vaginal smear or presence of vaginal plug) was designated as GD 0.


The characterization details of the test item was provided by the study Sponsor
by a Certificate of Analysis (CoA) and Test Item Data Sheet (TIDS). The stability and homogeneity of the test item in the vehicle was established under Study No. G24817 at concentrations of 1 and 100 mg/mL. The test item was found to be stable in the vehicle for 48 hours when stored at room temperature at both the concentrations.
During the conduct of the experiment, homogeneity and active ingredient analysis was carried out from the dose formulation samples collected prior to initiation of treatment and 2 days before termination of treatment. The results of analysis of formulations were within the acceptable limits of 80-120 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 20 %.


The following parameters and endpoints were evaluated in the study: maternal parameters (mortality, clinical signs, body weight, body weight gain, food consumption, gross pathology, gravid uterine weight, number of corpora lutea, implantation sites, early and late resorptions, pre and post implantation loss) and fetal parameters (total number of live fetuses, total number of dead fetuses, sex ratio, fetal weight, anogenital distance, and external, visceral and skeletal observations). Approximately half the number of the fetuses from each litter were examined for visceral malformations and variations and the remaining half were evaluated for skeletal malformations and variations. In addition, from each dam, the thyroid and liver were weighed, and thyroid gland subjected to microscopic evaluation. Thyroid hormones levels were determined from the blood samples collected at terminal sacrifice (on GD 20). Main findings from the study are summarized below: 
• Mortality, clinical signs, and gross necropsy changes: There were no unscheduled deaths. Treatment related clinical signs of discolouration pink of tail (7/24) and ears (12/24) were observed at 800 mg/kg/day. Grossly at necropsy, tail discolouration (1/24 and 3/24 at 400 and 800 mg/kg/day, respectively) and ear discolouration (3/24) at 800 mg/kg/day observed was without any histological correlates.
• Body weight and food consumption: At 800 mg/kg/day, treatment related lower overall mean maternal body weight gains were noted during GD 6 to 20 (-59%) and GD 0 to 20 (-47%) with associated lower food consumption. The body weight corrected to gravid uterine weight and gains were also lower at 800 mg/kg/day by -18% and -143%, respectively. At 400 and 200 mg/kg/day, 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 and these decreases were below 10% at 200 mg/kg/day and marginally over 10% at 400 mg/kg/day and considered treatment related non-adverse effects. The food consumption at 200 mg/kg/day was comparable to the vehicle control group, while at 400 mg/kg/day the food consumption was lower during the initial part of treatment (GD 6 to 12) which reversed back to normal food consumption and hence considered treatment related nonadverse effects.
• Maternal developmental parameters: At 800 mg/kg/day, statistically significant decreases in mean uterine weights associated with lower litter weights were noted. Other maternal parameters, namely implantations, early and late resorptions and post-implantation loss were comparable across the tested doses. No gross pathological changes in the placenta were
observed at any dose level. 


 • Litter Parameters: The total number of fetuses were 350, 324, 337 and 315 at 0, 200, 400 and 800 mg/kg/day dose groups, respectively with the mean litter size in a range 14.3 to 15.2 per female, with no treatment-related or statistically significant differences present. No dead fetuses were observed in any of the test item treated groups. No significant changes in sex ratio were observed. Significant decreases in male and female fetal weights were observed at 800 (15 to 16%) mg/kg/day dose group and these effects were attributed secondary to maternal lower body weight gains and food consumption.
• Thyroid hormone levels (T3, T4 and TSH), thyroid and liver weights, and thyroid histology: No test item related changes in serum T3, serum T4, serum TSH were observed at any dose level. Lower thyroid and liver weights were observed at 800 mg/kg/day and considered as secondary effects associated with the test item related decreased body weights. Thyroid histology, no microscopic changes were observed at any dose level.


• Fetal examination: Fetal morphology (external, visceral and Skeletal) were 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.


 

Toxicity to reproduction: other studies

Description of key information

No other studies available

Additional information

No other studies available

Justification for classification or non-classification

Based on the available data and according to the CLP criteria, the test substance should not be classified as toxic to reproduction.

Additional information