Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive Toxicity Study:

A reproduction /developmental toxicity screening test was performed to evaluate and assess the effects of the the test chemical, following repeated oral dosing to males for 30 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 chemical on male and female reproductive performance such as gonadal function, mating behaviour, 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 [corn oil], animals in G2, G3 and G4 groups were administered with test chemical at dose levels of 250, 500 and 1000 mg/kg body weight for low dose, mid dose 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 30 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 once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. Serum was collected from adult males and lactation day 13 pups representing each per litter were used for screening of 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 anogenital 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 31 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 except dark green colored fecal matter at all the tested dose group animals of either sex during treatment period which was due to dark green color of the test chemical and no mortality or morbidity was observed. No treatment related changes in body weight, per cent change in body weight with respect to day 1 and feed consumption of either sex was noted during the pre-mating and mating 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, all the females were confirmed with mating within 14 days of cohabitation period with the first male only. The mating index for the males and females was 100% for all the tested dose groups. A total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of confirmed pregnancy) of 83.3%, 91.7%, 83.3% and 100.0%; total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of siring a litter) of 83.3%, 91.7%, 83.3% and 100.0%. A total of 10, 11, 10 and 12 females from vehicle control, low dose, mid dose and high dose groups respectively, were found to be pregnant with a pregnancy rate of 83.3%, 91.7%, 83.3% and 100.0%; total of 2, 1, 2 and 0 females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-pregnant [no evidence of implantations] with a per cent of 16.7%, 8.3%, 16.7% and 0.03% during necropsy 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. During gross pathological examination, green colour appearance of tail and non-glandular stomach was observed at all the tested dose group males and green colour appearance of tail and non-glandular stomach in 42% of animals from G2 (250 mg/kg) females, 33% of animals from G3 (500 mg/kg) females and 83% of animals from G4 (1000 mg/kg) females was noted. These changes can not to be considered as adverse treatment related as the appearances were due to colour of the test chemical. There were no gross pathological lesions noticed at necropsy of pups (both live and dead).During histopathological examination, no treatment related histopathological findings noticed in the microscopic evaluation of collected reproductive organs and gross pathological tissues like, tail and non-glandular stomach. Thus, based on all the observations and results, it was concluded that the No Observed Adverse Effect Level (NOAEL) of the test chemical was found to be 1000 mg/kg bw.

Link to relevant study records
Reference
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 a study report.
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
The above study was performed to evaluate and assess the effects of the test chemical on the reproductive and developmental parameters of the Sprague Dawley rats.
GLP compliance:
yes
Specific details on test material used for the study:
Details on test material
- Molecular weight (if other than submission substance): No Data Available
- Substance type: Organic
- Physical state: Dark Green Liquid
- Impurities (identity and concentrations): 33.39% Pure (by GC)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Details on test animals and env. conditions
TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: (P) x 9 weeks
- Weight at study initiation: (P) Males: 236.64 g to 296.64 g; Females: 204.64 g to 260.61 g
- Fasting period before study: No Data Available
- 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 animals were acclimatized for nineteen days (including fourteen days of oestrus cycle evaluation) to experimental room conditions and females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment and were observed for clinical signs once daily.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 to 23.6oC
- Humidity (%): 40 to 69%,
- 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: 30 May 2018
To: 20 July 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
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 there by adding little volume of the vehicle into the beaker, mixed well using glass rod and transferred into measuring cylinder. This 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 25, 50 and 100 mg/mL of test item for low, mid and high dose groups respectively.
Chromatographic Conditions:
Column: BDS Hypersil C18 100 × 4.6 mm, 5 µm
Flow rate: 1.0 mL/min.
Injection volume: 10 µL
Wavelength: 254 nm
Run time: 16 minutes
Column temperature: 40°C

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): The test item was not miscible with distilled water and clearly miscible with corn oil at the concentration of 100 mg/mL. Hence corn oil was used as vehicle for test item formulation preparation. Also, corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: Concentration of 0, 25, 50 and 100 mg/mL of test item for low, mid and high dose groups respectively.
- Amount of vehicle (if gavage): 100 ml
- Lot/batch no. (if required): A1712001
- Purity: No Data Available
Details on mating procedure:
Details of mating
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: No Data Available
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: Every morning, the vaginal smear of each female was examined for presence of sperm in the vaginal smear. 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.
- Further matings after two unsuccessful attempts: [no / yes (explain)]: No
- After successful mating each pregnant female was caged (how): No Data Available
- Any other deviations from standard protocol: No Data Available

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 there by adding little volume of the vehicle into the beaker, mixed well using glass rod and transferred into measuring cylinder. This 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 25, 50 and 100 mg/mL of test item for low, mid and high dose groups respectively.
Chromatographic Conditions:
Column: BDS Hypersil C18 100 × 4.6 mm, 5 µm
Flow rate: 1.0 mL/min.
Injection volume: 10 µL
Wavelength: 254 nm
Run time: 16 minutes
Column temperature: 40°C
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 30 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
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
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control Group
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Low Dose Group
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Mid Dose Group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High Dose Group
No. of animals per sex per dose:
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.
Control animals:
yes, concurrent vehicle
Details on study design:
No Data Available
Positive control:
No Data Available
Parental animals: Observations and examinations:
Parental animals observation 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.

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
- Time Schedule: 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.

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 normal 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):
No Data Available
Litter observations:
The duration of gestation was recorded and calculated from day 0 of pregnancy. The day of littering was considered as lactation day 1. The number of pups born (dead and live) in a litter, sex, live births and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 were recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4) and the ratio of AGD to the cube root of pup body weight was calculated. All survived male pups were examined for appearance of nipples/areolae on postnatal day 13 (lactation day 13). The litter was observed daily in order to note the number of alive, dead and cannibalized pups. All the dead and sacrificed pups were examined and subjected to gross pathological examination. Fertility index for dams, sires and pup live birth index, mean litter size per group, survival index and sex ratio at birth were calculated.
Postmortem examinations (parental animals):
Postmortem examinations (Parent Animal):
SACRIFICE:
- Male animals: The males were sacrificed after completion of 30 days of treatment, females were sacrificed on lactation day 14 and pups were sacrificed on lactation day 13. The animals were fasted overnight, water was provided ad libitum during fasting. The next day, the body weight of all the fasted animals was recorded prior to exsanguination. During necropsy, the males were randomized.
- Maternal animals: The vaginal smear of females on the day of necropsy (lactation day 14) was performed and stage of oestrus cycle was recorded. The animals were euthanized using deep CO2 Asphyxiation and subjected to gross necropsy, external and internal gross pathological examination.

GROSS NECROPSY
- Gross necropsy consisted of Epididymides, Levator ani plus bulbocavernosus muscle complex, Cowper’s glands, Glans penis, ovaries, Prostate and Seminal vesicles and coagulating glands, Testes, Uterus with cervix, Thyroid along with parathyroid glands and other gross lesions in other organs of respiratory, digestive and nervous system were observed.

HISTOPATHOLOGY / ORGAN WEIGHTS: Detailed histopathological examination was performed on the ovaries, testes and epididymides of the animals from the control and the high dose group including all macroscopically abnormal tissues of all animals, sacrificed at termination. The histopathological examination was extended to lower dose groups for gross findings like, tail and non-glandular stomach to cover all gross lesions encountered. The tissues were embedded in paraffin wax, sectioned at 4 to 6 micrometers and stained with haematoxylin and eosin. The testes were sectioned at 3 to 4 micrometers and stained with Hematoxylin and Eosin stain and also with Periodic Acid-Schiff (PAS) and Hematoxylin stain. PAS stain aided spermatogenesis evaluation.
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. The thyroid along with parathyroid was collected from one male and one female pup per litter on lactation day 13. The thyroid along with parathyroid from adults and pups were preserved in 10% v/v Neutral Buffered Formalin. The thyroid along with parathyroid from adults was weighed post fixation. The histopathological examination of thyroid from pups and adults was not conducted as there were no treatment related effects noted in T4 levels of adult males and lactation day 13 pups.
Statistics:
The data was subjected to various statistical analyses using SPSS software version 22.
For Body weight, Percent change in body weight, Feed consumption, Copulatory interval, Gestation length, Organ weights, Anogenital distance ratio, Mean pup weight, Live birth Index, Pup survival index, One-way ANOVA with Dunnett’s post test was used. For, Pre/post implantation loss, Pre/post natal loss, No. of resorptions per dam, Corpora lutea per dam, Implantations per dam, No. of live/dead pups/dam, Sex ratio and Litter size, Kruskal-Wallis Test was used. For, Pregnancy rate, No. of litters with/without resorptions, No. of dams with/without live young born, No. of dams with/without dead pups , Chi-square test was used.
Reproductive indices:
Male Mating Index, Male Fertility Index, Female Mating Index, Female Fertility Index, Precoital Interval (Days), Mean Gestational Length, Gestation Index (%), Mean number of corpora lutea, Mean number of Implantations, Pre-Implantation Loss (%), Pre-natal Loss/Post-Implantation Loss (%), Pre-natal Loss (No.), Post-natal Loss on lactation day 4/13, Post-natal Loss on lactation day 4/13 (%)
Offspring viability indices:
Mean Litter Size per Group, Live Birth Index (%) per dam, Precoital Interval (Days), Pup Survival index (%) on lactation day 4, Sex ratio, Percentage of male/female offspring (%), Ano-genital Distance to cube root of the body weight Ratio (AGD ratio).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The detailed clinical examination of animals did not reveal any changes at any of the tested dose group animals of either sex during the experimental period. There were no treatment related clinical signs of toxicity at any of the tested dose group animals of either sex during the experimental period. However, dark green colored fecal matter was observed at all the tested dose group animals. This coloration is due to dark green color of the test item but not any adverse effects of test chemical.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
There were no treatment related mortality/morbidity observed at any of the tested dose group animals of either sex during the experimental period.
Body weight and weight changes:
no effects observed
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 doe groups of either sex during pre-mating and mating periods when compared with vehicle control group was noted.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in feed consumption at all the tested doe groups of either sex during pre-mating and mating periods when compared with vehicle control group was noted. However, statistical significant reduction in average feed consumption during week 2 of pre-mating period at all the tested dose group females and statistical significant reduction in average feed consumption during week 2 of pre-mating period at G2 (250mg/kg) and G3 (500 mg/kg) males was noted when compared with vehicle concurrent control group. These changes may not to be considered as treatment related as the reduction is not consistent 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:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in serum T4 levels in adult males and lactation day 13 pups at any of the tested dose groups when compared with vehicle control group. However, statistical significant increase in serum T4 levels in adult males was noted at G2 (250 mg/kg) and G3 (500 mg/kg) was noted when compared with vehicle control group males. This change is not considered as treatment related as the change is not dose dependant
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 noticed in the microscopic evaluation of collected reproductive organs. Neutrophils infiltration at submucosa of non-glandular stomach was observed in one male from G4 (1000 mg/kg) group. This lesion considered as spontaneous, incidental because of lack of consistency. One male from vehicle control group revealed cytoplasmic vacuolation in Sertoli cells of testes. This lesion considered as spontaneous and incidental. 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 histopathological examination was extended to lower dose groups for gross findings like, tail and non-glandular stomach. There were no treatment related changes noted in these tissues at all the dose groups during microscopic evaluation. Reproductive organs (Testes, Epididymides and Ovaries) did not show any pathological findings in G4 (1000 mg/kg) group animals.

Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no changes observed in the oestrus cyclicity at any of the tested dose group females during pre-mating treatment, mating treatment and on lactation day 14.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
A total of twelve pairs were started for mating from each group, all the females were confirmed with mating within 14 days of cohabitation period with the first male only. The mating index for the males and females was 100% for all the tested dose groups. A total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of confirmed pregnancy) of 83.3%, 91.7%, 83.3% and 100.0%; total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of siring a litter) of 83.3%, 91.7%, 83.3% and 100.0%. There were no treatment related changes observed in the copulatory interval and number of conceiving days at any of the tested dose group animals when compared with vehicle control group animals.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive performance
Remarks on result:
other: Not Specified
Critical effects observed:
not specified
System:
other: Not Specified
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs and external anomalies observed in any of the pups of tested dose group animals during lactation period.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no treatment related changes observed in pup survival index of each litter at any of the tested dose group animals during lactation period when compared with vehicle control group animals.
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 on lactation day 1, 4, 7 and 13 at any of the tested dose groups when compared with vehicle control group dams. However, statistical significant reduction in mean male pup weight on lactation day 7 at all the tested dose groups; statistical significant reduction in mean female pup weight on lactation day 7 and 13 at G2 (250 mg/kg) and G3 (500 mg/kg) was noted when compared with vehicle control group. This observation was not considered as treatment related as the occurrences were not consistent, no changes were noted in pup observations during this period and also the changes are not dose dependent.
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:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in serum T4 levels in adult males and lactation day 13 pups at any of the tested dose groups when compared with vehicle control group. However, statistical significant increase in serum T4 levels in adult males was noted at G2 (250 mg/kg) and G3 (500 mg/kg) was noted when compared with vehicle control group males. This change is not considered as treatment related as the change is not dose dependant and no changes were noted in absolute and relative weight of thyroid in these groups.
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no changes observed in ano-genital distance ratio of both male and female pups recorded on lactation day 4 at any of the tested dose group litters when compared with vehicle control group litters. There were no occurrences of nipples in male pups of dams at any of the tested dose group litters and vehicle control group litters observed on lactation day 13.
Histopathological findings:
no effects observed
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
haematology
clinical biochemistry
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Not Specified
Critical effects observed:
not specified
System:
other: Not Specified
Reproductive effects observed:
not specified
Treatment related:
not specified
Conclusions:
Based on all the observations and results, it was concluded that the No Observed Adverse Effect Level (NOAEL) of the test chemical was found to be 1000 mg/kg bw.
Executive summary:

A reproduction /developmental toxicity screening test was performed to evaluate and assess the effects of the the test chemical, following repeated oral dosing to males for 30 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 chemical on male and female reproductive performance such as gonadal function, mating behaviour, 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 [corn oil], animals in G2, G3 and G4 groups were administered with test chemical at dose levels of 250, 500 and 1000 mg/kg body weight for low dose, mid dose 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 30 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 once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. Serum was collected from adult males and lactation day 13 pups representing each per litter were used for screening of 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 anogenital 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 31 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 except dark green colored fecal matter at all the tested dose group animals of either sex during treatment period which was due to dark green color of the test chemical and no mortality or morbidity was observed. No treatment related changes in body weight, per cent change in body weight with respect to day 1 and feed consumption of either sex was noted during the pre-mating and mating 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, all the females were confirmed with mating within 14 days of cohabitation period with the first male only. The mating index for the males and females was 100% for all the tested dose groups. A total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of confirmed pregnancy) of 83.3%, 91.7%, 83.3% and 100.0%; total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of siring a litter) of 83.3%, 91.7%, 83.3% and 100.0%. A total of 10, 11, 10 and 12 females from vehicle control, low dose, mid dose and high dose groups respectively, were found to be pregnant with a pregnancy rate of 83.3%, 91.7%, 83.3% and 100.0%; total of 2, 1, 2 and 0 females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-pregnant [no evidence of implantations] with a per cent of 16.7%, 8.3%, 16.7% and 0.03% during necropsy 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. During gross pathological examination, green colour appearance of tail and non-glandular stomach was observed at all the tested dose group males and green colour appearance of tail and non-glandular stomach in 42% of animals from G2 (250 mg/kg) females, 33% of animals from G3 (500 mg/kg) females and 83% of animals from G4 (1000 mg/kg) females was noted. These changes can not to be considered as adverse treatment related as the appearances were due to colour of the test chemical. There were no gross pathological lesions noticed at necropsy of pups (both live and dead).During histopathological examination, no treatment related histopathological findings noticed in the microscopic evaluation of collected reproductive organs and gross pathological tissues like, tail and non-glandular stomach. Thus, based on all the observations and results, it was concluded that the No Observed Adverse Effect Level (NOAEL) of the test chemical was found to be 1000 mg/kg bw.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data is from Klimisch 1 source which is a study report.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproductive Toxicity Study:

A reproduction /developmental toxicity screening test was performed to evaluate and assess the effects of the the test chemical, following repeated oral dosing to males for 30 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 chemical on male and female reproductive performance such as gonadal function, mating behaviour, 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 [corn oil], animals in G2, G3 and G4 groups were administered with test chemical at dose levels of 250, 500 and 1000 mg/kg body weight for low dose, mid dose 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 30 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 once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. Serum was collected from adult males and lactation day 13 pups representing each per litter were used for screening of 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 anogenital 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 31 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 except dark green colored fecal matter at all the tested dose group animals of either sex during treatment period which was due to dark green color of the test chemical and no mortality or morbidity was observed. No treatment related changes in body weight, per cent change in body weight with respect to day 1 and feed consumption of either sex was noted during the pre-mating and mating 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, all the females were confirmed with mating within 14 days of cohabitation period with the first male only. The mating index for the males and females was 100% for all the tested dose groups. A total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of confirmed pregnancy) of 83.3%, 91.7%, 83.3% and 100.0%; total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of siring a litter) of 83.3%, 91.7%, 83.3% and 100.0%. A total of 10, 11, 10 and 12 females from vehicle control, low dose, mid dose and high dose groups respectively, were found to be pregnant with a pregnancy rate of 83.3%, 91.7%, 83.3% and 100.0%; total of 2, 1, 2 and 0 females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-pregnant [no evidence of implantations] with a per cent of 16.7%, 8.3%, 16.7% and 0.03% during necropsy 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. During gross pathological examination, green colour appearance of tail and non-glandular stomach was observed at all the tested dose group males and green colour appearance of tail and non-glandular stomach in 42% of animals from G2 (250 mg/kg) females, 33% of animals from G3 (500 mg/kg) females and 83% of animals from G4 (1000 mg/kg) females was noted. These changes can not to be considered as adverse treatment related as the appearances were due to colour of the test chemical. There were no gross pathological lesions noticed at necropsy of pups (both live and dead).During histopathological examination, no treatment related histopathological findings noticed in the microscopic evaluation of collected reproductive organs and gross pathological tissues like, tail and non-glandular stomach. Thus, based on all the observations and results, it was concluded that the No Observed Adverse Effect Level (NOAEL) of the test chemical was found to be 1000 mg/kg bw.

Effects on developmental toxicity

Description of key information

Developmental Toxicity Study:

A reproduction /developmental toxicity screening test was performed to evaluate and assess the effects of the the test chemical, following repeated oral dosing to males for 30 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 chemical on male and female reproductive performance such as gonadal function, mating behaviour, 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 [corn oil], animals in G2, G3 and G4 groups were administered with test chemical at dose levels of 250, 500 and 1000 mg/kg body weight for low dose, mid dose 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 30 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 once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. Serum was collected from adult males and lactation day 13 pups representing each per litter were used for screening of 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 anogenital 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 31 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 except dark green colored fecal matter at all the tested dose group animals of either sex during treatment period which was due to dark green color of the test chemical and no mortality or morbidity was observed. No treatment related changes in body weight, per cent change in body weight with respect to day 1 and feed consumption of either sex was noted during the pre-mating and mating 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, all the females were confirmed with mating within 14 days of cohabitation period with the first male only. The mating index for the males and females was 100% for all the tested dose groups. A total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of confirmed pregnancy) of 83.3%, 91.7%, 83.3% and 100.0%; total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of siring a litter) of 83.3%, 91.7%, 83.3% and 100.0%. A total of 10, 11, 10 and 12 females from vehicle control, low dose, mid dose and high dose groups respectively, were found to be pregnant with a pregnancy rate of 83.3%, 91.7%, 83.3% and 100.0%; total of 2, 1, 2 and 0 females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-pregnant [no evidence of implantations] with a per cent of 16.7%, 8.3%, 16.7% and 0.03% during necropsy 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. During gross pathological examination, green colour appearance of tail and non-glandular stomach was observed at all the tested dose group males and green colour appearance of tail and non-glandular stomach in 42% of animals from G2 (250 mg/kg) females, 33% of animals from G3 (500 mg/kg) females and 83% of animals from G4 (1000 mg/kg) females was noted. These changes can not to be considered as adverse treatment related as the appearances were due to colour of the test chemical. There were no gross pathological lesions noticed at necropsy of pups (both live and dead).During histopathological examination, no treatment related histopathological findings noticed in the microscopic evaluation of collected reproductive organs and gross pathological tissues like, tail and non-glandular stomach. Thus, based on all the observations and results, it was concluded that the No Observed Adverse Effect Level (NOAEL) of the test chemical was found to be 1000 mg/kg bw.

Link to relevant study records
Reference
Endpoint:
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 a study report.
Qualifier:
according to
Guideline:
other: OECD 421 (Reproduction/Developmental Toxicity Screening Test)
Principles of method if other than guideline:
The above study was performed to evaluate and assess the effect of the test chemical on the developmental parameters ofthe Sprague Dawley rats.
GLP compliance:
yes
Specific details on test material used for the study:
- Molecular weight (if other than submission substance): No Data Available
- Substance type: Organic
- Physical state: Dark Green Liquid
- Impurities (identity and concentrations): 33.39% pure (by GC)
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Details on test animals and env. conditions
TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: (P) x 9 weeks
- Weight at study initiation: (P) Males: 236.64 g to 296.64 g; Females: 204.64 g to 260.61 g
- Fasting period before study: No Data Available
- 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 animals were acclimatized for nineteen days (including fourteen days of oestrus cycle evaluation) to experimental room conditions and females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment and were observed for clinical signs once daily.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 to 23.6oC
- Humidity (%): 40 to 69%,
- 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: 30 May 2018
To: 20 July 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
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 there by adding little volume of the vehicle into the beaker, mixed well using glass rod and transferred into measuring cylinder. This 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 25, 50 and 100 mg/mL of test item for low, mid and high dose groups respectively.
Chromatographic Conditions:
Column: BDS Hypersil C18 100 × 4.6 mm, 5 µm
Flow rate: 1.0 mL/min.
Injection volume: 10 µL
Wavelength: 254 nm
Run time: 16 minutes
Column temperature: 40°C

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): The test item was not miscible with distilled water and clearly miscible with corn oil at the concentration of 100 mg/mL. Hence corn oil was used as vehicle for test item formulation preparation. Also, corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: Concentration of 0, 25, 50 and 100 mg/mL of test item for low, mid and high dose groups respectively.
- Amount of vehicle (if gavage): 100 ml
- Lot/batch no. (if required): A1712001
- Purity: No Data Available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 there by adding little volume of the vehicle into the beaker, mixed well using glass rod and transferred into measuring cylinder. This 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 25, 50 and 100 mg/mL of test item for low, mid and high dose groups respectively.
Chromatographic Conditions:
Column: BDS Hypersil C18 100 × 4.6 mm, 5 µm
Flow rate: 1.0 mL/min.
Injection volume: 10 µL
Wavelength: 254 nm
Run time: 16 minutes
Column temperature: 40°C
Details on mating procedure:
Details of mating
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: No Data Available
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: Every morning, the vaginal smear of each female was examined for presence of sperm in the vaginal smear. 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.
- Further matings after two unsuccessful attempts: [no / yes (explain)]: No
- After successful mating each pregnant female was caged (how): No Data Available
- Any other deviations from standard protocol: No Data Available
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 30 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Frequency of treatment:
The test item or vehicle was administered to animals through oral (gavage) route once daily.
Duration of test:
No Data Available
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle Control
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Low Dose Group
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Mid Dose Group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High Dose Group
No. of animals per sex per dose:
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.
Control animals:
yes, concurrent vehicle
Details on study design:
No Data Available
Maternal examinations:
Maternal animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity. 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. 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). Feed consumption was measured for all maternal 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. Oestrus cycles were monitored for two weeks after five days of acclimatization to evaluate its normal 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. Maternal animals were sacrificed on lactation day 14 and pups were sacrificed on lactation day 13. The animals were fasted overnight, water was provided ad libitum during fasting. The next day, the body weight of all the fasted animals was recorded prior to exsanguination. The vaginal smear of females on the day of necropsy (lactation day 14) was performed and stage of oestrus cycle was recorded. The animals were euthanized using deep CO2 Asphyxiation and subjected to gross necropsy, external and internal gross pathological examination. Detailed histopathological examination was performed on the ovaries, testes and epididymides of the animals from the control and the high dose group including all macroscopically abnormal tissues of all animals, sacrificed at termination. The histopathological examination was extended to lower dose groups for gross findings like, tail and non-glandular stomach to cover all gross lesions encountered. The tissues were embedded in paraffin wax, sectioned at 4 to 6 micrometers and stained with haematoxylin and eosin.
Ovaries and uterine content:
The number of corpora lutea and implantation sites for females were recorded. Ovaries and uterus with cervix were weighed and subjected to gross pathology.
Fetal examinations:
The duration of gestation was recorded and calculated from day 0 of pregnancy. The day of littering was considered as lactation day 1. The number of pups born (dead and live) in a litter, sex, live births and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 were recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4) and the ratio of AGD to the cube root of pup body weight was calculated. All survived male pups were examined for appearance of nipples/areolae on postnatal day 13 (lactation day 13). The litter was observed daily in order to note the number of alive, dead and cannibalized pups. All the dead and sacrificed pups were examined and subjected to gross pathological examination. Fertility index for dams, sires and pup live birth index, mean litter size per group, survival index and sex ratio at birth were calculated. Blood samples were collected from all the animals for measurement of serum total T4 levels on the following schedule:

a. Two pups per litter on lactation day 4 based on the following conditions:
- two female pups.
- no pups were eliminated when litter size dropped equal or below 10 pups/litter.
- only one pup was used for blood collection in case of one pup was available above 10 pups/litter.
b. Two male pups per litter at termination (lactation day 13)

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 data was subjected to various statistical analyses using SPSS software version 22.
For Body weight, Percent change in body weight, Feed consumption, Copulatory interval, Gestation length, Organ weights, Anogenital distance ratio, Mean pup weight, Live birth Index, Pup survival index, One-way ANOVA with Dunnett’s post test was used. For, Pre/post implantation loss, Pre/post natal loss, No. of resorptions per dam, Corpora lutea per dam, Implantations per dam, No. of live/dead pups/dam, Sex ratio and Litter size, Kruskal-Wallis Test was used. For, Pregnancy rate, No. of litters with/without resorptions, No. of dams with/without live young born, No. of dams with/without dead pups , Chi-square test was used.
Indices:
Male Mating Index, Male Fertility Index, Female Mating Index, Female Fertility Index, Precoital Interval (Days), Mean Gestational Length, Gestation Index (%), Mean number of corpora lutea, Mean number of Implantations, Pre-Implantation Loss (%), Pre-natal Loss/Post-Implantation Loss (%), Pre-natal Loss (No.), Post-natal Loss on lactation day 4/13, Post-natal Loss on lactation day 4/13 (%), Mean Litter Size per Group, Live Birth Index (%) per dam, Precoital Interval (Days), Pup Survival index (%) on lactation day 4, Sex ratio, Percentage of male/female offspring (%), Ano-genital Distance to cube root of the body weight Ratio (AGD ratio).
Historical control data:
No Data Available
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related clinical signs of toxicity observed at any of the tested dose group animals of either sex during the experimental period. However, dark green colored fecal matter was observed at all the tested dose group animals. This coloration is due to dark green color of the test item but not any adverse effects of test item. The detailed clinical examination of animals did not reveal any changes at any of the tested dose group maternal animals during the experimental period.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
There were no treatment related mortality/morbidity observed at any of the tested dose group animals of either sex during the experimental period.
Body weight and weight changes:
no effects observed
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 doe groups of maternal animals during pre-mating and mating periods when compared with vehicle control group was noted. Also, there were no changes observed in the gestation body weight and percent change in gestation body weight during gestation period at any of the tested dose group animals when compared with vehicle control group animals. There were no changes observed in the lactation body weight and percent change in lactation body weight at any of the tested dose group animals when compared with vehicle control group animals.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in feed consumption at all the tested doe groups of either sex during pre-mating and mating periods when compared with vehicle control group was noted. However, statistical significant reduction in average feed consumption during week 2 of pre-mating period at all the tested dose group females and statistical significant reduction in average feed consumption during week 2 of pre-mating period at G2 (250mg/kg) and G3 (500 mg/kg) males was noted when compared with vehicle concurrent control group. Also, there were no changes observed in the feed consumption during gestation period at all the tested dose groups when compared with vehicle control group animals. However, statistical significant reduction in average feed consumption during gestation day 0 to 7 at all the tested doe groups and during gestation day 14 to 20 at G4 (1000 mg/kg) was noted when compared with vehicle control group animals. These changes can not to be considered as treatment related as the reduction is not consistent during the experimental period and also no changes in mean body weight and percent change in body weight was noted during this period. There were no changes observed in the feed consumption during lactation period at any of the tested dose group animals when compared with vehicle control group animals.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in serum T4 levels in adult males and lactation day 13 pups at any of the tested dose groups when compared with vehicle control group. However, statistical significant increase in serum T4 levels in adult males was noted at G2 (250 mg/kg) and G3 (500 mg/kg) was noted when compared with vehicle control group males. This change is not considered as treatment related as the change is not dose dependant and no changes were noted in absolute and relative weight of thyroid in these groups.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no effects observed in absolute weight of epididymis, testes, Prostate and Seminal vesicles and coagulating glands (PSC) and thyroid weights along with parathyroid and its relative weight with respect to terminal body weight at any of the tested dose group males when compared with vehicle control group males. There were no effects observed in absolute weight of thyroid weights along with parathyroid and its relative weight with respect to terminal body weight at any of the tested dose group females when compared with vehicle control group females
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Green colour appearance of tail and non-glandular stomach was observed at all the tested dose group males and green colour appearance of tail and non-glandular stomach was observed in five animals from G2 (250 mg/kg) group females, four animals from G3 (500 mg/kg) group females and ten animals from G4 (1000 mg/kg) group females was noted. These changes can not to be considered as adverse treatment related as the appearances were due to colour of the test item. There were no gross pathological lesions noticed at necropsy of pups (both live and dead).
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no treatment related effects noted in uteri observations like pre and post-implantation losses at all the tested dose groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no treatment related effects and no occurrences of resorptions at all the tested dose group was noted.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no treatment related effects and no occurrences of resorptions at all the tested dose group was noted.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no treatment related changes observed in the number of pups and pup survival index of each litter at any of the tested dose group animals during lactation period when compared with vehicle control group animals.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no changes observed in the gestation length at any of the littered tested dose group animals when compared with vehicle control group animals.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 10, 11, 10 and 12 females from vehicle control, low dose, mid dose and high dose groups respectively, were found to be pregnant with a pregnancy rate of 83.3%, 91.7%, 83.3% and 100.0%; total of 2, 1, 2 and 0 females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-pregnant
Other effects:
not specified
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings
Remarks on result:
other: Not Specified
Abnormalities:
not specified
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes observed in mean pup (male and female) weight on lactation day 1, 4, 7 and 13 at any of the tested dose groups when compared with vehicle control group dams. However, statistical significant reduction in mean male pup weight on lactation day 7 at all the tested dose groups; statistical significant reduction in mean female pup weight on lactation day 7 and 13 at G2 (250 mg/kg) and G3 (500 mg/kg) was noted when compared with vehicle control group. This observation was not considered as treatment related as the occurrences were not consistent, no changes were noted in pup observations during this period and also the changes are not dose dependent.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no treatment related changes observed in the number of pups and pup survival index of each litter at any of the tested dose group animals during lactation period when compared with vehicle control group animals.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no treatment related changes observed in the sex ratio at any of the tested dose group animals during lactation period when compared with vehicle control group animals.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There were no changes observed in ano-genital distance ratio of both male and female pups recorded on lactation day 4 at any of the tested dose group litters when compared with vehicle control group litters. There were no occurrences of nipples in male pups of dams at any of the tested dose group litters and vehicle control group litters observed on lactation day 13.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
other: Ano-Genital Distance, Nipple retention and Sex Ratio
Abnormalities:
not specified
Developmental effects observed:
not specified
Treatment related:
not specified
Conclusions:
Thus, based on all the observations and results, it was concluded that the No Observed Adverse Effect Level (NOAEL) of the test chemical was found to be 1000 mg/kg bw.
Executive summary:

A reproduction /developmental toxicity screening test was performed to evaluate and assess the effects of the the test chemical, following repeated oral dosing to males for 30 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 chemical on male and female reproductive performance such as gonadal function, mating behaviour, 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 [corn oil], animals in G2, G3 and G4 groups were administered with test chemical at dose levels of 250, 500 and 1000 mg/kg body weight for low dose, mid dose 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 30 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 once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. Serum was collected from adult males and lactation day 13 pups representing each per litter were used for screening of 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 anogenital 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 31 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 except dark green colored fecal matter at all the tested dose group animals of either sex during treatment period which was due to dark green color of the test chemical and no mortality or morbidity was observed. No treatment related changes in body weight, per cent change in body weight with respect to day 1 and feed consumption of either sex was noted during the pre-mating and mating 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, all the females were confirmed with mating within 14 days of cohabitation period with the first male only. The mating index for the males and females was 100% for all the tested dose groups. A total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of confirmed pregnancy) of 83.3%, 91.7%, 83.3% and 100.0%; total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of siring a litter) of 83.3%, 91.7%, 83.3% and 100.0%. A total of 10, 11, 10 and 12 females from vehicle control, low dose, mid dose and high dose groups respectively, were found to be pregnant with a pregnancy rate of 83.3%, 91.7%, 83.3% and 100.0%; total of 2, 1, 2 and 0 females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-pregnant [no evidence of implantations] with a per cent of 16.7%, 8.3%, 16.7% and 0.03% during necropsy 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. During gross pathological examination, green colour appearance of tail and non-glandular stomach was observed at all the tested dose group males and green colour appearance of tail and non-glandular stomach in 42% of animals from G2 (250 mg/kg) females, 33% of animals from G3 (500 mg/kg) females and 83% of animals from G4 (1000 mg/kg) females was noted. These changes can not to be considered as adverse treatment related as the appearances were due to colour of the test chemical. There were no gross pathological lesions noticed at necropsy of pups (both live and dead).During histopathological examination, no treatment related histopathological findings noticed in the microscopic evaluation of collected reproductive organs and gross pathological tissues like, tail and non-glandular stomach. Thus, based on all the observations and results, it was concluded that the No Observed Adverse Effect Level (NOAEL) of the test chemical was found to be 1000 mg/kg bw.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data is from Klimisch 1 source which is a study report.
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

Developmental Toxicity Study:

A reproduction /developmental toxicity screening test was performed to evaluate and assess the effects of the the test chemical, following repeated oral dosing to males for 30 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 chemical on male and female reproductive performance such as gonadal function, mating behaviour, 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 [corn oil], animals in G2, G3 and G4 groups were administered with test chemical at dose levels of 250, 500 and 1000 mg/kg body weight for low dose, mid dose 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 30 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 once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. Serum was collected from adult males and lactation day 13 pups representing each per litter were used for screening of 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 anogenital 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 31 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 except dark green colored fecal matter at all the tested dose group animals of either sex during treatment period which was due to dark green color of the test chemical and no mortality or morbidity was observed. No treatment related changes in body weight, per cent change in body weight with respect to day 1 and feed consumption of either sex was noted during the pre-mating and mating 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, all the females were confirmed with mating within 14 days of cohabitation period with the first male only. The mating index for the males and females was 100% for all the tested dose groups. A total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of confirmed pregnancy) of 83.3%, 91.7%, 83.3% and 100.0%; total of 10, 11, 10 and 12 males from vehicle control, low dose, mid dose and high dose groups respectively, were found to be fertile with a fertile rate (with evidence of siring a litter) of 83.3%, 91.7%, 83.3% and 100.0%. A total of 10, 11, 10 and 12 females from vehicle control, low dose, mid dose and high dose groups respectively, were found to be pregnant with a pregnancy rate of 83.3%, 91.7%, 83.3% and 100.0%; total of 2, 1, 2 and 0 females from vehicle control, low dose, mid dose and high dose groups respectively were confirmed as non-pregnant [no evidence of implantations] with a per cent of 16.7%, 8.3%, 16.7% and 0.03% during necropsy 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. During gross pathological examination, green colour appearance of tail and non-glandular stomach was observed at all the tested dose group males and green colour appearance of tail and non-glandular stomach in 42% of animals from G2 (250 mg/kg) females, 33% of animals from G3 (500 mg/kg) females and 83% of animals from G4 (1000 mg/kg) females was noted. These changes can not to be considered as adverse treatment related as the appearances were due to colour of the test chemical. There were no gross pathological lesions noticed at necropsy of pups (both live and dead).During histopathological examination, no treatment related histopathological findings noticed in the microscopic evaluation of collected reproductive organs and gross pathological tissues like, tail and non-glandular stomach. Thus, based on all the observations and results, it was concluded that the No Observed Adverse Effect Level (NOAEL) of the test chemical was found to be 1000 mg/kg bw.

Justification for classification or non-classification

From the available information, it may be concluded that the test chemical might not induce any kind of the toxicity, thus it might not be classified under CLP regulation.