Polar modified Rice Bran Wax

Repeated dose toxicity: oral

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Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

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

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 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.

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
- 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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: mg/kg body weight/day

Remarks:

Vehicle Control and Recovery Vehicle Control

Doses / concentrations 2

Dose / conc.:

100 other: mg/kg body weight/day

Remarks:

Low Dose

Doses / concentrations 3

Dose / conc.:

300 other: mg/kg body weight/day

Remarks:

Mid Dose

Doses / concentrations 4

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.

Examinations

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).

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

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.