Polar modified Rice Bran Wax

Endpoint summary

Description of key information

NOAEL> 1000 mg/kg bw per day (according to OECD 408)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

28 day repeated dose toxicity study with 14 days recovery period

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 April 2020 to 02 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

OECD Guideline No. 407, “Repeated Dose 28-Day Oral Toxicity Study in Rodents” adopted on 03 October 2008.

GLP compliance:

no

Remarks:

This study was performed as a dose range finding study for a 90 day repeated dose toxicity study. Therefore GLP is not necessary. The main OECD 408 staudy was performed accroding to GLP

Limit test:

no

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 DEF2105728
- Expiration date of the batch: 16.01.2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29⁰C)
- Solubility and stability of the test substance in the solvent/vehicle: Corn oil was used as vehicle for the preparation of formulations as evidenced by the in-house solubility/suspendibility test results. The stability and homogeneity of the test item in dose formulations were not established under this dose range finding study.

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.

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
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 130.15 g to 149.91 g; Females: 123.11 g to 139.98 g
- Housing: Maximum of two animals of same sex 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 feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Polycarbonate rat tunnels were provided to the individually housed rat as an enrichment object.
- Diet: Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the acclimatization and 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: 5 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7ºC to 22.9ºC
- Humidity (%): 44% 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: 16 April 2020 To: 02 June 2020

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered through oral route using stainless steel (gavage) cannula as it is the probable route in human.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared daily before dose administration. The required quantity of test item was weighed separately and was transferred, mixed and triturated well in a mortar. A small quantity of vehicle was added and triturated 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 complete transfer of the contents to the measuring cylinder. Finally, the volume was adjusted to required mark in measuring cylinder with vehicle to get a 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: Corn oil was used as vehicle for the preparation of formulations as evidenced by the in-house solubility/suspendibility test results. The details of the vehicle used for formulation was recorded in the raw data. Corn oil is the universally accepted vehicle in preclinical formulations.
- Concentration in vehicle: G1 (Vehicle Control): 0 mg/mL; G2 (Low Dose): 10 mg/mL; G3 (Mid Dose): 30 mg/mL; G4 (High Dose): 100 mg/mL
- Amount of vehicle: 10 (mL/kg body weight)
- Lot no.: L12017004 and L32011001

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 Days

Frequency of treatment:

Once Daily

Dose / conc.:

0 other: mg/kg body weight/day

Remarks:

Vehicle Control and Recovery Vehicle Control

Dose / conc.:

100 other: mg/kg body weight/day

Remarks:

Low Dose

Dose / conc.:

300 other: mg/kg body weight/day

Remarks:

Mid Dose

Dose / conc.:

1 000 other: mg/kg body weight/day

Remarks:

High Dose and Recovery High Dose

No. of animals per sex per dose:

5 Males + 5 Females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 100, 300 and 1000 mg/kg body weight was selected as low (G2), mid (G3) and high dose (G4/G4R) levels for test item Licocare RBW 106 TP as per the sponsor suggestion.

- Rationale for animal assignment: The animals for the experiment were weighed and arranged in ascending order of their body weights. These body weight stratified rats were distributed to all the experimental groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the experiment did not exceed ±20% (Male: -6.62% to +6.24% and Female: -4.52% to +6.11%) of the mean body weights 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.

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before initiation of treatment and Weekly once thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 before initiation of treatment and once weekly thereafter. Fasting bodyweights were taken prior to sacrifice on the day of necropsy.

FOOD EFFICIENCY:
- Individual animal feed consumption were recorded weekly, coinciding with the body weights of the respective animals. Feed intake per rat (g/rat/day) was calculated using the amount of feed given, spillage and left over in each cage and the number of rats in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examination was performed for all the animals before start of the treatment and during week 4 of treatment for G1 and G4 group animals and during week 6 for recovery group animals (G1R and G4R). No treatment related changes were observed in high dose group animals during Week 4. Hence, the examination was not extended to extended to lower main dose groups.
- Dose groups that were examined: Main Groups G1 and G4 and Recovery Groups: G1R and G4R

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from all the rats of main groups on day 29 and from recovery group animals on day 43.
- Anaesthetic used for blood collection: Yes. Blood samples were collected from retro-orbital plexus puncture under mild isoflurane anesthesia with the help of a fine capillary tube.
- Animals fasted: Yes
- How many animals: 5 Males + 5 Females from each dose group.
- Parameters checked in table [No.10] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from all the rats of main groups on day 29 and from recovery group animals on day 43.
- Animals fasted: Yes
- How many animals: 5 Males + 5 Females from each dose group
- Parameters checked in table [No.11] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from all rats of main group animals and recovery group animals on day 29 and 43 respectively.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.12] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Neurological/Functional examination was carried out during Week 4 for vehicle control and high dose main group animals (G1 and G4) and during last week (week 6) for recovery group animals (G1R and G4R).
- Dose groups that were examined: Main Groups G1 and G4, Recovery Groups: G1R and G4R
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table No. 15)

HISTOPATHOLOGY: No

Statistics:

The data was subjected to statistical analysis. The computer printout of the data (in the form of appendix) was verified with the original raw data. After verification, the data was subjected to statistical analysis using SPSS software version 22. Body weight, body weight gain (percent change in body weight with respect to Day 1 body weight), feed consumption, organ weights and ratios, haematological and clinical chemistry estimations and urine analysis parameters (urobilinogen, pH, specific gravity and urine volume), FOB parameters (rearing, urination, defecation, excessive grooming, body temperature, grip strength, motor activity and hind limb foot splay) were subjected to statistical analysis. One-way ANOVA followed by Dunnett’s post test was done for different treatment groups comparing with the control group data. Comparison of means between recovery groups was done using student ‘t’ test. All analysis and comparisons were evaluated at the 95% level of confidence (p<0.05). Statistically significant changes obtained from the aforementioned tests were designated by the superscripts in the summary table throughout the report as stated below:

*: Statistically significant (p<0.05).

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were noted in any of the treated group animals in either sex.

Mortality:

no mortality observed

Description (incidence):

No mortality/morbidity were noted in any of the treated group animals in either sex.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item related changes in mean body weight or percent change in body weight with respect to day 1 was noted.

Food efficiency:

no effects observed

Description (incidence and severity):

No test item related changes in feed consumption were noted in any of the tested group animals in either sex.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ocular changes were observed during ophthalmoscopic examination in all groups examined.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse test item related changes were observed in haematology parameters. However, the following statistically significant changes were observed when compared to respective vehicle control groups.

In main group, statistically significant decrease in percent monocytes (G2M), PT (G4M), RBC (G3F), haemoglobin (G3F), MCHC (G3F); increase in reticulocyte counts (G3F), absolute reticulocyte counts (G3F); PT (G3F) was noted.

Decrease in PT is not of toxicological significance and also all the values are within the historical range of the species (13.9 to 26.3 seconds). All other variations noted are considered incidental in the absence of dose responsiveness.

In recovery groups, statistically significant increase in percent basophils (G4RF) and decrease in percent lymphocytes (G4RF) was noted. The noted changes are considered incidental in the absence of such changes in recovery males.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse test item related variations were observed in clinical chemistry parameters when compared to control group. However, the following statistically significant variations were noted.

In main group, decrease in total bilirubin (G2M, G4M), urea (G3F), BUN (G3F); increase in triglycerides (G4M) was noted.

Decrease in total bilirubin without any variation in absolute and relative weight of kidney in G2M and G4M is considered incidental; also all the values were within the historical range of the species (0.00 to 0.80 mg/dL). Increase in triglycerides in G4M without any variation in weight of kidneys is considered incidental and also all the values were within historical range of species (30.0 to 138.0). Variations noted in all other parameters is considered incidental due to lack of dose responsiveness.

In recovery groups, decrease in sodium (G4RF) noted is considered incidental in the absence of similar change in recovery males. These changes are within historical control data and without toxicological relevance.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item treatment related changes in urinalysis parameters were noted except for an isolated incidence of higher pH (G4RF) which is considered incidental in the absence of similar change in recovery males and other treated groups (G1, G2 G3 and G4)

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No test item related changes were observed in neurological/functional examination test. However, an isolated incidence of statistically significant lower movement counts was noted in G4R males which is incidental as similar variation was not noted in recovery females.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse test item related changes in organ weights (absolute and relative) were noted. However, the following statistically significant changes were noted in organ weights and its ratios.

Increase in absolute weight of spleen (G2M, G3F), heart (G3M), liver (G4F); decrease in relative weight of kidneys (G2M) and increase in relative weight of spleen (G3F) was noted.

Increase in absolute weight of liver in G4F without any associated variations in liver enzymes is considered incidental. All the other variations noted is considered incidental in the absence of dose responsiveness. All the variations are within historical control data and therefore without toxicological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathological changes were noted at any of the tested dose group animals in main groups and recovery group.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 other: mg/kg body weight/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food efficiency

gross pathology

haematology

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Key result

Critical effects observed:

no

Conclusions:

Based on the results discussed, the No Observed Adverse Effect Level (NOAEL) of the test item, Licocare RBW 106 TP was found to be 1000 mg/kg body weight/day, administered for a period of 28 consecutive days repeatedly by oral (gavage) route to Sprague Dawley rats under the experimental conditions and the doses employed.

Executive summary:

The objective of this study was to assess the toxic potential of test item Licocare RBW 106 TP when administered for a period of 28 consecutive days repeatedly by oral (gavage) route to Sprague Dawley rats (according to OECD 407, adopted on 03 October 2008). This study was conducted as a dose range finding study for a 90 days repeated dose oral toxicity study (according to OECD 408) and generates information on major toxic effects, target organs, possibility of cumulative effects and also to assess the reversibility of effects after 14 days of recovery period and an estimate of the No Observed Adverse Effect Level (NOAEL).

A total of 60 Sprague Dawley rats (30 males +30 females) were distributed to 4 main (G1, G2, G3 & G4) and 2 recovery groups (G1R & G4R). Each group consisted of 5 rats/sex. The rats allocated to groups G2, G3 and G4/G4R were administered with test item, Licocare RBW 106 TP at the doses of 100,300 and 1000mg/kg body weight/day respectively and G1/G1R group animals received vehicle (corn oil) alone. The vehicle/test item formulations were administered by oral (gavage) route at an equivolume of 10 mL/kgbody weight.

The stability and homogeneity of the test item in dose formulations were not established under this dose range finding study. However, freshly prepared test item formulations were administered to the animals and homogeneity was achieved by thorough stirring.

.

All the rats were observed once daily for clinical signs and twice daily for mortality and morbidity, weekly for detailed clinical examination, body weight and feed consumption; ophthalmoscopic examination was performed during acclimatization period for all the rats and during week 4 of dosing period for G1 and G4 groups and during week 6 for G1R and G4R groups. Functional observational battery was performed during week 4 of dosing period for G1and G4 groups and during week 6 for G1R and G4R groups.

At the end of treatment and recovery period, i.e., Day 29 and Day43, respectively, blood and urine samples were collected and analyzed from overnight fastedrats. Subsequently, the rats were sacrificed using CO₂asphyxiation followed by exsanguination and subjected to gross pathological examination. The study plan specified organs/tissues were collected, weighed and preserved.

No mortality and clinical signs were observed throughout the dosing and recovery period. No test item-related changes in body weight, percent change in body weight and feed consumption were observed throughout the experimental period. No test item-related neurological/functional abnormalities and no ocular changes were observed during examination.

No toxicologically significant changes noted in haematology, coagulation, clinical chemistry and urinalysis parameters.No test item-related effects in absolute and relative organ weights and no gross changes were observed.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 July 2020 to 08 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted on 25 June 2018

Deviations:

no

Principles of method if other than guideline:

- Principle of test: The test item Licocare RBW 106 TP was orally administered daily in graduated doses of 100, 300 and 1000 mg/kg body weight as low (G2), mid (G3) and high dose (G4/G4R) levels to several groups of experimental animals, for a period of 90 days. During the period of administration, the animals are observed closely for signs of toxicity. Animals were humanely killed during the test and necropsied at the conclusion of the test, after the full dosing period.

- Short description of test conditions:
1. Rationale for dose level selection: The doses of 100, 300 and 1000 mg/kg body weight were selected as low (G2), mid (G3) and high dose (G4/G4R) levels for test item Licocare RBW 106 TP based on the 28-day repeated dose toxicity study (BIO-CTX 145).

2. Formulation Preparation: The test item formulations were prepared freshly before dose administration on each treatment day. The required quantity of test item was weighed separately and transferred, mixed and triturated well in a mortar. A small quantity of vehicle was added and triturated 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 complete transfer of the contents to the measuring cylinder. Finally, the volume was adjusted to required mark in measuring cylinder with vehicle to get a 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.

3. Stability and homogeneity of the preparation: The stability of the test item Licocare RBW 106 TP in dose formulations were established under Analytical Department of Bioneeds India Private Limited, Study No.: BIO-ANM 1579. The test item formulations at the concentrations of 10 mg/mL and 100 mg/mL are stable for 6 hours at room temperature.

4. Route of Administration and Justification: The test item formulations and vehicle were administered through oral gavage using oral intubation cannula attached to disposable syringe. The oral route was selected as it is the intended route of administration in humans.

5. Actual doses (mg/kg body weight/day): G1/G1R (Vehicle Control): 0; G2 (Low Dose): 100; G3 (Mid Dose): 300; G4 (High Dose): 1000

5. Details of food and water quality: Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) and Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.


- Parameters analysed / observed:
1. Clinical Signs
2. Detailed Clinical Examination
3. Body Weight
4. Feed Consumption
5. Neurological/Functional Examination
6. Ophthalmological Examination
7. Vaginal Smear Examination
8. Clinical Pathology
a. Haematology
b. Clinical Chemistry
c. Urinalysis
9. Assessment of Thyroid Hormones T3 (Triiodothyronine), T4 (Thyroxine) and Thyroid Stimulating Hormone (TSH)
10. Anatomic Pathology
a. Necropsy and Gross Pathology
b. Tissue Collection, Organ Weights, Preservation and Histopathology
c. Histopathological Examination

GLP compliance:

yes (incl. QA statement)

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 DEF2105728
- Expiration date of the batch: 16.01.2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility and stability of the test substance in the solvent/vehicle: Corn oil was used as vehicle for the preparation of formulations as evidenced by the in-house solubility/suspendibility test results. The stability of the test item Licocare RBW 106 TP in dose formulations were established under Analytical Department of Bioneeds India Private Limited, Study No.: BIO-ANM 1579. The test item formulations at the concentrations of 10 mg/mL and 100 mg/mL are stable for 6 hours at room temperature.

FORM AS APPLIED IN THE TEST: The test item/vehicle formulations were administered to animals through oral gavage using stainless steel intubation cannula attached to a disposable syringe once daily.

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
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 07 weeks
- Weight at study initiation: Males: 130.09 g to 157.28 g; Females: 120.65 g to 149.25 g
- Fasting period before study: No
- Housing: Maximum of two animals of same sex 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 feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material. Polycarbonate rat tunnels were provided as an enrichment object to the individually housed animal.
- Diet: Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) were provided ad libitum to the animals throughout the acclimatization and 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: Six Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5°C to 23.0°C
- Humidity (%): 47% to 68%
- Air changes (per hr): 12-15 Air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 30 July 2020 To: 08 December 2020

Route of administration:

oral: gavage

Details on route of administration:

The test item formulations and vehicle were administered through oral gavage using oral intubation cannula attached to disposable syringe. The oral route was selected as it is the intended route of administration in humans.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared freshly before dose administration on each treatment day. The required quantity of test item was weighed separately and transferred, mixed and triturated well in a mortar. A small quantity of vehicle was added and triturated 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 complete transfer of the contents to the measuring cylinder. Finally, the volume was adjusted to required mark in measuring cylinder with vehicle to get a 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: Corn oil was used as vehicle for the preparation of formulations as evidenced by the in-house solubility/suspendibility test results. The details of the vehicle used for formulation was recorded in the raw data and presented in study report. Corn oil is the universally accepted vehicle for oral toxicity studies.
- Concentration in vehicle: G1 (Vehicle Control) - 0 mg/ml; G2 (Low Dose) - 10 mg/ml; G3 (Mid Dose) - 30 mg/ml; G4 (High Dose) - 100 mg/ml
- Amount of vehicle: 10 ml/kg body weight
- Lot no.: L32011001

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The Dose formulation samples of Lilocare RBW 106 TP were analyzed for homogeneity and dose concentration analysis by GC and the results are found within the range ± 15% recovery to the nominal concentration with ˂10% RSD.

Duration of treatment / exposure:

The animals were dosed with vehicle or test item formulations once a day for a period of 90 consecutive days.

Frequency of treatment:

Once Daily

Dose / conc.:

0 other: mg/kg bw/day

Remarks:

Vehicle Control

Dose / conc.:

100 other: mg/kg bw/day

Remarks:

Low Dose

Dose / conc.:

300 other: mg/kg bw/day

Remarks:

Mid Dose

Dose / conc.:

1 000 other: mg/kg bw/day

Remarks:

High Dose

No. of animals per sex per dose:

10 Males + 10 Females for main groups
5 Males + 5 Females for recovery groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 100, 300 and 1000 mg/kg body weight were selected as low (G2), mid (G3) and high dose (G4/G4R) levels for test item Licocare RBW 106 TP based on the 28-day repeated dose toxicity study (BIO-CTX 145).
- Rationale for animal assignment: The animals for the experiment were weighed and arranged in ascending order of their body weights. These body weight stratified rats were distributed to all the experimental groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the experiment did not exceed ±20% of the mean body weight of each sex (Male -10.90% to +11.84% and female -8.81% to +9.82%). The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analyzed statistically for mean body weight to rule out the statistical significant difference between groups within each sex.

- Rationale for selecting satellite groups: For observation after the treatment period, for the potential reversibility or persistence of any toxic effects.

- Post-exposure recovery period in satellite groups: 28 Days

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were subjected to detailed clinical examinations before initiation of the treatment and at weekly intervals thereafter during the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weight was recorded on the Day 1 before test item administration and weekly thereafter. Fasting body weight of all animals were recorded at terminal sacrifice.

FOOD EFFICIENCY:
- Cage wise feed consumption was recorded weekly coinciding with the body weight recordings of the respective animals. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examination was performed during Week 13 for vehicle control and high dose main group animals (G1 and G4) and during Week 17 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1/G1R & G4/G4R

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from all the rats on experimental day 91 and 119 for main and recovery groups respectively.
- Anaesthetic used for blood collection: Yes (Blood samples were collected from retro-orbital plexus puncture method under mild Isoflurane anesthesia with the help of a fine capillary tube.)
- Animals fasted: Yes
- How many animals: Main Groups (40 M + 40 F); Recovery Groups (10 M + 10 F)
- Parameters checked in table [No.10] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Time schedule for collection of blood: Blood samples were collected from all the rats on experimental day 91 and 119 for main and recovery groups respectively.
- Animals fasted: Yes
- How many animals: Main Groups (40 M + 40 F); Recovery Groups (10 M + 10 F)
- Parameters checked in table [No. 11] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: experimental day 91 and 119 for main and recovery groups respectively.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.11] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Neurological/Functional examination was carried out during Week 13 for vehicle control and high dose main group animals (G1 and G4) and during Week 17 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1/G1R & G4/G4R
- Battery of functions tested: sensory activity / grip strength / motor activity

OTHER:
- Assessment of Thyroid Hormones T3 (Triiodothyronine), T4 (Thyroxine) and Thyroid Stimulating Hormone (TSH): The blood samples for estimation of T3, T4 and TSH for all animals.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 19 )

HISTOPATHOLOGY: Yes (see table 20)

Statistics:

The data was subjected to statistical analysis. The computer printout of the data (in form of appendix) was verified with the original raw data. After verification, the data was subjected to statistical analysis using SPSS software, version 22. Body weight, percent change in body weight (with respect to day 1), feed consumption, organ weights and ratios, hematological and clinical chemistry estimations, urinalysis parameters (pH, specific gravity and urobilinogen) and FOB parameters (rearing, urination, defecation, excessive grooming, body temperature, movement count, hind limb foot splay and grip strength) was subjected to statistical analysis. One way ANOVA followed by Dunnett’s post test was done for different treatment groups. Student ‘t’ test was done for recovery groups comparing with the respective vehicle control group data. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). Statistically significant changes obtained from the aforementioned tests were designated by the superscripts in the summary table throughout the report as stated below:
*: Statistically significant (P<0.05).

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were noted in any of the treated group animals in either sex.

Mortality:

no mortality observed

Description (incidence):

No mortality/morbidity were noted in any of the treated group animals in either sex.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment related variations in the mean body weight or percent body weight change with respect to day 1 were observed at all the tested doses when compared to vehicle control group.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes in feed consumption were noted in both main group and recovery group animals. However, statistically significant lower feed consumption was noted during weeks 3, 4, 10, 12 in G3M, week 9 in G3M and G4M, week 4 in G4F, week 9 and 10 in G2F, G3F and G4F, week 12 in G4F, week 13 in G2F and G4F; higher feed consumption was observed during week 1 in G3F and G4RM. The noted changes are considered as incidental in the absence of dose responsiveness and/or there was no concomitant changes noted in body weights.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmological abnormalities were noted in main and recovery group animals.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant treatment related changes in hematology parameters were noted. However, the following statistically significant variations were noted.
Statistically significant increase in MCV and MCH (G2M, G3M), decrease in PT (G4F), increase in relative monocytes and APTT (G4RF) was observed.
Increase in MCV and MCH is considered incidental in the absence of dose responsiveness. Decrease in PT is toxicologically not significant and hence considered incidental. Increase in relative monocytes without any variations in absolute count and othere differential counts is considered incidental. Increase in APTT is also considered incidental as similar changes were not noted in other sex and also at the end of treatment in main groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant variations noted in clinical chemistry parameters at all the tested dose groups. However, the following statistically significant variations were noted.
Non-dose dependent significant decrease in urea (G3M), total bilirubin (G2M), BUN (G3M); increase in triglycerides (G2M), ALP (G3M) and calcium (G3M and G4M); increase in albumin (G3F) and albumin/globulin ratio (G3F) was observed. All the observed changes are considered incidental in the absence of dose responsiveness and/or there were no histopathological changes in the high dose group. Also many values were within the historical range of the species (Refer Annexure 5 for historical range). Few values that attained statistical significance were minimal in nature and also the changes noted could be due to random biological variation.
In recovey group, significant decrease in AST was observed only in G4R males which is considered incidental as similar change was not noted in other sex or at the end of treatment period. Also most of the values are within the historical range of the species; the significance attained is because of the higher values in control recovery group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No significant treatment related changes in urinary parameters were noted. However, an isolated incidence of statistically significant increase in specific gravity in G2F was noted which is considered incidental in absence of dose relationship.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No treatment related changes were observed in neurological/functional examination battery carried out during Week 13 for main groups (G1 and G4) and during Week 17 for recovery groups (G1R and G4R).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant variations noted in fasting body weights in all the groups tested. No adverse changes were observed in organ weights and its ratios, however, the following statistically significant changes were noted.
Absolute weights: Decrease in weight of adrenals (G4M), spleen (G4M) and brain (G3M) was noted.
Relative weights (relative to body weight): Decrease in spleen weight (G4M) was noted.
No statistically significant variations in organ weight or its ratios were observed in recovery group animals.
All the changes noted are considered as incidental and not related to treatment in the absence of dose responsiveness and/or absence of microscopic changes in the high dose main groups. Also, most of the values were within the historical range of the species (Refere Annexure 5 for Historical range).

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item related gross pathological lesions were observed.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related histopathological findings in the study.
Few microscopic findings observed in this study such as ultimobranchial cyst/s in thyroid gland, epithelial cyst/s in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats of this age.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormones - T3 (Triiodothyronine), T4 (Thyroxine) and Thyroid Stimulating Hormone (TSH):
No treatment related changes in thyroid hormones (T3, T4 and TSH) were noted. However, statistical significance increase in TSH was noted in G2M and G4M which is considered incidental in the absence of associated microscopic (or histopathological) or macroscopic changes in thyroid in the high dose group. Also, such changes in TSH was not noted in other sex (females) and hence, the noted variation is considered as incidental. Statistical significance decrease in T4 observed in G4RM is considered incidental as the same changes in T4 was not observed in other sex (recovery females), no significant variation in thyroid weight was noted and no macroscopic changes were observed in thyroid gland.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 other: mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

other: Thyroid Hormones - T3 (Triiodothyronine), T4 (Thyroxine) and Thyroid Stimulating Hormone (TSH)

Key result

Critical effects observed:

no

Conclusions:

Based on the observed results, the No Observed Adverse Effect Level (NOAEL) of test item, Licocare RBW 106 TP is 1000 mg/kg body weight/day when administered for a period of 90 consecutive days by oral (gavage) route to Sprague Dawley rats under the test conditions and doses employed.

Executive summary:

The objective of this study was to assess the toxic potential of the test item,Licocare RBW 106 TP when administered for a period of 90 consecutive days repeatedly by oral (gavage) to Sprague Dawley rats (according to OECD TG 408, adopted on 25^thJune 2018). This study provides information on major toxic effects, target organs, possibility of cumulative effects and also reversibility of effects after 28 days recovery period and an estimate of the No Observed Adverse Effect Level (NOAEL).

A total of 100 (50 males + 50 females) Sprague Dawley rats were distributed to four main and two recovery groups. Each main group (G1 to G4) consisted of 10 males and 10 females and recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G2, G3 and G4/G4R were administered with test item at the doses of 100, 300 and 1000 mg/kg body weight/day. The control group animals (G1and G1R) were administered with vehicle alone (Corn oil). All the vehicle and test item formulations were administered through oral (gavage) route at an equivolume of 10 mL/kg body weight.

The test item formulations at the concentrations of 10 mg/mL and 100 mg/mL were stable for 6 hours at room temperature. The test item formulations were prepared freshly and administered to the animals and homogeneity was achieved by thorough stirring. Formulation analysis for homogeneity and concentration verification was performed for all the dose formulations during week 1, month 2 and month 3 of the treatment. All the formulations were considerd acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was less than 10.

All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity. Detailed clinical examination, body weight and feed consumption was carried out weekly; ophthalmological examination was carried out before start of the treatment and during week 13 for main groups (G1 and G4), week 17 for recovery groups. Functional observation battery test was carried out during week 13 for main groups (G1 and G4) and week 17 for recovery groups. Hematology, coagulation, clinical chemistry and urinalysis were carried out on day 91 for main groups and on Day 119 for recovery groups. Vaginal smear examination, thyroid hormone assay (T3, T4 and TSH) was performed at termination. All the animals were sacrificed by excess of CO₂and

subjected to gross pathological examination and study plan specified organs/tissues were collected, weighed and preserved. The organs of control and high dose were subjected to histopathological examination.

No clinical signs of toxicity or mortality were observed. No treatment related changes in mean body weight and percent change in body weight (%) with respect to day 1, feed consumption, ophthalmological examination and functional observation battery test were observed. No treatment related changes were observed in hematology, coagulation, clinical chemistry and urinalysis parameters. No treatment related changes were observed in thyroid hormones (T3, T4 and TSH), vaginal smear examination, fasting body weights, organ weights and its ratios. No gross pathological changes were observed. No test item related histopathological findings were observed during microscopic examination.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 1- According to OECD TG 408 and GLP

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available