Polar modified Rice Bran Wax

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July 2020 to 23 December 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)

Justification for study design:

- Premating exposure duration for parental (P0) animals: 2 Weeks

- Basis for dose level selection: As per Federal Register / Vol. 84, No. 38 / Tuesday, February 26, 2019 / Rules and Regulations, available studies on rice bran wax oxidized includes an acute oral toxicity study, a dermal irritation study, an eye irritation study, a dermal sensitization study, and an Ames assay. No subchronic or chronic studies are available for rice bran wax oxidized. The test item Rice ban wax oxidized (Licocare RBW 106 TP) have low acute oral toxicity (greater than 2000 mg/kg body weight) and there were no acute dermal or inhalation studies submitted. The dermal and eye irritation studies showed the rice bran wax oxidized was not an irritant and also not considered a skin sensitizer in related studies. No endpoint of concern was identified for any of the acute, subchronic, chronic, or reproductive/ developmental studies conducted at any dose level including the limit dose of 1000 mg/kg/day.
Based on the above information, the doses of 0, 100, 300 and 1000 mg/kg body weight had been selected as control, low, mid and high dose groups respectively for Dose Range Finding Study for Reproduction/ Developmental Toxicity Screening Test of Licocare RBW 106 TP by Oral Gavage in Sprague Dawley Rats [Bioneeds Study No. BIO-DTX 028] 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, the same doses of 100, 300 and 1000 mg/kg body weight had been selected as low, mid and high dose groups respectively, in consultation with sponsor for present
Reproduction/Developmental Toxicity Screening Test of Licocare RBW 106 TP by oral gavage in Sprague Dawley Rats.

- Route of administration: Oral

- 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.

Total Number of Animals: 96 (48 Males + 48 Females)

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 lot/batch No.of test material: Clariant Plastics and Coatings (Deutschland) GmbH and DEF2105728
- Expiration date of the lot/batch: 16.01.2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)

- Stability under test conditions: The stability and homogeneity of the test item in dose formulations was established by the Analytical Department of Bioneeds India Private Limited (Bioneeds study no.: BIO-ANM 1579) before initiation of the treatment. The prepared test formulations were found to be stable with the active content for 6 hours at room temperature in corn oil with the mean % recovery range of 85 to 115%.

- Solubility 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 insoluble 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 was 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: 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 animals

- Females nulliparous and non-pregnant: yes

- Age at study initiation: 8 to 9 Weeks

- Weight at study initiation: Males: 210.65 g to 273.18 g; Females: 200.64 g to 216.69 g

- Fasting period before study: Animals were not fasted before study.

- 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 (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 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.

- Acclimatization 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): 19.7 to 22.9°C

- Humidity (%): 46 to 67%

- 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: 01 August 2020; To: 01 December 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared before dose administration and administered within the stability period.
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.

VEHICLE
- Justification for use and choice of vehicle: The corn oil was used as vehicle for test item formulations as per in-house solubility/suspendibility test results.
The test item was insoluble 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 was 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: 0 mg/mL (vehicle control), 10 mg/mL (low dose), 30 mg/mL (mid dose) and 100 mg/mL (high dose).

- Amount of vehicle: 10 mL/kg body weight

- Lot no. : L32011001

Details on mating procedure:

- M/F ratio per cage: 1:1

- Length of cohabitation: 14 Days + 3 Days (Females which were not mated with the first male were cohabitated with proven males of the same group)

- Proof of pregnancy: [sperm in vaginal smear] referred to as day 0 of pregnancy

- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility: Yes, 3 Days (Females which were not mated with the first male were cohabitated with proven males of the same group)

- Further matings after two unsuccessful attempts: no

- After successful mating each pregnant female was caged (how): 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and dose formulation analysis for dose concentration verification was done by Analytical Chemistry department of Bioneeds India Private Limited.

Sampling and analysis of formulations was performed during week 1 and 4 of the treatment. The samples were collected in duplicates (5 mL each) from top, middle and bottom layers for low, mid and high dose concentrations and in duplicates from single layer for vehicle control.

The prepared test item formulations were maintained under stirring conditions using magnetic stirrer during sampling.

The collected samples were transferred to Analytical Chemistry Department of Bioneeds India Private Limited for dose formulation analysis at established stability conditions. One set of aliquot of each formulation was analysed and second aliquot was stored as a backup purpose at established stability conditions. The second set of aliquots were discarded, as the analysis results of first set of samples were within the limits. The formulations were considered acceptable, as the mean results were found within the range ±15% recovery to the nominal concentration with ˂10% RSD.

Duration of treatment / exposure:

Males: 33 days
Females: treated with a maximum period of 65 days including pre-mating, mating, 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: 13 to 14 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: Based on the results obtained from the Dose Range Finding Study, the doses of 100, 300 and 1000 mg/kg body weight had been selected as low, mid and high dose groups respectively, in consultation with sponsor for present Reproduction/Developmental Toxicity Screening Test of Licocare RBW 106 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: -13.08 to 9.14%; Females: -13.45 to 5.60%) 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
- 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, 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: Feed consumption was measured for all the animals once in a week during premating 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 parental males:
The testes and epididymis were collected and preserved. The organs were weighed before preservation. Adherent tissue/fat from the organs was trimmed, and their wet weight was recorded. Paired organs were weighed together. The organ weight ratios as a percentage of body weight was determined and presented in the report. The testes and Epididymides, were preserved in modified Davidson’s fixative for 48 hours and then transferred to 10% NBF.

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, whether died during the study or sacrificed at termination.

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.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups.

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

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All males were sacrificed after completion of 33 days of treatment.
- Maternal animals: All females were sacrificed on lactation day 14.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [15] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were were sacrificed on PND 4/13.

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

Statistics:

The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22.
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.
The statistical analysis was followed but not limited to the parameters as mentioned below:
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)
Non Parametric - Kruskal-Wallis 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
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 evidence of mating / Total no. of males used for mating X
100
Male Fertility Index (%) : No. of males siring a litter / Total no. of males used for mating X 100
Female Mating Index (%) : No. of females with evidence of mating (or confirmed pregnancy) / Total
no. of females used for mating X 100
Female Fertility Index (%) : No. of females with confirmed pregnancy / Total no. of females used for
mating X 100
Precoital Interval (Days) : Date of confirmation mating – Date of initiation of cohabitation
Mean 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

Offspring viability indices:

Mean No. of Corpora Lutea : Sum of No. of corpora lutea in the group / Total No. of dams in the group X 100
Mean No. of Implantations : Sum of No. of implantations in the group / Total no. of dams in the group X 100
Pre-natal Loss/Post-Implantation Loss (%) : No. of Implantations - No. of Viable pups / No. of Implantations X 100
Pre-natal Loss (No.) : No. of Implantations – No. of Live Births
Postnatal Loss on Lactation Day 4/7/13 (No.) : Live births - pups alive at Lactation Day 4/7/13
Postnatal Loss on Lactation Day 4/7/13 (%) : No. of pups alive on Lactation Day 4/7/13 / No. of livepups at birth X 100
Mean Litter Size per Group : Total No. of pups delivered (live and stillborn) / No. of dams that delivered
Live Birth Index (%) per dam : No. of pups born alive / Total No. of pups born X 100
Pup Survival index (%) on Lactation Day 4 : Total No. of live pups (on Lactation Day 4) / No. of pups born X 100
Sex ratio (m/f) : No. of male offspring / No. of female offspring
Percentage of male/female offspring (%) : No. of male/female offspring / Total No. of offspring X 100
Ano-genital Distance to cube root of the body weight Ratio (AGD ratio) : Ano-genital Distance (mm) / Cube root of the body weight

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 any of the tested dose 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 in any of 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:

effects observed, non-treatment-related

Description (incidence and severity):

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

A single incidence of unilateral, follicular cyst in ovaries was observed in high dose female and was considered as an incidental finding in rats of this age

Other effects:

no effects observed

Description (incidence and severity):

Serum Thyroxine (T4) Hormone Levels - Adult Males: There were no changes observed in mean serum Thyroxine (T4) hormone levels at all the tested dose groups (adult males) when compared with vehicle control group. The examination was not extended to adult females as there were no changes noted in serum Thyroxine (T4) hormone levels of adult males.

Serum Thyroxine (T4) Hormone Levels - Pups: There were no changes observed in mean serum Thyroxine (T4) hormone levels of PND 13 pups (pooled samples) representing all the litters from all the tested dose group when compared with vehicle control group. The examination was not extednded to PND 4 pups as there were no changes noted in serum Thyroxine (T4) hormone levels of PND 13 pups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related irregularities observed in oestrus cyclicity of females from 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 and the mean length was comparable with vehicle control group. However, one female from group G2 noted with six day oestrus cycle during pre-mating period. This observed irregular cycle is considered as incidental and un-related to treatment as this female was noted with normal cycles further during mating period and also confirmed as pregnant.

Reproductive performance:

no effects observed

Description (incidence and severity):

Male Mating Index (%): A total of 11 (out of 12), 12 (out of 12), 11 (out of 12) and 11 (out of 12) males were confirmed with mating with a mating index of 91.7%, 100.0%, 91.7% and 91.7% from groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for male fertility index in any of the tested dose groups when compared with vehicle control group.
Male Fertility Index (%): A total of 10 (out of 12), 11 (out of 12), 10 (out of 12) and 10 (out of 12) males were confirmed as impregnating a female with a fertility index of 83.3%, 91.7%, 83.3% and 83.3% from groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for male fertility index in any of the tested dose groups when compared with vehicle control group.

Female Mating Index (%): All the 12 females from each tested dose groups (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 10 (out of 12), 11 (out of 12), 11 (out of 12) and 11 (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 83.3%, 91.7%, 91.7% and 91.7% from groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for female fertility indices in any of the tested dose 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 6.25, 7.42, 8.00 and 8.00 days for groups G1, G2, G3 and G4, respectively. There was no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group. The obtained values for this endpoint are within in-house historical control range [In-house historical control range: Mean: 7.3 days, ranging from 1.0 days to 19 days].

Gestation Length (Days): The mean gestation length [confirmation of mating to parturition] was 22.60, 22.82, 22.27 and 22.36 days for groups G1, G2, G3 and G4, respectively. There was no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group.

Gestation Index / Parturition Index (%): A total of 10 (out of 10), 11 (out of 11), 11 (out of 11), and 11 (out of 11) females were confirmed with live born pups with a gestation / parturition index of 100%, 100%, 100.0% and 100% from groups G1, G2, G3 and G4 respectively.

Pregnancy Index (%): A total of 10 (out of 12), 11 (out of 12), 11 (out of 12), and 11 (out of 12) females were confirmed with live born pups with a pregnancy index of 83.3%, 91.7%, 91.7% and 91.7% from groups G1, G2, G3 and G4, respectively. There was no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group.

Mean number of Implantations (No.): The mean number of implantations per litter was 11.30, 11.00, 11.27 and 11.27 from groups G1, G2, G3 and G4, respectively. There was no statistically significant differences noted for 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 0.70, 0.64, 0.82 and 0.82 with a percentage of 6.38, 5.88, 7.90 and 6.76 from groups G1, G2, G3 and G4, respectively. There was no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group.

Postnatal losses on Lactation Day 13 (No.) and (%): There were no postnatal losses noted in any of the litters from all the tested dose groups and vehicle control group.

Note: The data of 10 (out of 12 mated females), 11 (out of 12 mated females), 11 (out of 12 mated females) and 11 (out of 12 mated females) females confirmed with parturition from groups G1, G2, G3 and G4, respectively, were considered for mean calculations and subjected to statistical analysis to analyse the maternal endpoints.

Details on results (P0)

There is no indication of reproduction and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg body weight/day in P generation.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 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

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no external anomalies noted during daily observation of pups in any of the tested dose groups and vehicle control group litters during postnatal 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 in any of the tested dose groups and vehicle control group litters during postnatal 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.

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

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.

Other effects:

no effects observed

Description (incidence and severity):

Mean Pup Anogenital Distance and Anogenital Distance Ratio per Litter on Postnatal Day 4: There were no changes observed in mean pup [both male and female] anogenital distance measurement (mm) and ratio per litter recorded on PND 4 in any of the tested dose groups when compared with vehicle control group.

Nipple Retention in Male Pups per Litter on Postnatal Day 13: There were no occurrences of nipples in male pups examined on PND 13 in any of the tested dose group litters and vehicle control group litters.

Serum Thyroxine (T4) Hormone Levels - Pups: There were no changes observed in mean serum Thyroxine (T4) hormone levels of PND 13 pups (pooled samples) representing all the litters from all the tested dose group when compared with vehicle control group. The examination was not extednded to PND 4 pups as there were no changes noted in serum Thyroxine (T4) hormone levels of PND 13 pups.

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, the oral administration of the test item Licocare RBW 106 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 in Sprague Dawley Rats was to determine the possible health hazards likely to arise from repeated exposure of Licocare RBW 106 TP over a relatively limited period of time. This study was also conducted to obtain initial information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition and an estimate of the No Observed Adverse Effect Level (NOAEL)/No Observed Effect Level (NOEL) with repeated exposure once daily. The males were treated for a period of 33 days including pre-mating, mating and post-mating. The females were treated with a maximum period of 65 days including pre-mating, mating, 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 groups G2, G3 and G4 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 treatment. The prepared test formulations were stable with active content for 6 hours at room temperature in corn oil with the mean % recovery range of 85 to 115% (Reference: Bioneeds study no.: BIO-ANM 1579). Homogeneity and dose formulation analysis for dose concentration verification was performed during week 1 and 4 of dose administration period and the mean results were found within the range ±15% recovery to the nominal concentration with ˂10% RSD.

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 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 epididymides collected from groups G1 and G4 animals with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure in males.

All the males were evaluated for reproductive performance such as, mating and fertility indices. 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 the 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 females on gestation day (GD) 0, 7, 14 and 20 and on lactation day (LD) 1, 4, 7 and 13. Terminal body weights were recorded for all animals on the day of termination. At birth/litter observations 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 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. All the male pups were observed for retention of any nipples/areolae 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 animals from both sexes at all the tested dose groups. 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 both sexes. There were no changes noted in mean serum thyroxine hormone (T4) levels in any of the tested dose group males. The mean 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 dose group animals 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 indices 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 any of the tested dose groups.

For maternal toxicity endpoints, there were no test item-related 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 observed on PND 13. There were no gross pathological changes noted in any of the pups during scheduled sacrifice at all tested doses. 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.