Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311, Gajwel, Mandal, Medak District, Telangana

- Age at study initiation: 12 to 13 weeks
- Weight at study initiation:
Mean body weight Body weight range
G1 : 215.166 ± 15.01 191.21 to 244.60g
G2 : 215.756 ± 15.72 191.21 to 241.87g
G3 : 214.956 ± 13.81 189.93 to 239.41g
G4 : 215.421 ± 14.94 184.21 to 238.16g

- Housing:
Rats were housed in standard polysulfone rat cages (size: Length 425 mm x Breadth 266 mm x Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube.

i. Pre mating / Acclimatization: Two rats of the same sex per cage were housed.
ii. Mating: Female rats were cohabited with males in a 1:1 ratio in same cage.
iii. Post-mating / Treatment: After mating confirmation, females were housed individually.

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories, P.O.Box 44220, Madison Wi 53744-4220, was provided ad libitum to the animals.

- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 65 – 68 %,
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.

IN-LIFE DATES: From: 14 April 2016 To: 13 May 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dose formulations were prepared once in 3 – 4 days based on the stability results. The prepared dose formulations were divided into daily aliquots and stored in the experimental room (19 to 25 ºC) when not in use.

Required quantities of the test item were weighed in pre-calibrated beakers for each dose level separately and about 3 to 8 mL of vehicle [Milli-Q water] was added and stirred using a glass rod till a uniform solution was obtained and the volume was made up to the mark using the vehicle to get the final desired concentration.

The homogeneity of the Leomin AN Liq formulation during treatment/sampling was maintained by constant stirring using a magnetic stirrer.

Pre-calibration of the beaker to desired volume: Milli-Q water was measured in a graduated cylinder to the final volume of 150 mL. The measured water was transferred into a clean beaker (to be pre-calibrated) and upper and lower meniscus of water was marked on the beaker. Once these lines had been marked, the water was discarded and the beaker was dried. The upper meniscus was used to make up the volume while preparing the dose formulations.

For vehicle control groups, vehicle [Milli-Q water] was administered.

The weight of the test item, the volume of preparation and volume of administration varied depending on the requirement. The unused dose formulations were sent for disposal.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
by Liquid Chromatograph with Mass Spectrometer (LC-MS/MS)
Details on mating procedure:
During the mating period, female rats were cohabited with males in a 1:1 ratio. When sperm was detected in a vaginal smear or vaginal plug was observed in the morning, the animal was considered to be mated. This day was considered as day 0 of gestation.

The mated female rats obtained each day were assigned to the treatment groups and vehicle control groups by body weight stratification. This procedure was continued till the required numbers of Day 0 mated females were obtained (24 per group).
Duration of treatment / exposure:
The dose formulations of test substance was administered orally by gavage using disposable plastic syringe attached with a metal feeding/intubation cannula to rats of low dose (G2), mid dose (G3) and high dose (G4) groups once daily from GD 5 to GD19 of presumed gestation, at approximately the same time each day (varying by± 2 hours).

The dose volume ( at 10 mL/kg) to be administered was calculated based on the body weight of individual animals on first day of treatment
(on GD 5) and was adjusted according to the most recently recorded body weights recorded till GD 19. The animals in the vehicle control group (G1) were handled in an identical manner to the treatment group and were administered vehicle only.
Frequency of treatment:
Gestation days: 5 to 19
Duration of test:
14 April 2016 to 27 October 2016
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
due to mortality at 800 mg/kg bw/d at high dose of 800 mg/kg bw/d the dosing was reduced to 450 mg/kg bw/d
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Remarks:
reduced to 450 mg/kg bw/d on gestation day
- 10: rats no. Rs3891 to Rs3898 (8 rats)
- 9: rats no. Rs3899 to Rs3903 (5 rats)
- 8: rats no Rs3904 to Rs3913 (10 rats)
- 7: rat no. Rs3914
No. of animals per sex per dose:
24 day '0' pregnant rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:

As per the Sponsor’s suggestion based on the results of a combined repeated dose oral toxicity study with reproduction/developmental toxicity screening test the following doses are selected for definitive embryo-fetal developmental toxicity study with Leomin AN Liq in Wistar rats by oral route:
• G1 - Vehicle control - 0 mg/kg/day
• G2 - Low dose - 50 mg/kg/day
• G3 - Mid dose - 200 mg/kg/day
• G4 - High dose - 800/450 mg/kg/day

- Rationale for animal assignment (if not random): The mated female rats obtained each day were assigned to the treatment groups and vehicle control groups by body weight stratification. This procedure was continued till the required numbers of Day 0 mated females were obtained (24 per group).

Examinations

Maternal examinations:
Observations for clinical signs were performed twice a day - pre dose and post dose (within 1-2 hours of administration) during treatment days and once on non-treatment days.

Each rat was observed twice daily for morbidity and mortality i.e., once in the morning and once in the afternoon. Based on the assessment, as there were toxic signs of concern, the observation was carried out twice during weekends and public holidays.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [half per litter ]
- Skeletal examinations: Yes: [ half per litter ]
- Head examinations: Yes: [half per litter ]
Statistics:
The data on maternal body weight, body weight change, gravid uterine weight, corrected body weight gain, maternal food consumption, number of corpora lutea , number of implantations, total number of fetuses, male and female fetus number and weight was analyzed using ANOVA model, after testing for homogeneity for intra group variance using Levene’s test.

Incidence of pre-implantation loss, post implantation loss, Number of early, late and total resorptions were analyzed using Kruskal Wallis test.

Overall percentage of minor external, visceral and skeletal malformations, Sex ratio and number of dams with any resorptions were analyzed using
2 X 2 Contingency Table.

Statistically significant differences (p < 0.05), indicated by the aforementioned tests were designated by symbol ‘*’ throughout the report.
Indices:
Maternal Parameters:
- Mean no. of corpora lutea/group = total no. of corpora lutea/total no. of pregnant animals
- Mean no. of implantations/group = total no. of implantations/total no. of pregnant animals
- embryonic resorption index (%) = no. of early resorptions/no. of implantations x 100
- Fetal resorption index (%) = no. of late resorptions/no. of implantations x 100
- Pre-implantation loss/group (%) = no. of CL - no. of implantations/ no. of CL x 100
- Post-implantation loss/group (%) = no. of (early + late) resorptions/total no. of implantations x 100
- Implantation index (%) = no. of implantation sites/ no. of corpora lutea x 100

Litter Paramenters:
- Mean litter size/group = total no. of fetuses/total no. of pregnant animals
- Percentage of abnormal fetuses = no. of abnormal fetuses/total no. of fetuses x 100
- Percentage of live fetuses/group (live fetus index) = no. of live fetuses/total no. of fetuses x 100
- Percentage of dead fetuses/group (dead fetus index) = no. of dead fetuses/total no. of fetuses x 100
- Sex ratio (F:M) = no. of females/no. of males


Other:
- Corrected body weight (carcass weight) = terminal body weight (body weight on GD 20) - unopened uterine weight
- Correctred body weight gain = Corrected body weight - body weight on GD 5

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In high dose (800/450 mg/kg/day) group, there was treatment related clinical sign of hypoactivity during post dose observation in few rats (8/23) during GD 7- 9 and one rat Rs 3891 was found dead on GD 9 (when treated at 800 mg/kg/day). After the dose was reduced to 450 mg/kg/day, there were no clinical signs and all the rats were normal.
Mortality:
mortality observed, treatment-related
Description (incidence):
One animal at 800 mg/kg bw/d.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg/day, there was a significant reduction in mean body weights on GD 20 (-6%) as compared to vehicle control group. At 800/450 mg/kg/day, there was a significant decrease in mean body weight on GD 8 (-9%), GD 11 (-11%), GD 14 (-9%), GD 17 (-8%) and GD 20 (-9 %) as compared to vehicle control group.

At 200 mg/kg/day, there was a significant reduction in maternal body weight gain during treatment period GD 5 to 20 (-23 %), and for entire period of gestation GD 0- 20 (-21%), as compared to vehicle control group.
At 800/450 mg/kg/day, there was a significant reduction in maternal weight gain during GD 5 - 8 (-314%), during treatment period GD 5-20 (-37 %), and for entire period of gestation GD 0-20 (-31 %), as compared to vehicle control group.
There was significant reduction in corrected body weight (-7 to -8 %) and weight gain (-111 % to -140 %) at 200 mg/kg/day and 800/450 mg/kg/day respectively as compared to vehicle control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg/day, there was a significant reduction in maternal food consumption during GD 11-14 (-23%), GD 14-17 (-13%), during treatment period GD 5-20 (-12%) and for entire gestation period GD 0-20 (-10 %) as compared to vehicle control group.
At 800/450 mg/kg/day, there was a significant reduction in maternal food consumption during GD 5-8 (-48 %), GD 8-11 (-24 %), GD11-14 (-17 %), and during treatment period GD5-20 (-19 %) and for entire gestation period GD 0-20 (-15 %) as compared to vehicle control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were gross pathological findings of thickening of stomach- non glandular mucosa - multifocal in 8/24 rats at 200 mg/kg/day and 17/23 at 800/450mg/kg/day. The rat found dead had scanty ingesta in stomach and intestine.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There were no treatment-related changes observed in litter parameters at all the doses tested. At 50 mg/kg/day there was a significant increase in fetal weight of total live fetuses and female fetuses and a significant decrease in number of female fetuses as compared to vehicle control group these findings were considered incidental and not attributed to treatment.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MATERNAL BODY WEIGHT AND WEIGHT GAIN

The maternal group mean body weights were unaffected by the administration of test item Leomin AN Liq at the dose of 50 mg/kg/day.

At 200 mg/kg/day, there was a significant reduction in mean body weights on GD 20 (-6%) as compared to vehicle control group. At
800/450 mg/kg/day, there was a significant decrease in mean body weight on GD 8 (-9%), GD 11 (-11%), GD 14 (-9%), GD 17 (-8%) and GD 20
(-9 %) as compared to vehicle control group.

At 200 mg/kg/day, there was a significant reduction in maternal body weight gain during treatment period GD 5 to 20 (-23 %), and for entire period of gestation GD 0- 20 (-21%), as compared to vehicle control group.

At 800/450 mg/kg/day, there was a significant reduction in maternal weight gain during GD 5 - 8 (-314%), during treatment period GD 5-20
(-37 %), and for entire period of gestation GD 0-20 (-31 %), as compared to vehicle control group.

There was significant reduction in corrected body weight (-7 to -8 %) and weight gain (-111 % to -140 %) at 200 mg/kg/day and 800/450 mg/kg/day respectively as compared to vehicle control group.

FOOD CONSUMPTION
At 50 mg/kg/day, the maternal food consumption was comparable to vehicle control group.

At 200 mg/kg/day, there was a significant reduction in maternal food consumption during GD 11-14 (-23%), GD 14-17 (-13%), during treatment period GD 5-20 (-12%) and for entire gestation period GD 0-20 (-10 %) as compared to vehicle control group.

At 800/450 mg/kg/day, there was a significant reduction in maternal food consumption during GD 5-8 (-48 %), GD 8-11 (-24 %), GD11-14 (-17 %), and during treatment period GD5-20 (-19 %) and for entire gestation period GD 0-20 (-15 %) as compared to vehicle control group.

The significant reduction in maternal body weights along with concomitant reduction in food consumption at 200 and 800/450 mg/kg/day was considered as treatment-related finding.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 50 mg/kg bw/day
Based on:
other: treatment-related significant reduction in maternal body weight, corrected body weight gain and food consumption
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 800 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 50 mg/kg/day there was a significant increase in fetal weight of total live fetuses and female fetuses and a significant decrease in number of female fetuses as compared to vehicle control group these findings were considered incidental and not attributed to treatment.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
There was an incidence of small fetus at 200 and 800/450mg/kg/day.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed during skeletal examination of fetuses of dams treated up to 800/450 mg/kg/day.
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed during visceral examination of fetuses of dams treated up to 800/450 mg/kg/day.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
MATERNAL DATA

No treatment-related changes were observed in maternal parameters at 50, and 200 mg/kg/day. There was significant decrease in mean gravid uterine weight (-15%) and non- significant increase in late resorptions, pre and post implantation loss at 800/450 mg/kg/day as compared to vehicle control group. No treatment-related changes were observed in other maternal parameters (mean numbers of corpora lutea, early resorptions, and dams with any resorption).

LITTER DATA
There were no treatment-related changes observed in litter parameters at all the doses tested. At 50 mg/kg/day there was a significant increase in fetal weight of total live fetuses and female fetuses and a significant decrease in number of female fetuses as compared to vehicle control group these findings were considered incidental and not attributed to treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 800 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

Maternal toxicity is 50 mg/kg/day due to treatment-related significant reduction in maternal body weight, corrected body weight gain and food consumption at 200 and 800/450 mg/kg/day and significant reduction in uterine weight at 800/450 mg/kg/day.

Fetal developmental toxicity is 800/450 mg/kg/day in Wistar rats as the litter data parameters were comparable to vehicle control group up to the high dose of 800/450mg/kg/day.

Teratogenicity is 800/450 mg/kg/day as there were no signs of teratogenicity in any of the tested dose levels up to the high dose of
800/450 mg/kg/day

in Wistar rats when Leomin AN Liq was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.
Executive summary:

The objective of this study was to evaluate theembryo-fetal developmentaltoxicity of test item Leomin AN Liq when administered to pregnant Wistar rats by oral route during gestation days (GD) 5 to GD19.The results of this study helped to establish the No Observed Adverse Effect Level (NOAEL) of the test item.

 

In this study, each group (G1, G2, G3 and G4) consisted of 24 presumed pregnant Wistar rats (gestation day 0). Day `0' of gestation for each individual female rat in the study was considered as the day on which vaginal smear was found positive for sperm.

 

The test item, Leomin AN Liq was dissolved in vehicle [Milli-Q water] and administered orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 50, 200 and 800/450 mg/kg/day for low (G2), mid (G3) and high(G4) dose group rats, respectively. The rats in the vehicle control (G1) group received the vehicle alone. A constant dose volume of 10 mL/kg body weight was administered to all groups.

 

High dose group (G4) rats were treated at the dose of 800 mg/kg/day initially (From 25 April 2016 to 29 April 2016). The rats exhibited treatment related clinical sign of hypoactivity, one rat was found dead on GD 9 and there was drastic reduction in body weight and food intake. Considering the treatment related findings and in consultation with the sponsor, the high dose was reduced from 800 to 450 mg/kg/day
(30 April to 12 May 2016). The dose for the high dose group is presented as 800/450mg/kg/day.
  

 

 The dose formulation solutions were analyzed for active ingredient concentrations at the initiation and termination of treatment. The results of analysis of formulations revealed that the analyzed concentrations were within the acceptable limits.

 

The mated females were observed twice daily for clinical signs, mortality and morbidity. Body weights were recorded on GD0, 3, 5, 8, 11, 14, 17 and 20. About 200 g (food input) was provided on Day ‘0’. The food left over was recorded and replenished to a known weight on GD 3, 5, 8, 11, 14 and 17. The food left over was also recorded on Day 20 of presumed gestation. The intermittent body weight gain and food intake was calculated and presented for rats found pregnant at caesarean section.

 

Caesarean section was performed for all the surviving rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the animal were removed (by laparotomy) and the contents were examined.The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. The number of corpora lutea was counted on each ovary. All the fetuses were sexed, weighed and examined for external malformations.Approximately half the number of fetuses from each dam was examined for visceral malformations and the remaining half was evaluated for skeletal malformations.

 

Results of the study are presented below:

 

·       There was no mortality and clinical signs in the low dose and mid dose groups. In the high dose group there was a treatment-related clinical sign of hypoactivity and in addition, one rat (Rs3891) was found dead on GD 9 (when treated at the dose of 800 mg/kg/day). After the dose was reduced to 450 mg/kg/day, there were no clinical signs and all the rats were normal.

·       There was treatment-related significant reduction in maternal body weights, corrected body weight gain and food consumption in mid and high dose groups as compared to vehicle control group.

·       Gross necropsy finding of thickening of stomach non-glandular mucosa multifocal was observed in 8/24 and 17/23 rats of mid and high dose groups respectively.

·       Maternal data parameter - uterine weight was significantly reduced in high dose group. The other maternal data parameters were comparable to vehicle control group up to the high dose.

·        Litter data parameters were comparable to vehicle control group at all the doses tested.

·       Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs ofteratogenicity in any of the tested doses.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

 

·          Maternal toxicity is 50 mg/kg/day due to treatment-related significant reduction in maternal body weight, corrected body weight gain and food consumption at 200 and 800/450 mg/kg/day and significant reduction in uterine weight at 800/450 mg/kg/day. 

 

·          Fetal developmental toxicity is 800/450 mg/kg/day in Wistar rats as the litter data parameters were comparable to vehicle control group up to the high dose of 800/450 mg/kg/day.

 

·          Teratogenicity is 800/450 mg/kg/day as there were no signs of teratogenicity in any of the tested dose levels up to the high dose of 800/450 mg/kg/day

 

in Wistar rats when Leomin AN Liqwas administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.