[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 June 2020 TO 30 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

As per OECD Guideline No. 421, “Reproduction/Developmental Toxicity Screening Test”, adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

- Premating exposure duration for parental (P0) animals: 14 Days

- Basis for dose level selection: Dose Range Finding Study for Reproduction/ Developmental Toxicity Screening Test of Licocare RBW 300 FL TP by Oral Gavage in Sprague Dawley Rats [Bioneeds Study No. BIO-DTX 021] was conducted with the doses of 0, 100, 300 and
1000 mg/kg body weight for vehicle control (G1), low dose (G2), mid dose (G3) and high dose (G4) respectively, in consultation with sponsor. There were notest item-related effects noted at all the tested dose groups (100, 300 and 1000 mg/kg body weight) for both reproduction and developmental toxicity end points when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 29 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13, under the experimental conditions employed.

Based on the results obtained from the Dose Range Finding Study [Bioneeds Study No. BIO-DTX 021], the doses of 0, 100, 300 and 1000 mg/kg body weight were selected as control, low, mid and high dose groups in consultation with sponsor for present Reproduction/Developmental Toxicity Screening Test of Licocare RBW 300 FL TP by oral gavage in Sprague Dawley Rats.

- Route of administration: The vehicle and test item formulations were administered through oral (gavage) route as it is the probable route of exposure to human.

- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: Rat-Sprague Dawley (standard laboratory rodent species and strain used for toxicity assessment and also recommended by various regulatory authorities).

The corn oil was used as vehicle for test item formulations as per in-house solubility/suspendibility test results.

The test item was in-soluble in distilled water and not suspended uniformly in 0.5 % w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL. The test item is uniformly suspended in corn oil at the concentration of 200 mg/mL as per in-house solubility/suspendibility test results.
Corn oil is a routinely used vehicle of choice for the oral toxicity studies and accepted universally.

Number of Animals: 96 (48 Males + 48 Females); 12 Males + 12 Females / Group

Test material

Test material information

Constituent 1

Test material form:

solid: flakes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Clariant Plastics and Coatings (Deutschland) GmbH and DEF2114527
- Expiration date of the batch: 05.11.2022 (as per retest COA)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (15 to 25°C)
- Stability under test conditions: The test item formulation was stable up to 48 hours at room temperature in corn oil at low dose, 4 mg/mL and high dose, 200 mg/mL.
To ensure the stability of test item in vehicle, the test item formulations were prepared freshly on each day of dose administration and used.

- Solubility and stability of the test substance in the solvent/vehicle: The corn oil was used as vehicle for test item formulations as per in-house solubility/suspendibility test results.
The test item was in-soluble in distilled water and not suspended uniformly in 0.5 % w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL. The test item is uniformly suspended in corn oil at the concentration of 200 mg/mL as per in-house solubility/suspendibility test results.
Corn oil is a routinely used vehicle of choice for the oral toxicity studies and accepted universally.

FORM AS APPLIED IN THE TEST (if different from that of starting material): The test item/vehicle were administered to animals through oral gavage using stainless steel intubation cannula attached to a disposable syringe once daily.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred
- Females: nulliparous and non-pregnant: Yes
- Age at study initiation: 10 Weeks
- Weight at study initiation: Males: 251.36 to 298.09 g; Females: 202.65 to 249.31 g
- 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. During acclimatization, two animals of same sex were housed.
Pre mating - Per cage two animals of the same sex and group were housed.
Cohabitation Period (mating) - Per cage two animals (one male and one female) of the same group were housed.
Post-mating - After confirming presence of sperm in the vaginal smear and/or vaginal plugs (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 for main group females from gestation day 20 onwards.

- Diet :Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.

- Water : 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 five days 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. Veterinary examination of all the animals was performed on the day of receipt and on day of randomization and grouping.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 to 22.9oC
- Humidity (%): 43 to 66%
- 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: 06 June 2020 To: 27 August 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

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 in aluminium foil, transferred to mortar and triturated well in a mortar using pestle. A small quantity of vehicle was added and grinded well until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.
The test formulations were maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test item formulations.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The corn oil was used as vehicle for test item formulations as per in-house solubility/suspendibility test results.
The test item was in-soluble in distilled water and not suspended uniformly in 0.5 % w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL. The test item is uniformly suspended in corn oil at the concentration of 200 mg/mL as per in-house solubility/suspendibility test results.
Corn oil is a routinely used vehicle of choice for the oral toxicity studies and accepted universally.

- Concentration in vehicle: G1 (Vehicle Control): 0 mg/mL; G2 (Low Dose): 10 mg/mL; G3 (Mid Dose): 30 mg/mL; G4 (High Dose); 100mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- batch no.: L32011001

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 Days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- No unsuccessful mating
- After successful mating each pregnant female was caged: 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 during gestation and lactation periods. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and dose formulation analysis for Calcium content in test item. Sampling and analysis of formulations was performed during week 1 and week 5 of the treatment. The samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. Exact volume of test item formulation samples was included in study report.

Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

Duration of treatment / exposure:

The males were treated for a period of 29 days including pre-mating, mating and post-mating. The females were treated with a maximum period of 63 days including pre-mating, mating, and gestation and up to lactation day 13.

Frequency of treatment:

Once daily

Details on study schedule:

- Age at mating of the mated animals in the study: 10 to 11 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low Dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

Mid Dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High Dose

No. of animals per sex per dose:

12 Males + 12 Females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose Range Finding Study for Reproduction/ Developmental Toxicity Screening Test of Licocare RBW 300 FL TP by Oral Gavage in Sprague Dawley Rats [Bioneeds Study No. BIO-DTX 021] was conducted with the doses of 0, 100, 300 and
1000 mg/kg body weight for vehicle control (G1), low dose (G2), mid dose (G3) and high dose (G4) respectively, in consultation with sponsor. There were no
test item-related effects noted at all the tested dose groups (100, 300 and 1000 mg/kg body weight) for both reproduction and developmental toxicity end points when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 29 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13, under the experimental conditions employed.

Based on the results obtained from the Dose Range Finding Study [Bioneeds Study No. BIO-DTX 021], the doses of 0, 100, 300 and 1000 mg/kg body weight were selected as control, low, mid and high dose groups in consultation with sponsor for present Reproduction/Developmental Toxicity Screening Test of Licocare RBW 300 FL TP by oral gavage in Sprague Dawley Rats.

- Rationale for animal assignment: The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ±20% (Males: -11.41 to 9.35%; Females: -8.88 to 8.65%) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment. Body weight of the animals were analyzed statistically for mean body weight to rule out the statistical significant difference between groups within each sex.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule: Once Daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed at receipt, on the first day of dosing, at least weekly thereafter (may vary by ± 1 or 2 days) 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:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
-Time Schedule: Feed consumption was measured for all animals once a week during premating and weekly 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.

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, which may induce pseudopregnancy. The status of oestrus cyclicity of females was determined on termination day (lactation day 14).

Sperm parameters (parental animals):

Parameters examined in all parental males: The paired organ weights of testes and epididymidis were recorded
Detailed histopathological examination was performed on the testes and epididymidis with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure of the animals from high dose group and the control group including all macroscopically abnormal tissues of all animals sacrificed at termination.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
a. Pup Observations: The day of parturition was considered as postnatal day
(PND) 1 for each pup. Individual pups from each litter were observed for external examination or clinical signs once daily and for mortalities twice daily.
b. Pup weight: Individual live pup weight from each litter was recorded on PND 1 (within 24 hours of parturition), 4, 7, and 13. The mean pup weight per litter (sex-wise) is reported.
c. Anogenital Distance: The Anogenital Distance of each live pup from each litter was measured on PND 4. The mean pup Anogenital Distance and its ratio was reported per litter (sex-wise) as mentioned below:
Anogenital Distance (AGD) Ratio = Anogenital Distance (mm) / Cube root of PND 4 pup weight X 100
d. Observation of Male Pups for Nipples/Areolae Retention: The number of nipples/areolae in male pups were counted on PND 13 and presented litter wise.
e. Blood collection for total T4 (Thyroxine) Level estimation: Blood samples were collected from the all the animals for measurement of serum total T4 levels on the following schedule:
- Two pups per litter on lactation day 4 based on the following conditions:
- two female pups in order to retain more male pups for nipple retention on PND 13 except in the event that removing these pups leaves no remaining females for assessment at termination.
- no pups were eliminated when litter size dropped to equal or below
10 pups/litter.
- only one pup was eliminated and used for blood collection for possible serum T4 assessments from litters with only one pup available above the normal litter size,
- Two pups per litter (preferably same sex) at termination (lactation day 13).
Note: Serum was pooled by litter for T4 analysis on both lactation day 4 and lactation day 13.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All the survived P males were sacrificed after completion of 29 days of treatment. 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 necropsy. The animals were humanely sacrificed using CO2 asphyxiation, subjected to gross necropsy, external and internal gross pathological examination. During necropsy, the males were randomized.
- Maternal animals: The females confirmed with mating but not littered were sacrificed on gestation day 25. The survived littered females 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 necropsy. 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 humanely sacrificed using CO2 asphyxiation, subjected to gross necropsy, external and internal gross pathological examination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera, special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histopathological examination was performed on the ovaries, testes and epididymidis with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure of the animals from high dose group and the control group including all macroscopically abnormal tissues of all animals, whether died during the study or sacrificed at termination.

The other preserved organs including thyroids were not examined as there were no treatment related changes observed in the high dose group. All gross lesions, organs and tissue samples were processed, embedded in paraffin, sectioned at a thickness of 3 to 5 micrometers and stained with hematoxylin 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.

At the time of termination, terminal (fasting) body weights and wet weights of the organs fom all P animals were determined as soon as possible after dissection to avoid drying. These organs were preserved under appropriate conditions. Paired organs were weighed combined.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on lactation day 13
- The dead pups and pups which were sacrificed on PND 4/13 were examined for gross abnormalities with particular attention to the external reproductive genitals and findings were recorded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid along with parathyroid was collected and weighed from one male and one female pup per litter on PND 13.
The histopathological examination of thyroid collected from pups was not conducted as there were no pathological changes observed in thyroid of adult animals.

Statistics:

The raw data was subjected to computer statistical processing.
All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests were designated by the superscripts throughout the report as stated below:
* Statistically significant (P<0.05) change than the vehicle control group.
Note: Data of non-pregnant females was presented in the individual animal data, but excluded for mean calculations and statistical analysis. The data of females mated but not littered and lactation data of females with total litter loss is presented in individual animal data and considered for mean calculations and statistical analysis where applicable.
The statistical analysis was followed but not limited to the parameters as mentioned below table:
The Parametric - One-way ANOVA with Dunnett’s post test was followed to the parameters as mentioned below:
• Body weight (weekly body weights and gestation/lactation body weights)
• Percent Change in body weight (weekly body weights and gestation/lactation body weights)
• Feed consumption (weekly and gestation/lactation)
• Copulatory interval
• Gestation length
• Absolute/relative organ weights
• Mean pup weight per litter
• Mean pup anogenital distance ratio per litter
• Serum T4 values (adults/pups)
The Non Parametric - Kruskal-Wallis method was followed to the parameters as mentioned below:
• Implantations/litter
• No. of pups/litter
• Sex ratio/litter at birth and during lactation period
• Litter size at birth and during lactation period
• No. of early resorptions/litter
• No. of late resorptions/litter
• Post-implantation loss/litter
• Postnatal loss/litter
The Cross Tabs - Chi-square test was followed to the parameters as mentioned below:
• Pregnancy rate
• No. of dams with/without live pups
• No. of dams with/without dead pups
• No. of litters with/without resorptions

Reproductive indices:

Male Mating Index (%): No. of males with confirmed mating / Total No. of males cohabited x 100
Female Mating Index (%): No. of sperm-positive females / Total No. of females cohabited x 100
Male Fertility Index (%): No. of males impregnating a female / Total No. of males cohabited x 100
Female Fertility Index (%): No. of pregnant females / No. of sperm-positive females x 100
Pre-coital Interval (Days): Date of confirmation of mating – Date of initiation of cohabitation
Gestational Length: Date of parturition – Date of positive evidence of mating (GD 0)
Gestation Index (%): No. of females with live born / No. of females with evidence of pregnancy x 100
Parturition Index (%): No. of females littered / No. of females with evidence of pregnancy x 100

Offspring viability indices:

Sex ratio (m/f): No. of male offspring / No. of female offspring
Live Birth Index (%) per litter: No. of pups born alive / Total no. of pups born x 100
Pup Survival index (%) on LD 4/7/13: Total no. of live pups on LD 4/7/13 / No. of pups born/4/7/13 x 100
Sex ratio (m/f) on LD 4/7/13: No. of male offspring on LD 4/7/13 / No. of female offspring on LD 4/7/13
Post-Implantation Loss (%): No. of Implantations - No. of Viable pups / No. of Implantations x 100
Post-implantation Loss (No.): No. of implantations- No. of live births
Post-natal loss on LD 13 (%): No. of pups alive on postnatal day 13 / No. of live pups at birth x 100
Post-natal Loss on LD 13 (No.): Live births - pups alive at lactation day 13
Anogenital Distance (AGD) Ratio: Anogenital Distance (mm) / Cube root of PND 4 pup weight x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity noted in any of the tested dose groups [G2 (100 mg/kg body weight/day), G3 (300 mg/kg body weight/day), G4 (1000 mg/kg body weight/day)] and vehicle control group [G1 (0 mg/kg body weight/day] animals of both sexes throughout the experimental period.

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity noted in any of the tested dose groups [G2 (100 mg/kg body weight/day), G3 (300 mg/kg body weight/day), G4 (1000 mg/kg body weight/day)] and vehicle control group [G1 (0 mg/kg body weight/day] animals of both sexes throughout the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 of both sexes during the experimental period in dosed groups when compared with vehicle control group.
Gestation Body Weight: There were no changes noted in mean body weight and percent change in mean body weight gain during gestation period in any of the tested dose groups when compared with vehicle control group.
Lactation Body Weight: There were no changes noted in mean body weight and percent change in mean body weight gain during lactation period in any of the tested dose groups when compared with vehicle control group.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no changes noted in mean feed consumption measured during pre-mating period (first 2 weeks) of both sexes from all the tested dose groups when compared with vehicle control group.
Gestation Feed Consumption: There were no changes noted in mean feed consumption during gestation period in any of the tested dose groups when compared with vehicle control group.
Lactation Feed Consumption: There were no changes noted in mean feed consumption during lactation period in any of the tested dose groups when compared with vehicle control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological findings noted in high dose group animals of both sexes.

Other effects:

no effects observed

Description (incidence and severity):

Serum Thyroxine (T4) Hormone Levels - Adults: There were no changes observed in serum Thyroxine (T4) hormone levels at all the tested dose groups when compared with vehicle control group.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no irregularities observed in the oestrus cyclicity of females in any of the tested dose groups during pre-mating and mating treatment periods. The mean length of oestrus cycle per female during pre-mating and mating treatment period was unaffected by the test item administration in any of the tested dose groups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no test item-related changes observed in both absolute and relative organ weights of testes and epidydimis at any of the tested dose group animals of both the sexes when compared with concurrent vehicle control group.

Reproductive performance:

no effects observed

Description (incidence and severity):

Male Mating Index (%): A total of 12 (out of 12), 12 (out of 12), 12 (out of 12) and 12 (out of 12) males were confirmed with mating with a mating index of 100.0%, 100.0%, 100.0% and 100.0% from group G1, G2, G3 and G4, respectively. There were no statistically significant differences noted in male fertility index at any of the tested dose groups when compared with vehicle control group.

Male Fertility Index (%): A total of 12 (out of 12), 12 (out of 12), 11 (out of 12) and 12 (out of 12) males were confirmed with impregnating a female with a fertility index of 100.0%, 100.0%, 91.7% and 100.0% from group G1, G2, G3 and G4, respectively. There were no statistically significant differences noted in male fertility index at any of the tested dose groups when compared with vehicle control group.

Female Mating Index (%): All the 12 females from each tested dose group (G2, G3 and G4) and vehicle control group (G1) were confirmed with mating with a mating index of 100% for each group.

Female Fertility Index (%): A total of 12 (out of 12), 12 (out of 12), 11 (out of 12) and 12 (out of 12) females were confirmed with pregnancy (presence of implantations / presence of live or dead fetuses / evidence of parturition) with a fertility index of 100.0%, 100.0%, 91.7% and 100.0% from group G1, G2, G3 and G4, respectively. There were no statistically significant differences on female fertility indices of dosed groups when compared with vehicle control group.
Pre-coital Interval (Days): A total of 12 pairs were left for cohabitation initially from each tested dose group and vehicle control group. The mean pre-coital interval was 5.58, 7.00, 7.42 and 6.33 days for groups G1, G2, G3 and G4, respectively. There was no statistically significant difference for this parameter in dosed groups when compared with the vehicle control group and also the obtained values are within in-house historical control data.

Gestation Length (Days): The mean gestation length [confirmation of mating to parturition] was 22.00, 22.00, 22.09 and 22.33 days for groups G1, G2, G3 and G4, respectively. There was no statistically significant differences in this parameter in any of the dosed groups when compared with the vehicle control group.
Gestation Index / Parturition Index (%): A total of 12 (out of 12), 12 (out of 12), 11 (out of 11), and 12 (out of 12) females were confirmed with live born pups with a gestation / parturition index of 100%, 100%, 100.0% and 100% from group G1, G2, G3 and G4 respectively.

Pregnancy Index (%): A total of 12 (out of 12), 12 (out of 12), 11 (out of 11), and 12 (out of 12) females were confirmed with live born pups with a pregnancy index of 100.0%, 100.0%, 91.7% and 100.0% from group G1, G2, G3 and G4, respectively. There was no statistically significant difference in this parameter in any of the dosed groups when compared with the vehicle control group.

Mean number of Implantations (No.): The mean number of implantations per litter was 11.67, 10.92, 10.09 and 11.25 from group G1, G2, G3 and G4, respectively. There was no statistically significant difference in this parameter in any of the tested dosed groups when compared with the vehicle control group.

Post implantation loss per litter (No.) and (%): The post implantation losses per litter was 0.58, 0.67, 0.82 and 0.67 with a percentage of 4.63, 5.64, 8.56 and 6.76 from group G1, G2, G3 and G4, respectively. There was no statistically significant difference in this parameter in any of the dosed groups when compared with the vehicle control group and also the obtained values are within in-house historical control data.

Post-natal losses on Lactation Day 13 (No.) and (%): There was no post-natal loss noted in any of the litters from all the dosed and vehicle control groups.

Details on results (P0)

The test item Licocare RBW 300 FL TP did not produce any indication of reproduction and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg body weight/day under experimental conditions employed.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 ca. mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food efficiency

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

other: no adverse effect observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no external anomalies noted were noted during daily observation of pups from treated and vehicle control group litters during post-natal period. All the pups were noted with normal behaviour during daily observations.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no test item-related mortalities were noted during daily observation of pups from treated and vehicle control group litters during post-natal period. All the pups were noted with normal behaviour during daily observations.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes observed in mean pup [both male and female] weight per litter recorded on Postnatal Day (PND) 1, 4, 7 and 13 in any of the tested dose group litters when compared with vehicle control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes observed during necropsy in any of the pups of both sexes in any of the tested dose groups and vehicle control group.

Histopathological findings:

not examined

Description (incidence and severity):

The thyroid along with parathyroid was collected from one male and one female pup per litter on PND 13.
The histopathological examination of thyroid collected from pups was not conducted as there were no pathological changes observed in thyroid of adult animals.

Other effects:

no effects observed

Description (incidence and severity):

Serum Thyroxine (T4) Hormone Levels - Pups: There were no changes observed in serum Thyroxine (T4) hormone levels of PND 13 pups from all the tested dose group litters when compared with vehicle control group.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

other: no adverse effect obsrved

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, the oral administration of the test item Licocare RBW 300 FL TP did not produce any indication of reproduction and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg body weight/day under experimental conditions employed. Therefore, the no-observed-adverse-effect-level (NOAEL) of test item is considered at 1000 mg/kg body weight/day for both reproduction and developmental toxicity endpoints.

Executive summary:

The objective of this Reproduction/Developmental Toxicity Screening Test with Licocare RBW 300 FL TP by oral route in Sprague Dawley Rats conducted as per
OECD Test Guideline 421 adopted on 29 July 2016, was to evaluate male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition with repeated exposure once daily. The males were treated for a period of 29 days including pre-mating, mating and post-mating. The females were treated with a maximum period of 63 days including pre-mating, mating, and gestation and up to lactation day 13.

A total of 96 (48 males + 48 females) Sprague Dawley rats were selected for the study and distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 12 males and 12 females. The animals in group G1 were administered with vehicle alone [Corn Oil], the animals in G2, G3 and G4 groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight for low, mid and high doses respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight.

The stability and homogeneity of test item in dose formulations were established before initiation of the treatment. The test item was stable up to 48 hours at room temperature in corn oil with the concentration of 4 mg/mL and 200 mg/mL.Homogeneity and dose formulation analysis for dose concentration verification was performed during week 1 and 5 of the dose administration period and the mean results were within the range of 85 to 115% recovery to the nominal concentration and the relative standard deviation (% RSD) was less than 10%.

All the animals were observed once daily for clinical signs, twice daily for mortality and morbidity. The body weight and feed consumption was recorded once weekly (except during cohabitation) for all the animals. The serum thyroxine hormone (T4) levels were estimated for all males by ELISA method. The gross pathology and organ weighing were performed on the day of termination for all animals. Detailed histopathological examination was conducted on ovaries, testes and epididymis from the groups G1 and G4 animals with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure in males.

All males were evaluated for reproductive performance such as, mating and fertility index. All the dams were evaluated for reproductive performance such as, mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length. All females were evaluated for oestrus cyclicity during premating and mating treatment periods. The stage of oestrus cyclicity was recorded at termination. The body weight and feed consumption was recorded for all the females during gestation day 0, 7, 14 and 20 and on lactation days 1, 4, 7 and 13. The terminal body weight for all animals was measured on the day of the termination (LD 14).
At birth/litter observation parameters such as, number of live/dead pups born, litter size, sex ratio, live birth index per litter and pup survival index were observed / calculated for all litters. The total number of implantations per litter was recorded during necropsy and the post-implantation and post-natal losses were calculated.

The pups were observed once daily for external examinations and twice daily for mortalities till termination [Postnatal Day (PND) 13], weighed individually on PND 1, 4, 7 and 13, measured for Anogenital Distance (AGD) to calculate AGD ratio on PND 4, observed for retention of any nipples/areolae in male pups on PND 13. All the pups were observed for gross pathological observations at termination and analyzed the serum collected from PND 13 pups for thyroxine hormone (T4) levels by using ELISA method.

There were no clinical signs of toxicity and no mortality/morbidity noted in any of the tested dose groups of either sex. There were no changes noted in mean body weight, percent change in mean body weight gain and mean feed consumption in any of the tested dose groups of either sex. There were no changes noted in mean serum thyroxine hormone (T4) levels in any of the tested dose group of males. The absolute and relative organ weights did not reveal any changes in any of the tested dose groups. No macroscopic changes noted during conduct of necropsy in any of the vehicle control and tested dosed groups of either sex. Histopathological examination conducted for group G4 animals of both sexes did not reveal any test item-related microscopic changes.

For reproduction toxicity endpoints, there were no effects noted in male mating and fertility indexes in any of the tested dose groups. No effects were noted in female mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length in all dosed groups.

For maternal toxicity endpoints, there were no irregularities noted in oestrus cyclicity and no changes in mean cycle length in any of the tested dose groups. There were no test item-related changes noted in mean body weight, percent change in mean body weight gain and mean feed consumption in any of the tested dose groups during gestation and lactation periods. There were no changes observed in birth parameters and litter observations during lactation period. The number of implantations,
post-implantation and post-natal losses were unaffected by the test item in all treated groups.

For developmental toxicity endpoints, there were no external anomalies noted and no test item-related mortalities among pups were observed during post-natal period at doses tested. There were no changes noted in mean pup weight and mean pup Anogenital Distance ratio per litter in either sex at doses tested. There were no occurrences of nipples in male pups of vehicle control and treated groups on PND 13. There were no gross pathological changes noted in any of the pups during scheduled sacrifice at any doses tested. There were no changes noted in serum thyroxine (T4) hormone levels measured for PND 13 (from all litters) pups at all the tested dose groups.