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Repeated dose toxicity: oral

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Administrative data

sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: nanoform

Test animals

Details on species / strain selection:
Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this strain.
Details on test animals or test system and environmental conditions:
Hylasco Biotechnology Pvt. Ltd., Plot 4B, AKP, Turkapally Village, Shameerpet Mandal, RR Dist, Telangana 500078
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 6 weeks
Body weight range at the start of treatment: Males: 189.68 to 249.91 g & Females : 156.56 to 192.12 g
At the commencement of the treatment, the weight variation of rats used did not exceed ± 20 % of the mean body weight in each sex and group.
Conditions: Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 20 to 24°C and relative humidity was between 49 to 68%. The photoperiod was 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12-15 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.
Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in recovery group of each sex was housed individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week. During the experimental period, animals were housed in a single experimental room of barrier area.
Bedding: Steam sterilized corn cob was used as bedding and changed along with the cage atleast twice a week.
Diet: Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum.
Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001,India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (± 3 hours) each day for a period of 90 consecutive days. Similarly, the vehicle was administered to rats in vehicle control/vehicle control recovery group once daily orally for 90 consecutive days.
The vehicle or the dose formulations were not administered to recovery groups for 28 days following the 90-day treatment period. The dose formulation and the vehicle were administered at an equivolume of 10 mL/kg/day. The dose volume was calculated for individual animals on the first day of treatment and was adjusted according to the most recent body weights recorded during the treatment period
CMC (carboxymethyl cellulose)
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during 2nd (Day 34) and 3rd (Day 63) month of the treatment period and analysed in-house. For each set, duplicate samples from top, middle and bottom layers were drawn from each preparation and in case of control, duplicate samples were drawn from middle layer.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19462. One set of samples was analyzed for concentration. The back up samples were discarded as analysis results of the first set of samples were within the acceptable limits. Formulations were considered acceptable as overall mean results of all the layers and mean of each layer were within ± 15.0 % of the claimed concentration and relative standard deviation (% RSD) was less than 10.0 %.
Duration of treatment / exposure:
90 Days
Frequency of treatment:
Doses / concentrationsopen allclose all
Dose / conc.:
111 mg/kg bw/day (nominal)
Dose / conc.:
333 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Control animals:
Details on study design:
No. of groups : 6
Vehicle control (G1)
Low dose (G2)
Mid dose (G3)
High dose (G4)
Vehicle control recovery (G1R)
High dose recovery (G4R)
No. of rats/group: Main groups: 10 males + 10 females
Recovery groups: 5 males + 5 females
Total = 100 (50 males + 50 females)


Observations and examinations performed and frequency:
Observations and examinations performed and frequency
Morbidity and Mortality: All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon except during holidays wherein the observation was done once daily as there were no clinical signs observed. Clinical Signs: Each rat was observed for checking general clinical signs twice once daily during treatment period once daily during the recovery period. On the days of scheduled detailed clinical examination, clinical signs were included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.
Detailed Clinical Examination: Detailed clinical examination was done prior to the test item administration on Day 1 and at weekly intervals thereafter (± 2 days) during treatment period. During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presen ce of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour (e.g. self-mutilation, walking backwards). On the days of detailed clinical examination, observation for general clinical signs (first post-dose) was not performed except on Day 1. Ophthalmological Examination: Ophthalmological examination of all animals was performed with an ophthalmoscope prior to start of the treatment, at the end of the treatment period for main groups (Day 84) and at the end of recovery period (Day 117)for recovery groups. Before examination, my driasis was induced using a 1 % solution of Tropicamide.
Functional Observation Battery Tests (FOB)
The following neurological examination was performed during the 12th week (Day 84) of treatment period for main groups and towards the end of recovery period (Day 117) for recovery groups.
Home Cage Observations: Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.
Observations during Removal of Animal from Home Cage and Handling: The objective of this phase of neurological examination was to observe the subject’s response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following examinations:
ease of removal from home cage
handling reactivity
palpebral closure
eye examination
skin/fur examination
perineum wetness
muscle tone and
extensor thrust response
The observations were recorded using scores/ranks.
Open Field Observation: Rat was placed (one at a time) in an open arena, on a flat surface with a clea
n absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each group.
During this observation period, rat was evaluated as it moves about freely/unperturbed and the fol
lowing observations were made and observations were recorded using score/ranks:
mobility score
arousal level
clonic or tonic movements
stereotypic behaviour
bizarre behaviour
abnormal vocalizations
Functional Tests: Functional testing includes motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.
Motor Activity: The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large ambulatory movement) in seconds, horizontal counts, a mbulatory counts were monitored. The Opto-Varimex 4 motor activity measurement system provided the data at 1 minute interval and the data was analyzed in blocks of 10 minutes interval and the same was reported.
Sensory Reactivity Measurements: After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/ranks.
approach response
touch response
click response
tail-pinch response
pupil response
aerial righting reflex
Landing Hindlimbs Footsplay: The landing hind limbs foot splay was performed by dropping the rat onto a horizontal surface of the table top from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat were gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on to a SOP format, which contains the details such as Study no., Animal no, Group and Sex. A clean recording SOP format was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values is presented in the report along with the individual footsplay values.
Grip Performance: Hindlimbs and forelimbs grip performance was tested using computerized dual grip strength meter (Model: Columbus Instruments). Three trials were conducted for each rat i.e., three trials each for forelimb and hind limbs. Averages of three trials for both forelimb and hindlimbs are calculated and presented in the report along with the individual grip strength values.
Physiological Observations: Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer. At the end of the functional test, body weight of each rat was measured.
Body Weight: Individual body weights (g) was recorded prior to test item administration on Day 1 and weekly thereafter (± 2 day) for all groups of rats during treatment and recovery period. Fasting body weight was recorded prior to sacrifice for all animals.
Food Consumption: The food consumption was measured at weekly intervals (± 2 days) during treatment and recovery period. The cage wise average food consumption (g/rat/day) was calculated and presented in the report.
Oestrous Cycle Evaluation: Vaginal smear was examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy.
Clinical Pathology Investigations
Blood Collection: At the end of the treatment and recovery periods, all rats were fasted overnight (water allowed) and approximately 4.0 mL of blood was collected under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture: After analysis and data review by the analyst, the residual samples were disposed. Haematology, Coagulation, Clinical Chemistry, Hormone Analysis & Urinalysis Parameters were done as study plan.
Sacrifice and pathology:
All rats from toxicity groups at the end of the scheduled period (Day 91 and 119) were subjected to detailed necropsy (examination of external surfaces of the body, all orifices; cranial, thoracic and abdominal cavities and their contents) and findings were recorded. Terminal fasting body weights were recorded for all animals immediately prior to terminal sacrifice and used in calculation of relative organ weights. All rats sacrificed at term were fasted overnight (water allowed), euthanized with isoflurane (as per the random numbers generated for the study), exsanguinated and subjected for gross examination.
At sacrifice, sperm motility was evaluated using the sperm samples collected from the right vas deferens, immediately after necropsy using Hamilton-Thorne TOX-IVOS sperm analyzer for all rats. For morphological evaluation of sperms, smears were made using semen samples collected from right vas deferens of all rats immediately after necropsy and fixed with acetone for evaluation by manual method. Initially, sperm morphology was assessed for control and high dose rats. The right epididymis was collected and frozen for enumeration of cauda epididymal sperm reserves. As the high dose group did not show any test item-related effect, the analysis was not extended to low, mid dose and recovery rats. Unused frozen samples were discarded at the time of final report preparation On completion of gross pathology examination, the tissues/organs noted in following table were collected from all rats. The below listed organs were weighed from all terminally sacrificed animals. The organ weight ratios (organ to body weight and brain weight) as percentage of fasting body weight
were determined and presented in the report. Paired organs were weighed together, and combined weights were presented.
Histopathological examination was carried out on the preserved organs of vehicle control (G1) and high dose group animals (G4). Examination of the testes also included a qualitative assessment of stages of spermatogenesis. In addition, all gross lesions from all the animals were examined microscopically. In the absence of test item-related changes, the tissues from low (G2), mid dose (G3) and recovery (G1R and G4R) groups were not evaluated.
The tissues were processed for routine paraffin embedding and 4-5-micron sections were stained with Haematoxylin and Eosin stain. In addition, testes were sectioned at 3-4 μm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived
For comparative statistics, data was evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be no
nhomogeneous or of nonnormal data was subjected for transformation and ANOVA was done on transformed data. When ANOVA was significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data captured outside of Provantis™: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0. All quantitative variables neurological observations (neuromuscular observation/body temperature/body weights) and T3, T4, TSH was tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modelling by treatment groups. Non-optimal (nonnormal or heteroschedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test was found significant.
For two groups, the comparisons of the mean between treatment and control group was done using‘t’ test.
Descriptive statistics (Mean, SD & Numbers) was presented by Treatment group and Day. All hypothesis testing were carried out at the 5% (2-sided) significance level. Significant differences are designated throughout the report as below:
*: Statistically significant difference from the control group at p < 0.05

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs and mortalities observed throughout the treatment and recovery period in either sex at all the doses tested. Light orange coloured faeces were observed at 111 and 333 mg/kg bwt/day doses and dark orange colour faeces were observed at 1000 mg/kg bwt/day doses in both sexes. This could be due to physical nature of the test item
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Significantly lower body weight gain was observed from Days 22-29 at 333 mg/kg/day and during Days 64-71 at 1000 mg/kg/day in males and significantly higher body weight gain during Days 57-64 at 111 mg/kg/day in females. Significantly higher body weight gain was observed during Days 85-90, 90-97 and 90-118 at 1000 mg/kg/day recovery males and during Days 57-64 and 104-111 at 1000 mg/kg/day recovery females
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The food consumption was significantly lower in males during Days 1-8 at 333 and during Days 15-22 at 111 and 333 mg/kg/day doses.
In females, the food consumption was significantly lower during Days 15-22 at all the doses in main toxicity groups and during Days 43-50 at 1000 mg/kg/day recovery females. Significantly higher food consumption was observed during Days 64-71 at all the doses in main toxicity groups and during Days 71-77 and 111-118 at 1000 mg/kg/day recovery females.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
Effect level:
<= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
ophthalmological examination
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:

Applicant's summary and conclusion

As there were no treatment-related adverse effects observed up to the highest dose the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the test item C.I. Pigment Orange 36 is considered to be 1000 mg/kg/day under the test conditions and doses employed
Executive summary:


The purpose of this 90 day repeated dose toxicity study was to evaluate the systemic toxicity profile of the test item, in Wistar rats when administered orally by gavage for a period of 90 consecutive daysand to assess the reversibilityof any effects during a subsequent 28days recovery period. This study was also intended to provide the information on major toxic effects, target organs and an estimation of a No Observed Adverse Effect Level (NOAEL). 

The test item was weighed and suspended in vehicle,i.e.,0.5% Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q®Water and administered to rats at the graduated dose levels of 111, 333 and 1000 mg/kg/day for low dose (G2), mid dose (G3) and high dose (G4 )/ high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1)/vehicle control recovery (G1R) groups received vehicle Carboxymethylcellulose alone. The dose volume administered was 10 mL/kg body weight. Each main group in the experiment was comprised of 10 male and 10 female rats and recovery groups comprised of 5 male and 5 female rats.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G19462 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable and homogeneous in the vehicle up to 24 hours when stored at room temperature.

During the conduct of this study, the prepared dose formulations and vehicle (Carboxy methylcellulose Sodium salt (medium viscosity) were analyzed for homogeneity and active ingredient (a.i.) concentration on Day 1 and during 2ndmonth (Day 34) and 3rdmonth (Day 63) of the treatment.The results indicated thatthe percent agreement of the analyzed concentrations were in the range, 85% to 115% of the claimed concentrations and the overall % RSD from six replicates at each dose level was<10.0%. This indicates that the prepared dose formulation met the acceptance criteria for concentration and % RSD.

Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and at the end of recovery period for recovery groups. The body weights and food consumption were measured during in-life phase of the experiment. Neurological examinations were conducted towards the end of treatment (Day 84) for main groups and towards the end of recovery period (Day 117) for recovery groups.The clinical laboratory investigations such as haematology, coagulation, clinical chemistry, hormone analysis and urine analysis were performed at termination. Vaginal smear was examined in the female rats and the stage ofoestrous cycle was recordedprior to necropsy.

All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratios were derived as percent fasting body weights and brain weight. Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose(G4) group animals. Histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis.In addition, gross lesions from all the animals were examined microscopically.There were no test item-related histopathological changes observed in any organ/tissue in high dose group (G4); hence, histopathological evaluation was not carried out in thelower dose (G2and G3) and recovery groups (G1R and G4R).

Under the experimental conditions employed, the following results were obtained:

·        Clinical Signs and Mortality:Orangecolour faecal matter(light to dark) were observed at all the tested doses in both sexes. This could be due to physical nature of the test item. There were no mortality observed at any of the doses tested in both sexes.

·        Ophthalmological Examination:Ophthalmological examination did not reveal any ocular abnormalities.

·        Neurological Findings:No treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested.

·        Body Weights:Treatment did not affect body weight at all the tested doses in either sex.

·        Food Consumption:Treatment did not affect food consumption at all the tested doses in either sex.

·        Haematology, Coagulation, Clinical Chemistry and urine parameters:There were no test item related alterations observed at any of the tested dose levels in either sex.

·        Thyroid Hormone Profile:Thyroid hormone profile (TSH, T4 and T3) was not affected in both sexes across the treated groups when compared to the concurrent vehicle control group.

·        Terminal Fasting Body Weights and Organ Weights:No significant changes in terminal fasting body weights and organ weights attributed to test item were observedat any of the tested dose levels in either sex.

·        Sperm Parameter:There were no test item-related changes in any of the sperm parameters.

·        Gross pathology:There were no test item-related gross pathological changes observed in both sexes. Orange colouration of intestinal contents (ileum, cecum, colon and rectum) observed in both sexes at all doses at the end of treatment period was attributed to the test item colour.

·        Histopathology:There were no test item-related microscopic lesions in any evaluated organs or tissues of male and female rats at the end of treatment period at tested dose levels.

No Observed Adverse Effect Level (NOAEL):

As there were no treatment-related adverse effects observed up to the highest dose the No Observed Adverse Effect Level (NOAEL)for systemic toxicityof the test item is considered to be 1000 mg/kg/day under the test conditions and doses employed.