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Description of key information

NOAEL (90 day, rat): 1000 mg/kg bw per day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 June 2020 to 13 November 2020
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:
The study was performed in accordance with OECD Guideline No. 408, “Repeated Dose 90-Day Oral toxicity Study in Rodents”, adopted on 25th June 2018.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL

- 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 test item Licocare RBW 300 FL TP in dose formulations is stable at the concentrations of 4 mg/mL and 200 mg/mL for 48 hours at room temperature.


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: 7 Weeks

- Weight at study initiation: Males: 130.33 to 159.35g; Females: 121.49 to 139.87g

- 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 (e.g. ad libitum):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): 19.2°C to 23.5°C
- Humidity (%): 46% to 69%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 06 June 2020 To: 08 October 2020
Route of administration:
oral: gavage
Details on route of administration:
The test tem 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 (if other than water): Corn oil was used as vehicle for the preparation of formulations as evidenced by the in-house solubility/suspendibility test results. Corn oil is the universally accepted vehicle for oral toxicity studies.
- Concentration in vehicle: G2: 10 mg/mL, G3: 30 mg/mL; G4/G4R: 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot no: L32011001
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and Dose Formulation Analysis for Calcium content in test item was done as per validated analytical methods detailed in the Study No. ARL/2020/AMVP/002.
Formulation analysis for homogeneity and concentration verification were performed for all the dose formulations in week 1, month 2 and month 3 of the treatment. The prepared formulations were sampled in duplicate sets (each from top, middle and bottom layers) from each dose level of prepared formulations. From vehicle control, (5mL) samples were drawn from middle layer only in duplicate sets during scheduled sampling.

For homogeneity and concentration verification analysis, the prepared formulations were sampled in duplicate sets (5mL each from top, middle and bottom layers) from all the dose groups during week 1, month 2 and month 3 of the treatment period. From vehicle control, samples were drawn from middle layer only in duplicate sets during scheduled sampling.

One set of aliquot of each formulation was analysed and formulations were considered acceptable, as the results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10.0%.
Duration of treatment / exposure:
90 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:
Main Groups: 10 Males + 10 Females per dose
Recovery Groups: 5 Males + 5 Females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels of 100 (G2), 300 (G3) and 1000 (G4/G4R) mg/kg body weight/day was considered for low, mid and high dose groups based on results obtained from dose range finding study for reproduction/developmental toxicity screening test in Sprague Dawley rats (BIO-DTX 021). In addition, vehicle control group (G1/G1R) are included which were administered vehicle alone.

- 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 -15.85% to +11.50% and female -10.84% to +8.73%). The grouping was done two days 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.
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: Day 1 before initiation of treatment and weekly Once

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 before initiation of treatment and weekly Once

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 12 for vehicle control and high dose main group animals (G1 and G4) and during Week 17 for recovery group animals (G1R and G4R). No treatment related ocular changes were observed in high dose main group animals. Hence, the examination was not extended to low and mid dose groups (G2 and G3) of main study animals.

- Dose groups that were examined: G1/G1R and 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: 10 Males + 10 Females from all the main groups and 5 Males + 5 Females from all the recovery groups.

- 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 on experimental day 91 and 119 for main and recovery groups respectively.

- Animals fasted: Yes

- How many animals: 10 Males + 10 Females from all the main groups and 5 Males + 5 Females from all the recovery groups.

- Parameters checked in table [No.11] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected from all the rats on 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. 12] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes

- Time schedule for examinations: Neurological/Functional examination was carried out during Week 12 for vehicle control and high dose main group animals (G1 and G4) and during Week 17 for recovery group animals (G1R and G4R). As no apparent treatment related effects noted in high dose main group animals, the examination was not carried out for low and mid dose groups (G2 and G3).

- Dose groups that were examined: G1/G1R and G4/G4R

- Battery of functions tested: sensory activity / grip strength / motor activity

Assessment of Thyroid Hormones T3 (Triiodothyronine), T4 (Thyroxine) and Thyroid Stimulating Hormone (TSH) : Yes

- Time schedule for examinations: Blood samples were collected from all the rats on experimental day 91 and 119 for main and recovery groups respectively.

- How many animals: 10 Males + 10 Females from all the main groups and 5 Males + 5 Females from all the recovery groups.

- Dose groups that were examined: G1/G1R, G2, G3 and G4/G4R

- Parameters checked in table [No.16, 17 and 18] were examined.
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:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related variations in the mean body weight or percent body weight change with respect to day 1 at all the tested doses. However, statistically significant higher mean body weights were noted in G3M (days 15, 22), G4M (days 8, 15, 22, 29, 36), G3F (days 64, 71, 78, 85, 90) and higher percent change in body weight with respect to day 1 was noted in G3M (during days 1-8), G4M (during days 1-8, 1-15), G3F (during days 1-64 to 1-90). The observed higher body weights noted in G4M was only during intital weeks which was up to 11% but considered incidental because of inconsistency between sexes and also no such variation was noted in high dose recovery groups during treatment period only. Higher body weight noted in females is considered incidental due to lack of dose dependency. Also, except few, the body weight is within the historical range of the species.
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 higher feed consumption was noted in G4M during weeks 1 to 4, G3M during week 4, G3F during weeks 9, 11 to 13, G4RM during week 16. The noted change is considered incidental as the consumption is slightly higher and is normal and no such variation is noted in recovery groups.
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.

In main groups, decrease in MCHC (G3M), APTT (G2M, G3M, G4M), absolute and percent eosinophils (G4F), percent eosinophils (G3F); increase in MCV (G3F), MCH (G3F) was noted. Decrease in APTT is not of toxicological significance and all values are within historical range of species; and all other varaitions noted is considered incidental in the absence of dose responsiveness. Also, except few, all the values are within the historical range of the species. Few values that have varied could be due to random biological variation.

In recovery groups, increase in Plateletes (G4RM), PT (G4RF); decrease in APTT (G4RM) was noted. Decrease in APTT is not of toxicological significance and all values are within historical range of species; and all other varaitions noted is considered incidental as no such variations were noted in other sex or at the end of treatment period. Also, except few, all the values are within historical range of the species. Few values that have varied could be due to random biological variation since no dose dependency was observed.
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.

In main groups, increase in total cholesterol (G4M), high density lipoprotein (G3M, G4M), albumin (G4M), sodium (G3M), chloride (G3M); decrease in creatinine (G3F, G4F) was noted.

Decrease in creatinine in G3 and G4 females without any associated microscopic or macroscopic changes in asscociated organ-kidney is considered incidental. All other variations noted are considered incidental in the absence of dose dependency. Also, except few, all the values are within the historical range of the species. Few values that have varied could be due to random biological variation.

In recovery groups, increase in albumin, calcium and A/G ratio was noted in G4RM and increase in glucose was noted in G4RF. The noted changes are considered incidental as no such variatons were noted at the end of treatment period. Also, except few, all the values are within the historical range of the species. Few values that have varied are due to random biological variation.
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 urobilinogen in G3M was noted which is considered incidental in absence of dose relationship.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes were observed in neurological/functional examination battery carried out during Week 12 for main groups (G1 and G4) and during Week 17 for recovery groups (G1R and G4R). However, an isolated incidence of statistically significant increase in body temperature in G4M was noted. This isolated variation without any associated changes in any other neuro-muscular parameters is considered incidental. Also, all the values were within historical range of the species.
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 except for significant increase in G3F which is incidental due to lack of dose responsiveness. No adverse changes were observed in organ weights and its ratios, however, the following statistically significant changes were noted.

Absolute weights: Increase in thymus (G2M), epididymides (G3M, G4M), heart (G2M, G3M, G4M, G3F), liver (G2M, G3F, G4F), prostate+seminal vesicles and coagulation glands (G3M), adrenals (G3F, G4RF), spleen (G3F), kidneys (G3F, G4RF), brain (G4RM) was noted.

Relative weights (relative to fasting body weight): Decrease in testes (G3M), kidneys (G3M), brain (G3M, G3F), pituitary (G2M, G3M), liver (G3F), lungs (G2F, G3F) was noted.

Variations without any associated macroscopic or microscopic changes in high dose groups is considered incidental. All other differences 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, except few, all the values are within the historical range of the species.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No 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 ectopic thymus in thyroid gland, epithelial cysts 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 as they are within historical control data.
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 T3 was noted in G4M and G4RM which is considered incidental in the absence of associated micorocopic (or histopathological) or macroscopic changes and dose dependency in thyroid in the high dose group. Also, such changes in T3 was not noted in other sex-females and also no variations were noted in T4 or TSH and hence, the noted variation is considered as incidental.
Details on results:
The test item Licocare RBW 300 FL TP when administered for a period of 90 consecutive days repeatedly by oral (gavage) to Sprague Dawley rats did not reveal any major toxic effects, target organs, possibility of cumulative effects and there were no reversibility of effects (after 28 days recovery period).
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
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: No effect observed
Conclusions:
Based on the observed results, the No Observed Adverse Effect Level (NOAEL) of test item, Licocare RBW 300 FL 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.Based on the observed results, the No Observed Adverse Effect Level (NOAEL) of test item, Licocare RBW 300 FL 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 300 FL 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 25th June 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 4 mg/mL and 200 mg/mL were stable for 48 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 12 for main groups (G1 and G4), week 17 for recovery groups. Functional observation battery test was carried out during week 12 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, blood collection for 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 specifired 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 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

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on a 90 day oral toxicity study (according to OECD 408) the NOAEL is set as 1000 mg/kg bw per day.

The available data on the subchronic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.