Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from an experimental study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
According to OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylenediamine, salt with phosphoric acid
EC Number:
238-914-9
EC Name:
Ethylenediamine, salt with phosphoric acid
Cas Number:
14852-17-6
Molecular formula:
C2H8N2.xH3O4P
IUPAC Name:
ethane-1,2-diamine; phosphoric acid
Test material form:
solid: crystalline
Remarks:
White crystals
Details on test material:
Name (as per report): Ethylenediamine, salt with phosphoric acid
CAS No.: 14852-17-6
Molecular Weight: 158.093 g/mol
Molecular Formula: C2-H8-N2.x-H3-O4-P
SMILES: C(CN)N.P(=O)(O)(O)O
Purity: 98.04%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) x 10 wks
- Weight at study initiation: (P) Males: 225.03 g to 282.17 g; Females: 200.24 g to 234.48 g
- Fasting period before study: No
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
i) Pre-Mating: Two animals of same sex and group per cage were housed.
ii) Mating: During mating, two animals (one male and one female) of same group were housed.
iii) Post mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: Healthy and young adult males and females were acclimatized for five days initially to experimental room conditions and observed for clinical signs once daily.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 to 22.9°C
- Humidity (%): 44 to 65%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle.
IN-LIFE DATES:
From:
To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled Water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulations were freshly prepared before dose administration on each treatment day. The required quantity of test item was weighed into a clean beaker and small volume of the vehicle was added into the beaker, mixed well using glass and transferred into measuring cylinder. Rinsing procedure was repeated until complete transfer of test item formulation into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of 3.75, 7.5 and 15 mg/mL of test item for low, mid and high dose groups respectively

DIET PREPARATION
- Rate of preparation of diet (frequency): No Data Available
- Mixing appropriate amounts with (Type of food): No Data Available
- Storage temperature of food: No Data Available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Distilled water was used as a vehicle.
- Concentration in vehicle: 3.75, 7.5 and 15 mg/mL of test item for low, mid and high dose groups respectively
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): No Data Available
- Purity: No Data Available
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation:
- Proof of pregnancy: The female was placed with the same male until pregnancy occurs by evidence of sperm in vaginal smear until two weeks have elapsed. Day ‘0’ pregnancy was confirmed by the presence of sperm in the vaginal smear.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: No Data Available
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: No Data Available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Method
Instrument : Agilent HPLC 1200 series with VWD detector
Column: C18, 250 × 4.6 mm, 5 µm
Flow rate: 1.0 mL/min
Injection volume: 20 µL
Oven temperature: 25ºC
Run time: 15 minutes
Wavelength: 190 nm

Mobile Phase: Milli-Q water: Acetonitrile (90:10 % v/v)
Diluent: Milli-Q water
Duration of treatment / exposure:
The males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 33 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).
Frequency of treatment:
The test item or vehicle was administered to animals through oral (gavage) route once daily.
Details on study schedule:
No Data Available
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group
Dose / conc.:
37.5 mg/kg bw/day (actual dose received)
Remarks:
Low Dose Group
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
Mid Dose Group
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group
No. of animals per sex per dose:
12 males and 12 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses of 0, 37.5, 75 and 150 mg/kg body weight for vehicle control, low dose, mid dose and high dose groups, respectively were selected based on the available literature (Ethylenediamine, Salt with Phosphoric Acid, End Point Summary, ECHA and OECD SIDS Initial Assessment Report, Ethylenediamine, Bern, Switzerland November 6-9, 2001).
- Rationale for animal assignment (if not random): The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20% (-15.14% to +15.31% for males and -12.50% to +8.41% for females) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analyzed statistically for mean body weight to rule out the statistical significant difference between groups within each sex.
- Fasting period before blood sampling for clinical biochemistry: Yes, all the animals were fasted before sampling of blood.
- Other: No Data Available
Positive control:
No Data Available

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity.
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All the animals were subjected to detailed clinical examinations on day 1 before treatment and weekly thereafter during treatment. These observations were made outside the home cage and preferably at the same time. Signs noted included, but not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period and on day 14 (fasting body weight).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, feed consumption was measured for all animals once a week during premating and once for males during post mating period. Feed consumption was not measured during mating period for both males and females. Thereafter, feed consumption for females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No Data Available

OTHER: No Data Available
Oestrous cyclicity (parental animals):
Oestrus cycles were monitored for two weeks after five days of acclimatization to evaluate its oestrus cyclicity (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa. Oestrus cyclicity was also monitored on the day of sacrifice for females.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: Testis weight and epididymis weight.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No Data Available
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. External Genitalia was observed.
GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No Data Available

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No Data Available
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, were sacrificed after completion of 33 days of treatment.
- Maternal animals: All surviving females were sacrificed on lactation day 14 and pups were sacrificed on lactation day 13.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [A] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
The pups were sacrificed on lactation day 13 and the sacrificed pups and dead pups were examined for gross abnormalities and the findings were recorded.
Statistics:
The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data. The statistical methods that were used were One-way ANOVA with Dunnett’s post test, Kruskal-Wallis Test and Chi-square test.
Reproductive indices:
Percent change in body weight (pre mating, mating, gestation and lactation periods), Copulatory interval, Gestation length, Corpora lutea per dam, Implantations per dam, No. of live/dead pups/dam, Pregnancy rate, Pre/post implantation loss, Pre/post natal loss, No. of resorptions per dam, Corpora lutea per dam, Implantations per dam, Litter size.
Offspring viability indices:
Anogenital distance ratio, Mean pup weight, Live birth Index, Pup survival index, No. of litters with/without resorptions, No. of dams with/without live young born and No. of dams with/without dead pups.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted at all the tested dose group [37.5, 75 and 150 mg/kg body weight/day] animals of either sex during the experimental period.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
There were no mortality/morbidity observed at all the tested dose group [37.5, 75 and 150 mg/kg body weight/day] animals of either sex during the experimental period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in mean body weight and percent change in body weight with respect to day 1 at all the tested dose group [37.5, 75 and 150 mg/kg body weight/day] animals of either sex during the experimental period when compared with vehicle control group. However, statistical significant decrease in percent change in body weight on day 7 and 14 with respect to day 1 at group G4 [150 mg/kg body weight/day] males and statistical significant decrease in percent change in body weight on day 7 with respect to day 1 at group G4 [150 mg/kg body weight/day] females was noted when compared with concurrent vehicle control group animals. This observation is considered as incidental and not treatment related as there was no effect on mean body weight and mean feed consumption during this period, and also the change is not consistent during the experimental period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no changes in mean feed consumption at all the tested dose group [37.5, 75 and 150 mg/kg body weight/day] animals of either sex when compared with vehicle control group during the experimental period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no treatment related changes observed in serum thyroxine hormone (T4) levels in adult males collected at termination and lactation day 13 pups at all the tested dose groups [37.5, 75 and 150 mg/kg body weight/day] when compared with vehicle control group.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related histopathological findings were noticed during microscopic examination conducted for epididymides, levator ani plus bulbocavernosus muscle complex, cowper’s glands, glans penis, prostate and seminal vesicles and coagulating glands, testes and thyroid along with parathyroid [in males]; ovaries, uterus with cervix and thyroid along with parathyroid [in females].
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings.
The observed finding like Interstitium, Mononuclear cells infiltrate prostate gland from group G4 [one male with minimal] and concretions in prostate gland from group G1 and G4 [one male with minimal from each group] considered as spontaneous and incidental.
The occurred finding like Placental Scar characterized by brown-pigmented nodules at the uterine-mesometrial boundary in uterus of all the pregnant females from group G1 and G4 were considered as spontaneous and incidental.
Reproductive organs and accessory sex glands, testes, epididymides, prostate, seminal vesicles with coagulating glands, cowper’s glands, levator ani plus bulbocavernosus muscle complex, glans penis [males] and ovaries and uterus with cervix [females] did not reveal any histopathological findings at high dose group [150 mg/kg].
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no effects noted in the oestrus cyclicity of females at all the tested dose groups [37.5 mg/kg, 75 mg/kg and 150 mg/kg] evaluated during pre-mating period, co-habitation period and also on the day of sacrifice (Lactation day 14).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings. No effects were observed in these examinations.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
A total of twelve pairs were started for mating from each group initially and among these a total of 11, 12, 12 and 11 females were confirmed with mating within 14 days of cohabitation period with the first male only. One female each from vehicle control and high dose groups was confirmed with mating after placing with proven male from same group.
The mating index for males was 91.7 %, 100.0 %, 100.0 % and 91.7 % for groups G1 (0 mg/kg body weight/day), G2 (37.5 mg/kg body weight/day), G3 (75 mg/kg body weight/day) and G4 (150 mg/kg body weight/day) respectively by considering 14 days of continuous pairing. The mating index for females was 100.0 % for all the vehicle control and tested dose groups (0, 37.5, 75 and 150 mg/kg body weight/day).
A total of 11, 11, 11 and 10 males from vehicle control [0 mg/kg body weight/day], low dose [37.5 mg/kg body weight/day], mid dose [75 mg/kg body weight/day] and high dose [150 mg/kg body weight/day] groups respectively, were found to be fertile with a fertility rate (with evidence of implantations in females) of 91.7%, 91.7%, 91.7% and 83.3%.
A total of 11, 11, 11 and 10 females from vehicle control [0 mg/kg body weight/day], low dose [37.5 mg/kg body weight/day], mid dose [75 mg/kg body weight/day] and high dose [150 mg/kg body weight/day] groups respectively, were found to be pregnant [with evidence of implantations] with a fertility rate of 91.7%, 91.7%, 91.7% and 83.3% and a total of 1, 1, 1 and 2 females from vehicle control [0 mg/kg body weight/day], low dose [37.5 mg/kg body weight/day], mid dose [75 mg/kg body weight/day] and high dose [150 mg/kg body weight/day] groups respectively, were confirmed as non-pregnant [with no evidence of implantations] at a percent of 8.3%, 8.3%, 8.3% and 16.7% were noted.
The females which were littered with live fetuses [11, 11, 11 and 10 females from vehicle control, low dose, mid dose and high dose groups respectively] only considered for mean calculations and statistical analysis for the following parameters.

Details on results (P0)

No test item related systemic and reproductive toxicity was was observed.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed in any of the pups at all the tested dose group [37.5, 75 and 150 mg/kg body weight/day] litters during lactation period observations.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no treatment related effects noted in pup survival index during lactation period, no changes were noted during daily observation of pups at all the tested dose group [37.5, 75 and 150 mg/kg body weight/day] litters when compared with vehicle control group litters.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes observed in mean pup (male and female) weight recorded on lactation day 1, 4, 7 and 13 at all the tested dose groups [37.5, 75 and 150 mg/kg body weight/day] when compared with vehicle control group. However, a slight reduction in mean pup weight was noted in both the sex at group G4 [150 mg/kg body weight/day]. This change cannot considered as adverse as there were no effects on mean ano-genital distance ratio was noted and also no abnormal findings noted in pups of either sex externally and viscerally.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no treatment related changes observed in serum thyroxine hormone (T4) levels in adult males collected at termination and lactation day 13 pups at all the tested dose groups [37.5, 75 and 150 mg/kg body weight/day] when compared with vehicle control group.
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects noted in pup ano-genital distance ratio of either sex recorded on lactation day 4 at all the tested dose groups [37.5, 75 and 150 mg/kg body weight/day] when compared with vehicle control group. However, statistical significant decrease in mean ano-genital distance ratio recorded on lactation day 4 at group G3 [75 mg/kg body weight/day] was noted when compared with vehicle control group. This observation is considered as incidental and not treatment related as there was no effect on pup weight during this period and also the effect was not occurred in dose dependent manner.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no occurrences of nipples in male pups at all the tested dose group litters [37.5, 75 and 150 mg/kg body weight/day] and also at vehicle control group litters observed on lactation day 13.
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological changes were noted during conduct of necropsy in any of the pups of either sex [dead and sacrificed on PND 4 and 13] from all the tested dose group [37.5, 75 and 150 mg/kg body weight/day] and vehicle control group litters.
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Based on all the observations and results, it was concluded that the NOAEL for the test chemical was 150 mg/kg bw.
Executive summary:

A reproductive and developmental toxicity screening test, according to OECD TG 421 (Reproductive/Developmental Screening Test) was performed using male and female Sprague Dawley rats. The test chemical was evaluated for possible adverse effects following repeated oral dosing to males for 33 days, to females for two weeks pre-mating period, during mating period, during pregnancy (gestation) and up to lactation day 13 to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, parturition, and early neonatal development. A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 12 males and 12 females. The animals in G1 group were administered with vehicle [distilled water], animals in G2, G3 and G4groups were administered with test item at dose levels of 37.5, 75 and 150 mg/kg body weight/day for low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 33 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed). All animals were observed for clinical signs of toxicity once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. The serum collected from adult males and lactation day 13 pups representing each per litter were screened for Thyroxine hormone (T4) levels. Females were observed for oestrus cyclicity during pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice. The females were observed for copulatory interval and all the adult animals were observed for mating and fertility index. Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. The dams were observed for body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index throughout the lactation period. The pups were observed for clinical signs and external examinations once daily from lactation day 1 to 13. The both male and female pup weights were recorded separately on lactation days 1, 4, 7 and 13. The ano-genital distance of each pup was measured on lactation day 4. The male pups were observed for retention of nipples/areolae on lactation day 13. Gross pathology and organ weighing were performed on day 34 for males and on lactation day 14 for dams. Gross pathology was performed on lactation day 4/13 for pups. The number of corpora lutea and implantation sites for dams were recorded during necropsy. All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed at all the tested dose group [37.5 mg/kg, 75 mg/kg and 150 mg/kg] animals of either sex during treatment period. No treatment related changes in body weight, percent change in body weight with respect to day 1 and feed consumption of either sex was noted during the treatment period. No treatment related changes were observed in organ weights (both absolute and relative) at all the tested dose groups of either sex. A total of twelve pairs were started for mating from each group initially and among these a total of 11, 12, 12 and 11 females were confirmed with mating within 14 days of cohabitation period with the first male only. One female each from vehicle control and high dose groups was confirmed with mating after placing with proven male from same group. The mating index for males was 91.7 %, 100.0 %, 100.0 % and 91.7 % for groups G1 (0 mg/kg body weight/day), G2 (37.5 mg/kg body weight/day), G3 (75 mg/kg body weight/day) and G4 (150 mg/kg body weight/day) respectively by considering 14 days of continuous pairing and for females 100.0 % for all the groups. A total of 11, 11, 11 and 10 males from vehicle control [0 mg/kg], low dose [37.5 mg/kg], mid dose [75 mg/kg] and high dose [150 mg/kg] groups respectively, were found to be fertile with a fertility rate (with evidence of implantations in females) of 91.7%, 91.7%, 91.7% and 83.3%. A total of 11, 11, 11 and 10 females from vehicle control [0 mg/kg], low dose [37.5 mg/kg], mid dose [75 mg/kg] and high dose [150 mg/kg] groups respectively, were found to be pregnant [with evidence of implantations] with a fertility rate of 91.7%, 91.7%, 91.7% and 83.3% and a total of 1, 1, 1 and 2 females from vehicle control [0 mg/kg], low dose [37.5 mg/kg], mid dose [75 mg/kg] and high dose [150 mg/kg] groups respectively, were confirmed as non-pregnant [with no evidence of implantations] at a percent of 8.3%, 8.3%, 8.3% and 16.7% were noted. Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weight and feed consumption during gestation and lactation at all the tested dose groups. All pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, ano-genital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group. There were no gross pathological changes noticed during conduct of necropsy in any of the adult animals of either sex at all the tested dose group [37.5 mg/kg, 75 mg/kg and 150 mg/kg] and vehicle control group animals during conduct of necropsy. No gross pathological changes were noted during conduct of necropsy in any of the pups of either sex [dead and sacrificed on PND4 and 13] from all the tested dose group [37.5 mg/kg, 75 mg/kg and 150 mg/kg] and vehicle control group litters. During histopathological examination, no treatment related histopathological findings noticed in the microscopic evaluation of collected reproductive organs and thyroid along with parathyroid at high dose [150 mg/kg] group males and females. Based on all the observations and results, it was concluded that the NOAEL for the test chemical was 150 mg/kg bw.