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Description of key information

90-Day Repeated Dose Toxicity: Oral Route - Swathi (2020)

Under the conditions of the study, based on the observed results and considering the decrease in body weight, feed consumption at 5 000 (G4) and 12 000/4 000 ppm (G5) and microscopic findings observed in brain and thymus which included neuronal necrosis in piriform cortex and/or cornu ammonis (CA1 &2) of the brain and depletion of lymphocytes in thymus at 5 000 (G4) and at 12 000/4 000 ppm (G5) in both sexes, the No Observed Adverse Effect Level (NOAEL) of the test material is 2 000 ppm in the diet (163.12 mg/kg bw/day males; 199.52 mg/kg bw/day females) when administered for a period of 91 consecutive days by oral (dietary) route to Sprague Dawley rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Column 2 of REACH Annex VIII, information requirement 8.6.1, the short term toxicity study (28 days) does not need to be conducted if a reliable sub-chronic (90 days) or chronic study is available. A reliable sub-chronic toxicity study is available.
Critical effects observed:
not specified
Endpoint:
chronic toxicity: oral
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 June 2018 to 22 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
Section 4: Health Effects (2008)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
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 and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks
- Weight at receipt: 135.16 g to 149.86 g - male; 112.86 to 129.76 g - female
- Fasting period before study: no
- Housing: Maximum of two animals of same sex (two rats in one cage + one rat in another cage) were housed in standard polypropylene cages (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilised paddy husk was provided as bedding material.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: The animals were acclimatised for a period of five days to experimental room conditions and were observed for clinical signs once daily. Veterinary examination and body weight recording of all animals were performed on the day of receipt and on the day of randomisation. Ophthalmoscopic examination of all animals was performed on the day of grouping.

ENVIRONMENTAL CONDITIONS
- Temperature: 19.2 – 22.9 °C
- Humidity: 46 – 63 % (except on one day where the highest recorded humidity was 76 %)
- Air changes: 12 – 15 air changes per hour
- Photoperiod: 12 hours fluorescent light and 12 hours dark cycle
Route of administration:
oral: feed
Details on route of administration:
The test material was administered orally as admixture with the diet. Doses were expressed as parts per million (ppm) in diet. The oral route was chosen because this route is a possible route of human exposure.
Vehicle:
unchanged (no vehicle)
Remarks:
The test material was admixtured with powder diet.
Details on oral exposure:
DIET PREPARATION
- The test material was admixtured with powder diet. As per the information provided by the Sponsor, the test material mixed well with powder diet, hence, powder diet was used for preparation of test material admixture diet.
- Rate of preparation of diet (frequency): Fortified diets were prepared daily and used. The following procedure was followed when 0.5 kg of fortified diet was prepared: The required quantity of test material was weighed and initially mixed with a small volume of acetone. Then the same was transferred to the required quantity of feed in a stainless steel container and hand mixed using a stainless steel spoon for 5 minutes. The fortified diets were stored in polyethylene bags which were kept in stainless steel drums in study room at 22 ± 3 °C and used. The left out formulated diet was sent for disposal.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
14 consecutive days
Frequency of treatment:
All the rats received the experimental fortified diet specifically prepared for each group ad libitum, in stainless steel powder food hoppers for a minimum period of 14 consecutive days.
Dose / conc.:
250 ppm
Remarks:
(in diet)
Dose / conc.:
500 ppm
Remarks:
(in diet)
Dose / conc.:
750 ppm
Remarks:
(in diet)
Dose / conc.:
1 500 ppm
Remarks:
(in diet)
No. of animals per sex per dose:
3 animals per sex per dose
Control animals:
yes, plain diet
Details on study design:
Dose selection rationale:
- The doses of 0, 250, 500, 750 and 1500 ppm in diet were selected for vehicle control, low dose, low-mid dose, high-mid dose and high dose as recommended by the Sponsor.
- There are no repeated-dose toxicity studies that use the test material to base doses for a 14-day range-finding study on because the existing assessment of repeated-dose for the test material was based on an incorrect assumption.
- To choose doses for this study, two different toxicologists used two different approaches to decide on the dose series. This dose series was a compromise between the two assessors calculations. It was acknowledged that a 90-day study is 6-times the duration of a 14-day study, however, it is difficult to incorporate further adjustments given the substantial degree of uncertainty in the estimates.

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 the experimental groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the experiment did not exceed ±20 % (male: +10.13 % to -7.07 % and female: +6.78 % to -8.10 %) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analysed statistically for mean body weight to rule out any statistical significant difference between groups within each sex.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- All animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity throughout the experimental period.

BODY WEIGHT: Yes
- Individual animal body weight was recorded on the day of randomisation, prior to treatment (Day 1) and weekly thereafter. Fasting body weights of all animals were recorded at their scheduled terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Cage wise feed consumption was measured at weekly intervals.
- 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. Test material intake (in mg/kg bw/day) was calculated and presented.

CLINICAL PATHOLOGY
- Blood samples were collected from all animals on Day 16. The animals were fasted overnight before blood collection. Water was provided ad libitum during the fasting period. Blood samples were collected from the animals separately into tubes containing K2-EDTA and sodium heparin for haematology and clinical chemistry, respectively, and 3.2 % Sodium citrate tubes for Prothrombin time and activated partial thromboplastin time parameters. Blood samples were collected by retro-orbital plexus puncture method under mild Isoflurane anaesthesia with the help of a fine capillary tube.
- Haematology: The following Haematology parameters were estimated using an Advia 2120 Haematology system (Siemens Limited): Haemoglobin concentration (HGB) (g/dL), Haematocrit (HCT) (%), Erythrocyte count (RBC) (10^6 cells/µL), Total leukocyte count (WBC) (10^3 cells/µL), Mean corpuscular volume (MCV) (fL), Mean corpuscular hemoglobin (MCH) (pg), Mean corpuscular haemoglobin concentration (MCHC) (g/dL), Platelet count (PLT) (10^3 cells/µL), Mean platelet volume (MPV) (fL), Reticulocyte count (Retic) (%), Absolute reticulocyte count (10^9 cells/L), Differential leucocytes count (DLC) (%) and Absolute differential leucocytes count (DLC) (10^3 cells/µL). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by a coagulation analyser [Tulip Diagnostics (p) Ltd., India].
- Clinical Chemistry: The following clinical chemistry parameters were analysed using a “Rx Daytona+ Clinical Chemistry Analyser” (Randox Laboratories): Alanine aminotransferase (ALT) (U/L), Aspartate aminotransferase (AST) (U/L), Alkaline phosphatase (ALP) (U/L), Cholinesterase (U/L), Total Protein (g/dL), Albumin (g/dL), Total bilirubin (mg/dL), Glucose (mg/dL), Total Cholesterol (mg/dL), Creatinine (mg/dL), Urea (mg/dL), Triglycerides (mg/dL), Phosphorous (mg/dL), Calcium (mg/dL), Blood Urea Nitrogen (mg/dL), Globulin (g/dL) and Albumin/Globulin ratio (g/dL). Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using a Prolyte Na/K/Cl analyser (Diamond Diagnostics). The plasma was separated by centrifuging the blood samples at 5000 rpm for 10 minutes for determining the clinical chemistry parameters and 1500 rpm for 15 minutes for determining Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT).

URINALYSIS: Yes
- Urine was collected and analysed from all surviving rats on the day of necropsy (day 16). The animals were kept in urine collection cages overnight on day 15 and were fasted but water was provided ad libitum during this period. The overnight urine volume (mL) collected from these animals was measured. The volume of urine collected (mL), appearance and the colour was recorded by physical evaluation.
- The urine was analysed using an Urine Analyser for the following parameters: Blood (Ery/ µL), Bilirubin (mg/dL), Urobilinogen (mg/dL), Ketones (mg/dL), Protein (mg/dL), Glucose (mg/dL) and Leucocytes (Leu/µL). In addition nitrite, pH and specific gravity were also analysed. After analysis of the above parameters, the urine was subjected to centrifugation at 1500 rpm for 3 minutes. Then the urine sediment was subjected to microscopic examination.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- On day 16, all surviving animals of Group G1, G2, G3, G4 and G5 were subjected to necropsy and detailed gross pathological examination. The animals were fasted overnight (water was provided ad libitum) prior to scheduled necropsy. On the day of terminal sacrifice, the fasted body weight of all animals was recorded prior to exsanguination. The animals were euthanised using CO2 anaesthesia followed by exsanguination. Gross pathological changes were recorded for each rat. Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, with organs and tissues of each animal examined.
- Organ Weights: The following organs from all animals at the scheduled sacrifices were trimmed off any adherent tissue and fat, as appropriate, and weighed wet as soon as possible to avoid drying: Kidneys, Adrenals, Spleen, Heart, Liver, Thymus, Brain, Lungs, Testes/Ovaries and Epididymides/Uterus. Paired organs were weighed together. Relative Organ weights were calculated against fasting body weight.

HISTOPATHOLOGY:
- The tissues collected from all animals were preserved in 10 % Neutral Buffered Formalin except testes which were collected and preserved in Modified Davidson’s fluid. The collected organs will be archived for 2 years from the date of study completion as per instructions from Sponsor.
Statistics:
The raw data were subjected to statistical analysis. The computer printout of the data (in the form of appendix of the report) was verified with the original raw data. After verification, the data were subjected to statistical analysis using SPSS software version 22. Body weight, percent change in body weight with respect to day 1, feed consumption, hematological, clinical chemistry parameters, urinalysis, absolute organ weights and relative organ weights data were subjected to statistical analysis. One way ANOVA followed by Dunnett’s post test was done for different treatment groups comparing with the respective 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 denoted throughout the report: *: Statistically significant (P<0.05).
Clinical signs:
no effects observed
Description (incidence and severity):
- No clinical signs of toxicity were observed in any of the test material administered animals of either sex.
Mortality:
no mortality observed
Description (incidence):
- No mortality/morbidity was observed in any of the test material administered animals of either sex.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- No treatment related changes in mean body weight and percent change in body weight with respect to day 1 were observed in any of the treated group animals of either sex.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- No treatment related changes were observed in feed consumption in any of the treated group animals of either sex.
- The test material intake was as follows:
Males: 35.52, 68.94, 100.32 and 134.33 mg/kg/day for low dose, low-mid dose, high-mid dose and high dose groups, respectively.
Females: 34.23, 67.01, 101.37 and 132.36 mg/kg/day for low dose, low-mid dose, high-mid dose and high dose groups, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- The haematological parameters of test material administered animals (G2, G3, G4 and G5) were comparable with those of the vehicle control.
- In males, statistically significant decreases in haemoglobin in G4, haematocrit in G3 and G4, percent lymphocytes in G5, percent monocytes in G4 and increase in absolute neutrophils in G5 were noted. In females, statistically significant decrease in MCHC in G4 and increase in APTT values in G3, G4 and G5 were noted.
- Decrease in haematocrit in G3 and G4 males without any significant variations in Total Erythrocyte count is considered incidental. Increase in APTT values in G3, G4 and G5 females are considered as incidental and not related to treatment as all the values are within the historical range. All other variations noted are considered incidental due to lack of dose relationship and/or could be due to random biological variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- In males, statistical significant increases in triglycerides and sodium were observed in G5 which are considered incidental and not related to treatment due to lack of dose responsiveness.
Urinalysis findings:
no effects observed
Description (incidence and severity):
- No treatment related significant variations were observed in urinalysis parameters when compared to the control group.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no significant changes in fasting body weight, absolute and relative organ weights in any of the test material administered animals as compared to vehicle controls.
- A statistically significant decrease in absolute weight of testes in G4 males; a statistically significant decrease in absolute weight of brain in G5 females and an increase in relative weight of heart in G2 females were noted.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- No treatment related gross pathological lesions were observed in any of the treated group animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Remarks on result:
not measured/tested
Critical effects observed:
no
Conclusions:
Based on the results observed, it is concluded that the test material was well tolerated up to 1500 ppm in diet (males: 134.33 mg/kg/day and females: 132.36 mg/kg/day) and did not reveal any adverse changes when administered for a period of 15 consecutive days by oral (diet) route to Sprague Dawley rats under the experimental conditions employed. Hence, for the subsequent toxicity studies, dose levels up to 1500 ppm in diet (134.33 mg/kg/day for males/132.36 mg/kg/day for females) or above will be considered.
Executive summary:

The objective of this repeated oral (dietary) toxicity study was to generate information on health hazards likely to arise from repeated exposures to the test material for a period of two weeks to aid selecting doses for subsequent toxicity studies.

A total of 30 (15 males + 15 females) Sprague Dawley rats were distributed to five groups viz., Vehicle control (G1), Low dose (G2), Low-Mid dose (G3), High-Mid dose (G4) and High dose (G5). Each group consisted of 3 males and 3 females. The test material was admixture with powder diet and fed to rats at the doses of 250, 500, 750 and 1500 ppm. Control group rats were fed with basal diet throughout the treatment period without any test material.

All animals were observed once daily for clinical signs and twice daily for mortality. Body weight and feed consumption was measured at weekly intervals. At the end of the treatment period, blood and urine samples were collected and analysed. Subsequently, the animals were sacrificed and subjected to gross pathological examination and the organs were collected and preserved.

No mortality and clinical signs was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight with respect to day 1, feed consumption, haematology, clinical chemistry, urinalysis and organ weights (both absolute and relative) were observed.

There were no treatment related gross pathological changes observed at any of the tested doses in either sex during gross pathological examination.

Based on the results observed, it is concluded that the test material was well tolerated up to 1500 ppm in diet (males: 134.33 mg/kg/day and females: 132.36 mg/kg/day) and did not reveal any adverse changes when administered for a period of 15 consecutive days by oral (diet) route to Sprague Dawley rats under the experimental conditions employed. Hence, for the subsequent toxicity studies, dose levels up to 1500 ppm in diet (134.33 mg/kg/day for males/132.36 mg/kg/day for females) or above will be considered.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 July 2019 to 17 Janary 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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 and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: Yes. Females used were nulliparous and non-pregnant.
- Age at study initiation: 7 weeks.
- 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 sterilised paddy husk was provided as bedding material.
- Diet: Ad libitum.
- Water: Ad libitum. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: The animals were acclimatised for a period of five days to experimental room conditions prior to dosing and were observed for clinical signs once daily. Veterinary examination and body weight recording of all the animals were performed on the day of receipt and on the day of randomisation. Ophthalmoscopic examination of all the animals was performed on the day of grouping.

DETAILS OF FOOD AND WATER QUALITY: Contaminant analysis of food, water and bedding were provided.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 °C to 23.4 °C
- Humidity (%): Relative humidity 42 % to 67 %.
- Air changes (per hr): Air-conditioned room with adequate fresh air supply (12 - 15 Air changes per hour).
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle.

Route of administration:
oral: feed
Details on route of administration:
The test material was administered orally as admixture with diet. Doses were expressed as parts per million (ppm) in diet. The oral route was chosen because this route is a possible route of human exposure.
Vehicle:
unchanged (no vehicle)
Remarks:
The test material was admixtured with powder diet.
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: The test material was admixtured with powder diet. As per the information provided by the Sponsor, the test material mixed well with powder diet, hence, powder diet was used for preparation of test material admixture diet. Powder diet was used to prepare admixture in the 14-day and 28-day dose range finding studies and also to establish stability.
- Mixing appropriate amounts with: Fortified diets were prepared and used within the stability period.
The following procedure was followed when 5 kg of fortified diet was prepared on Day 1 of treatment.
The required quantity of test material was weighed and initially mixed with acetone. Then the same was premixed with a small amount of feed in a stainless steel container for 2 minutes. This premix was then added to the remaining amount of feed to obtain the desired dietary concentration and mixed in a blender for 10 minutes.
- Storage temperature of food: The test diets were stored in polyethylene bags which were kept in stainless steel drums in the study room at temperature 22 ± 3 ºC and used.

VEHICLE
- Amount of vehicle (if gavage): 60 mL. From the second week onwards, the volume of acetone used was 25 mL for all groups.
- Lot/batch no. (if required): F18A/0118/2402/21 and K18A/1918/3010/21
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Fortified diet analysis for homogeneity and dose concentration verification was conducted by the Analytical Department of Bioneeds during week 1, month 2 and month 3 of the treatment period as per methods detailed in the Bioneeds Study No. BIO-ANM 1032. For homogeneity and concentration verification analyses, feed samples were collected in duplicate from the top, middle and bottom layers of the container whereas the vehicle control feed sample (in duplicate) was collected only from the middle layer of container.
The collected samples were transferred to the Analytical Department for analysis. One set of each sample of each formulation was analysed. The second set of samples were discarded as the results obtained from the first set of samples were within the acceptance limits of ± 20 % of nominal concentrations with a Relative Standard Deviation (RSD) of ≤ 20 %.
Homogeneity was evaluated from the top, middle and bottom layers of the collected test formulations. The mean concentrations from all three sampled layers was used for the dose concentration verification.

The fortified diet prepared during week 1, month 2 and month 3 of the treatment period were analyzed for homogeneity and dose concentration verification. All results obtained were within the acceptable range of 80 to 120 % of nominal concentrations with a Relative Standard Deviation (RSD) of < 20 %.
Duration of treatment / exposure:
91 consecutive days
Frequency of treatment:
Daily
Dose / conc.:
750 ppm
Remarks:
Low dose
Dose / conc.:
2 000 ppm
Remarks:
Mid dose
Dose / conc.:
5 000 ppm
Remarks:
High dose
Dose / conc.:
12 000 ppm
Remarks:
Mid dose II (reduced to 4 000 ppm on Day 6)
No. of animals per sex per dose:
10 / sex / dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The doses of 750, 2 000, 5 000 and 12 000 ppm in diet for G2, G3, G4 and G5 groups, respectively were selected as per Sponsor recommendation based on the 14-day and 28-day dose range finding studies. The dose of 12 000 ppm was administered to the G5 group until Day 5. From Day 6 onwards, the dose level was reduced to 4 000 ppm because of a severe reduction in body weight and feed consumption and clinical signs. As few males and most females of Group 4 (5 000 ppm) also showed body weight losses after 3 days of treatment, the high dose level of 12 000 ppm was reduced to 4 000 ppm in consultation with the Sponsor.
- 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 a 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 -9.33 % to +9.90 % and female -11.32 % to +10.91 %). The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analysed statistically for mean body weight to rule out any statistical significant difference between groups within each sex.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity throughout the experimental period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were carried out on all animals prior to exposure to the test material and weekly thereafter. These observations were made outside the home cage in a standard arena.
Signs noted included but were not limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, unusual respiratory pattern and any unusual signs of urination or defecation. Responses with respect to body position, activity level and co-ordination of movement, as well as changes in gait, posture and reactivity to handling, placing or other environmental stimuli, as well as the presence of clonic or tonic movements, convulsions or tremors, stereotypies or bizarre behavior or aggression were also observed.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weight was recorded on day 1 prior to the test material administration and at weekly intervals of the experimental period. Fasting body weight of all the animals were recorded at terminal sacrifice.
Particular attention was given to body weight development of the high dose level animals during the first week of treatment. In addition to the weekly recording of body weight, animals of high dose groups and all the other dose groups were weighed on day 3 of treatment as per the sponsor suggestion.

FOOD CONSUMPTION AND COMPOUND INTAKE: Cage wise feed consumption was measured at weekly intervals. 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.
Particular attention was given to feed consumption of the high dose level animals during the first week of treatment. Visual inspection of the cages and feed hopers were done daily in order to determine the animals were eating or spilling the treated diet. In addition to the weekly recording of feed consumption, feed consumption for animals of the high dose group and all the other dose groups were also recorded on Day 3 of treatment as per the sponsor suggestion.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination was performed on all animals before the start of treatment (during acclimatisation) and during week 13 for G1 and G4 group animals. No treatment related ocular changes were observed in the high dose group animals, hence the examination was not extended to lower dose groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from all animals on experimental Day 92.
- Anaesthetic used for blood collection: Yes. Blood samples were collected by retro-orbital plexus puncture method under mild Isoflurane anesthesia with the help of a fine capillary tube. Blood samples were collected from the animals separately into tubes containing K2-EDTA for haematology and sodium citrate tubes for Prothrombin time and activated partial thromboplastin time parameters.
- Animals fasted: Yes. The animals were fasted overnight before blood collection. Water was provided ad libitum during the fasting period.
- How many animals: All animals.
- Parameters checked:
The following Haematology parameters were estimated using Advia Haematology System 2120 (Siemens Limited).
Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count, Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (Absolute DLC).
Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by a coagulation analyser [Tulip Diagnostics (p) Ltd., India].

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from all animals on experimental Day 92. Blood samples were collected from the animals separately into tubes containing sodium heparin.
- Animals fasted: Yes. The animals were fasted overnight before blood collection. Water was provided ad libitum during the fasting period.
- How many animals: All animals.
- Parameters checked: The following clinical chemistry parameters were analysed using the “Rx Daytona+ Clinical Chemistry Analyser” (Randox Laboratories).
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Triglycerides, Phosphorous, Calcium, High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL), Blood Urea Nitrogen (BUN-calculated), Globulin (calculated), Albumin/Globulin ratio (calculated).
Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Easylyte plus Na/K/Cl analyser (Medica Corporation). The plasma was separated by centrifuging the blood samples at 5 000 rpm for 10 minutes for determining the clinical chemistry and 1 500 rpm for 15 minutes for determining the Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) parameters.

URINALYSIS: Yes.
- Time schedule for collection of urine: Urine was collected from all rats on experimental Day 92.
- Metabolism cages used for collection of urine: Yes. The animals were kept in urine collection cages overnight.
- Animals fasted: Yes. Fasted overnight. Water was provided ad libitum during this period.
- Parameters checked: The volume of urine collected (mL), appearance and the colour was examined by physical evaluation.
The urine was analysed using “DIRUI H-100” (Dirui Industrial Company Ltd.) for the following parameters:
Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Leucocytes.
In addition, Nitrite, pH and Specific gravity were also analysed.
After analysis of the above parameters, the urine was subjected to centrifugation at 1 500 rpm for 3 minutes. Then the urine was subjected to microscopic examination for urinary sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Neurological/Functional examination was carried out during Week 13.
- Dose groups that were examined: Vehicle control and high dose group animals (G1 and G4). As no apparent treatment related effects were observed in the high dose group animals, the examination was not carried out for the low, mid dose and mid dose II groups (G2, G3 and G5).
- Battery of functions tested: The following Neurological/Functional observations were performed:
a) Home Cage Observations: Animals in the cage were observed for convulsions, tremors and palpebral closure and the scores were recorded.
b) Handling Observations: The animals were observed for the ease of removal from the cage and ease of handling. During handling observation, the animals were observed for the resistance, lacrimation, red and crusty deposits around eyes, nose and mouth, salivation, fur appearance, piloerection, palpebral closure, respiratory character, eye prominence and muscle tone.
c) Open Field Observations: Animals were placed in an open field arena for 2 minutes and observed for their mobility, gait, arousal, rearing, urination, defecation, stereotypies and excessive grooming.
d) Sensory Observations: Animals were observed for startle response, touch response, pupil response, response to nociceptive stimuli and righting reflex.
e) Neuromuscular Observations: As a part of neuromuscular observations, the animals were dropped from a height of approximately 30 cm with painted paws to record the Hind Limb Foot Splay.
f) Physiological Observations (Rectal temperature): Physiological temperature of the animals were recorded using a calibrated digital thermometer.
g) Motor Activity Assessment: Assessments of motor activity of the animals for 10 minutes were carried out using an actimeter.
h) Limb Grip Strength Assessment: Assessment of hind limb and fore limb grip strength was carried out using a grip strength meter.

IMMUNOLOGY: No

OTHER:
Vaginal Smear Examination
- A vaginal smear was examined for all the females on the day of sacrifice.
- Assessment of Thyroid Hormones T3 (Triiodothyronine), T4 (Thyroxine) and Thyroid Stimulating Hormone (TSH): Blood samples for estimation of T3, T4 and TSH were collected at termination at the same time when samples were collected for haematology and clinical chemistry. The collected blood samples were centrifuged at 5 000 rpm for 10 minutes, the serum was separated and stored frozen (-80 ºC) until analysed. The analysis was performed using commercially available ELISA kits procured from Cusabiotech Company. Before analysing the samples, the standards for each batch of kits were checked to confirm they were working proprel, as per the kits' instructions.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
At the end of treatment period, all animals were sacrificed under carbon dioxide anesthesia and gross pathological changes were recorded for each rat of all groups.
All animals were fasted overnight (water allowed) and on the day of necropsy (Day 92), they were weighed and then subjected to necropsy. All animals were euthanised by using excess CO2 anesthesia followed by exsanguination. A gross pathological examination was carried out for each rat. Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues. Animals were sacrificed as per random numbers and complete gross pathological examination was performed.
From all the animals, the following organs and tissues were collected, and trimmed off any adherent tissue, as appropriate. All organs/tissues mentioned below, from all animals, were preserved in 10 % v/v Neutral Buffered Formalin except testes and eyes which were preserved in modified Davidson’s fluid.
Eyes, Ovaries, Brain, Lungs, Spinal cord, Prostate gland, Stomach, Esophagus, Duodenum, Pharynx, Jejunum, Urinary bladder, Ileum with Peyer’s patches, Mesenteric Lymph nodes, Caecum, Mandibular Lymph nodes, Colon, Sciatic nerve, Rectum, Bone marrow smear, Liver, Epididymis, Kidneys, Aorta (thoracic), Adrenals, Skin (with mammary glands in Males and Females), Spleen, Sternum, Heart, Skeletal muscle, Thymus, Thyroid, Seminal vesicles with coagulating gland, Parathyroid, Pancreas, Vagina, Uterus and Cervix, Trachea, Testes, Salivary gland, Tongue, Nasal cavity, Femur (femoro-tibial joint), Pituitary, Harderian gland, Gross lesion.

- Organ Weights
On the day of scheduled necropsy, organs in the following list were collected from all animals, trimmed of adherent tissue and fat, as appropriate, and weighed wet as soon as possible to avoid drying.
List of the organs weighed:
Kidneys*, Adrenals*, Spleen, Heart, Liver, Thymus, Brain, Lungs, Testes*/Ovaries*, Uterus/Epididymis*, Thyroid and parathyroid$, Prostate + Seminal vesicles with coagulating gland, Pituitary gland$.
$: Weighed after fixation.
*: Paired organs were weighed together.
Organ weight ratio was calculated against fasting body weight.

- Bone Marrow Smear Examination: Bone marrow smear (from one femur) was prepared for all the animals at the time of necropsy but not evaluated as it was not warranted by haematology findings.


HISTOPATHOLOGY: Yes
Histopathological examination was carried out on the preserved organs of the vehicle control and high dose group (G1 and G4) animals. In addition, the brain and thymus were evaluated in the lower dose groups as they were identified as target organs in this study.
All organs and tissue samples were processed, embedded in paraffin, cut at a thickness of 4 to 6 micrometers and stained with haematoxylin and eosin. The bone marrow smear was fixed in methanol and stained with Giemsa.
Testes were sectioned at 3 - 4 µm thickness and in addition to haematoxylin and eosin stain, Periodic acid-Schiff and haematoxylin stain was also used for spermatogenesis evaluation.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify test material-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings.
Statistics:
Data were subjected to statistical analysis. The computer printout of the data was verified with the original raw data. After verification, the data were subjected to statistical analysis using SPSS software, version 22. Body weight, percent change in body weight with respect to day 1, feed consumption, absolute organ weights, organ weight relative to body weight, haematological and clinical chemistry parameters, urinalysis parameters (urine volume, pH, specific gravity and urobilinogen), FOB parameters (rearing, urination, defecation, excessive grooming, body temperature, movement count, hindlimb foot splay and grip strength) and thyroid hormone levels 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. All analysis and comparisons were evaluated at the 95 % level of confidence (P < 0.05).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were observed up to 5 000 ppm (G4) in either sex. Clinical signs were observed in a few rats of either sex at 12 000 ppm (G5). These consisted in dehydration from Day 4 onwards (G5M: 6/10, and up to 7/10; G5F: 6/10) and piloerection from Day 5 onwards (G5M: 5/10, and up to 6/10; G5F: 4/10, and up to 5/10).
Post dose reduction of G5 from 12 000 ppm to 4 000 ppm on Day 6, only piloerection was observed on Day 7 in two rats (G5M:1/10; G5F:1/10). All animals appeared normal from Day 8 onwards throughout the treatment period.
The detailed clinical examination of animals did not reveal any treatment related changes up to the highest dose tested in either sex.
Mortality:
no mortality observed
Description (incidence):
No signs of mortality were observed up to 5 000 ppm (G4) in either sex.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No treatment related variations in the mean body weight and body weight gain (percent change in body weight with respect to Day 1) were observed up to 2 000 ppm (G3) in either sex.
In males, statistically significant lower mean body weights were observed on Day 3 (G5), Day 8 and 15 (G4 and G5), Days 78 (G4), 85 (G4) and 91 (G4). Decreases in percent change in body weight with respect to day 1 (i.e. body weight loss) were noted during Days 1 - 3 (G5), 1 - 8 (G4 and G5), 1 - 15 (G4 and G5), 1 - 78 (G4), 1 - 85 (G4) and Day 1 - 91 (G4) of the treatment period. In females, statistically significant treatment related decreases in mean body weight were observed on Day 3, Day 8 (G4 and G5) and Days 15 to 91 (G4). Decreases in percent change in body weight with respect to day 1 (i.e. body weight loss) were noted during Days 1 - 3 (G4 and G5) and 1 - 8 (G4 and G5), and Days 1 - 15 and Day 1 - 91 in G4.
The body weights in G5 group animals recovered once the dose level was reduced from 12 000 to 4 000 ppm. Decreased body weights of up to 16 % and 23 %, and decreased body weight gains of up to 28 % and 51 % over the entire treatment period were noted in G4 (5 000 ppm) group males and females, respectively, which are considered treatment related.

The following statistically significant changes were noted:
Decrease in fasting body weight in the high dose group (G4 - 5 000 ppm); this is considered treatment related as the same was noted during the treatment phase of the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No treatment related changes in feed consumption were observed up to 2 000 ppm (G3) in either sex.
Statistically significant decreases in feed consumption were observed in week 1 (Days 1 - 3 and Days 3 - 8) in G4 and G5 males and from week 2 to week 13 in G4 and G5 group males.
In females, statistically significant decrease in feed consumption was observed in week 1 (Days 1 - 3 and Days 3 - 8) in G4 and G5 groups; week 2 to week 6 in G4 and G5 groups and from week 7 to week 13 in G4 group.
The significant lower feed consumptions are considered treatment-related because they were associated with decreases in body weights during many weeks.
Test material intake was as follows:
Males: 62.89, 163.12, 272.18 and 339.85 mg/kg/day for the 750 ppm (low dose), 2 000 ppm (mid dose I), 12 000/4 000 ppm (mid dose II) and 5 000 ppm (high dose) groups, respectively.
Females: 77.70, 199.52, 376.19 and 460.67 mg/kg/day for the 750 ppm (low dose), 2 000 ppm (mid dose I), 12 000/4 000 ppm (mid dose II) and 5 000 ppm (high dose) groups, respectively.
Test material intake was calculated only from the 2nd week onwards for mid dose II (G5, 12 000/4 000 ppm) because of the change in dose level during the first week.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmological abnormalities were noted in G1 and G4 group animals during week 13.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant variations were noted in haematology parameters.
In males, increases in percent neutrophils (G4: +53 %), absolute neutrophils (G5), prothrombin time (G3) and a decrease in percent lymphocytes (G4: -16 %) were noted.
In females, an increase in percent neutrophils (G4: +40 %) and decreases in percent lymphocytes (G4: -13 %), percent eosinophils (G3) and prothrombin time (G2, G4 and G5) were noted.
Variations in differential leucocyte counts in G4 animals (percent neutrophils and lymphocytes) are considered treatment related as they were associated with thymus atrophy. Increase in prothrombin time in G3 males is considered incidental in the absence of dose responsiveness. Decrease in prothrombin time in females (up to -12 %) is not considered adverse as none of the other coagulation or haematology parameters are affected, and no dose-response was apparent.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant variations in clinical chemistry parameters were noted.
In males, decreases in globulin (G3), triglycerides (G4) and calcium (G5) were noted. Increases in A/G ratio (G3), total bilirubin (G3, G4 and G5), AST (G4), ALP (G4), BUN (G4), urea (G4) and creatinine (G5) were observed.
In females, decereases in total protein (G4), albumin (G4), calcium (G4), potassium (G4) and cholinesterase (G3, G4 and G5) were noted; increases in urea, BUN, glucose, LDL, AST, ALP, sodium and chloride (G4) were observed.
Variations in globulin in G3 males, calcium in G5 males, A/G ratio in G3 males are considered incidental in the absence of dose responsiveness. The variations noted in G4 group (high dose - 5 000 ppm) were not associated with histopathological changes in associated organs, hence, they were not considered to be treatment related. Also, the changes noted could be 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. Statistically significant decreases in urine volume (G3M, G4M, and G5M), increases in urine volume (G3F and G4F) and increases in urobilinogen (G3M and G3F) were noted.
Decreases and increases in urine volume in males and females respectively are considered incidental because of the different direction of effect between sexes. Increase in urobilinogen is considered incidental in the absence of any dose responsiveness.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed in the neurological/functional examination battery and motor activity assessment carried out during Week 13 for G1 and G4 group animals. Statistically significant decreases in excessive grooming, hindlimb foot splay and average hindlimb grip strength were recorded in G4 females. These variations were not associated with any changes in other neurological parameters nor with any clinical signs, hence were considered as incidental and not related to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes were noted:
Absolute organ weights: Decreased thymus weight in G4M and G5M and increased in G3M; decreased spleen weight in G4M; decreased liver weight in G4M, G3F and G4F; decreased prostate + seminal vesicles with coagulating gland weight in G4M; decreased lungs weight in G2M, G3M and G5M, decreased brain weight in G4F and increased uterus weight in G2F and spleen weight in G5F.
Relative to body weight organ weights: Decreased relative thymus weight in G5M, decreased relative lungs weight in G2M and G3M and increased relative kidneys weight in G4M and G4F; increased relative adrenals, heart, brain, liver, lungs, thyroid and parathyroid and pituitary weights in G4F; increased relative uterus weight in G2F and G4F, and increased relative spleen weight in G5F.
Variations observed in thymus and brain weights in G4 and G5 were associated with microscopic changes and are considered as treatment related. All other variations noted in organ weights were considered incidental due to lack of dose responsiveness and/or because there were no associated changes in biochemical parameters and no microscopic changes in the high dose group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test material-related gross pathological changes were observed at necropsy. Single incidences of reduced size of testes and epididymides were observed in G2 and G3 males which microscopically correlated with tubular degeneration in testes and cell debris in epididymides. These findings were considered as spontaneous findings commonly observed in laboratory animals.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopically, test material-related changes were observed in the brain and thymus of both sexes at 5 000 ppm (G4) and at 12 000/4 000 ppm (G5). In the brain, minimal to mild neuronal necrosis characterised by cytoplasmic shrinkage with intense eosinophilia (red neuron) accompanied by karyorrhexis or karyolysis of nucleus in the piriform cortex and/or cornu ammonis (CA1&2) was observed.
In the thymus, minimal to moderate depletion of lymphocytes was observed at 5 000 ppm (G4) and at 12 000/4 000 ppm (G5) in both sexes. This depletion was characterised by loss of lymphocytes in the cortical region.
All other microscopic findings observed in this study were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats of this age.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Vaginal Smear Examination: Vaginal smear examination revealed normal stages of oestrous cyclicity in all the group females.

Thyroid Hormones - T3 (Triiodothyronine), T4 (Thyroxine) and Thyroid Stimulating Hormone (TSH): No treatment related changes in thyroid hormones (T3, T4 and TSH) were noted. T3 levels were statistically significantly decreased in G2 (750 ppm), G3 (2 000 ppm) and G4 (5 000 ppm) males but not in G5 (12 000/4 000 ppm) males and in G2 (750 ppm) and G4 (5 000 ppm) females. The recorded decrease in T3 levels appear to be due to particularly high values in control animals rather than a treatment-related effect. T4 levels were statistically significantly increased in G3 (2 000 ppm) and G4 (5 000 ppm) males and females, but not in G5 animals, and with no clear dose-relationship. Also in this case individual control values were at or below the lower limit of historical control ranges while all individual values of treated animals were well within historical control ranges. No significant difference from control values was noted in TSH levels in either sex.
In the absence of associated macroscopic or microscopic changes in thyroid of G4 group (high dose - 5 000 ppm), the observed changes are considered as incidental.
Key result
Dose descriptor:
NOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
163 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
199.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
4 000 ppm
System:
nervous system
Organ:
brain
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
4 000 ppm
System:
immune system
Organ:
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

SUMMARY OF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATION AND MORTALITY RECORD

Group, Sex & Dose

(ppm in Diet)

No. of

Animals

Clinical Sign(s)

(Total Number of Affected Animals /Total Number of Animals)

Mortality

(No. of Incidences/ No. of Animals)

G1, M & 0

10

N (10/10)

0/10

G2, M & 750

10

N (10/10)

0/10

G3, M & 2000

10

N (10/10)

0/10

G4, M & 5000

10

N (10/10)

0/10

G5, M & 12000/4000

10

2 (7/10), 117 (6/10)

0/10

G1, F & 0

10

N (10/10)

0/10

G2, F & 750

10

N (10/10)

0/10

G3, F & 2000

10

N (10/10)

0/10

G4, F & 5000

10

N (10/10)

0/10

G5, F & 12000/4000

10

2 (9/10), 117 (5/10)

0/10

M: Male; F: Female; N: Normal; 2: Dehydration; 117: Piloerection

Note: From Day 6 onwards, the dose level was reduced from 12 000 ppm in the diet to 4 000 ppm in the diet. The weighted average dose of G5 is 4 444 ppm.

SUMMARY OF BODY WEIGHTS (g) RECORD

Group, Sex & Dose

(ppm in diet)

Body Weight (g) on Days

1

3

8

15

22

29

36

43

50

57

64

71

78

85

91

G1, M & 0

Mean

167.35

175.44

193.21

229.18

243.13

280.24

291.28

300.36

326.58

345.21

362.62

373.22

383.04

389.77

396.32

±SD

8.55

10.75

17.18

19.41

18.56

27.49

26.04

37.99

38.13

39.85

40.61

39.92

40.55

42.60

44.35

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, M & 750

Mean

167.42

173.88

187.95

223.58

244.84

273.23

280.52

296.73

320.54

340.84

358.91

368.73

379.02

384.92

394.71

±SD

9.39

11.12

13.72

19.55

23.83

32.04

33.74

47.30

50.00

53.87

55.53

54.53

53.92

53.78

56.19

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, M & 2000

Mean

167.26

179.23

202.76

235.26

262.79

286.94

296.51

312.38

340.88

357.43

373.24

382.80

390.80

397.09

405.58

±SD

9.07

8.12

15.00

17.16

24.02

30.85

34.64

38.92

41.39

43.11

44.47

44.38

45.47

45.56

45.88

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, M & 5000

Mean

167.71

172.30

176.93*

207.19*

238.68

273.63

283.18

296.98

315.55

326.77

339.63

334.51

329.29*

331.54*

334.23*

±SD

8.98

10.33

11.43

14.01

16.30

20.52

26.92

38.53

43.69

44.99

46.70

51.36

50.38

53.94

55.85

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G5, M & 12000/4000

Mean

168.20

143.98*

156.69*

196.48*

231.97

271.81

283.32

293.57

316.94

335.24

350.91

360.09

368.94

373.04

378.09

±SD

8.53

7.05

13.17

18.16

16.47

24.06

26.52

28.19

27.18

30.94

31.89

31.71

31.43

31.72

33.57

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

M: Male; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

SUMMARY OF BODY WEIGHT (g) RECORD

Group, Sex & Dose

(ppm in diet)

Body Weight (g) on Days

1

3

8

15

22

29

36

43

50

57

64

71

78

85

91

G1, F & 0

Mean

152.82

158.16

170.72

190.25

203.11

215.62

224.74

236.61

246.64

256.06

263.87

268.68

274.80

277.42

280.46

±SD

9.38

9.49

10.22

11.53

12.07

10.77

10.85

9.86

12.83

12.78

12.36

12.19

12.43

12.88

12.70

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, F & 750

Mean

152.82

157.03

167.59

186.68

195.51

209.32

217.01

224.85

234.49

241.69

249.40

254.46

259.61

262.43

267.62

±SD

9.56

9.60

11.61

9.15

9.82

6.50

7.28

9.15

9.08

9.71

9.57

8.80

8.63

8.93

8.99

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, F & 2000

Mean

153.14

157.37

169.24

189.15

202.48

210.95

223.09

231.68

241.50

249.92

258.47

263.70

268.59

271.38

275.27

±SD

10.18

9.30

13.13

14.68

14.12

13.71

17.57

20.22

21.81

21.32

21.60

21.39

20.05

23.48

22.62

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, F & 5000

Mean

152.33

144.77*

155.24*

174.10*

187.68*

198.73*

204.68*

202.76*

208.08*

215.34*

222.29*

220.18*

215.26*

214.82*

214.23*

±SD

8.89

16.12

15.81

13.83

13.56

12.09

13.66

15.62

14.78

14.39

14.12

15.85

15.84

12.71

12.09

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G5, F & 12000/4000

Mean

152.54

135.41*

144.39*

179.10

196.47

208.46

214.05

224.73

238.87

245.57

253.17

257.87

262.92

265.81

268.03

±SD

8.79

9.09

9.02

10.96

9.87

12.30

9.22

20.24

24.07

25.18

24.87

25.02

25.63

23.68

22.15

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 F: Female; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose

(ppm in diet)

Percent Change in Body Weight (%) During Days

1 to 3

1 to 8

1 to 15

1 to 22

1 to 29

1 to 36

1 to 43

1 to 50

1 to 57

1 to 64

1 to 71

1 to 78

1 to 85

1 to 91

G1, M & 0

Mean

4.82

15.38

37.00

45.46

67.76

74.42

79.94

95.57

106.77

117.27

123.61

129.52

133.56

137.49

±SD

2.60

7.12

10.06

11.34

17.77

17.77

25.16

24.71

26.24

27.52

27.27

27.93

29.15

30.21

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, M & 750

Mean

3.88

12.35

33.87

46.62

63.65

68.05

78.06

92.12

104.41

115.19

121.09

127.24

130.81

136.74

±SD

3.96

7.02

13.32

16.09

21.26

22.56

32.32

32.94

36.28

37.13

36.87

36.60

36.95

38.80

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, M & 2000

Mean

7.21

21.18

40.62

57.00

71.46

77.14

86.61

103.74

113.68

123.13

128.91

133.70

137.54

142.53

±SD

1.79

5.13

6.23

9.85

15.41

17.35

20.00

22.19

23.55

24.11

24.61

25.26

26.08

25.17

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, M & 5000

Mean

2.72

5.49*

23.59*

42.42

63.27

68.82

76.88

87.95

94.65

102.27

99.14

96.04*

97.25*

98.76*

±SD

2.52

4.02

6.41

8.27

10.71

12.91

19.13

22.16

22.88

23.37

26.18

25.84

27.28

28.02

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G5, M & 12000/4000

Mean

-14.33*

-6.88*

16.69*

37.97

61.65

68.45

74.70

88.62

99.50

108.80

114.29

119.57

122.04

125.07

±SD

3.63

5.57

7.07

7.96

12.54

13.53

16.66

16.28

18.28

18.52

18.74

18.75

19.39

20.73

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

M: Male; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose

(ppm in diet)

Percent Change in Body Weight (%) During Days

1 to 3

1 to 8

1 to 15

1 to 22

1 to 29

1 to 36

1 to 43

1 to 50

1 to 57

1 to 64

1 to 71

1 to 78

1 to 85

1 to 91

G1, F & 0

Mean

3.61

11.93

24.82

33.31

41.53

47.49

55.46

61.97

68.17

73.27

76.42

80.41

82.13

84.12

±SD

4.98

6.88

9.34

10.71

10.50

10.19

13.05

13.45

13.95

13.75

13.81

13.64

13.90

13.79

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, F & 750

Mean

2.77

9.67

22.27

28.11

37.31

42.28

47.46

53.81

58.53

63.60

66.93

70.35

72.18

75.53

±SD

0.99

3.20

3.02

5.18

6.93

5.82

7.68

8.37

8.74

8.89

9.02

9.87

9.66

8.82

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, F & 2000

Mean

2.87

10.68

23.86

32.67

38.24

46.28

51.87

58.23

63.71

69.33

72.74

75.97

77.75

80.29

±SD

3.72

8.07

11.15

12.04

12.37

15.47

16.47

16.74

16.27

16.90

16.67

16.28

17.58

17.02

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, F & 5000

Mean

-5.18*

1.78*

14.23*

23.23*

30.56*

34.47*

33.26*

37.04*

41.84*

46.41*

44.93*

41.58*

41.33*

41.00*

±SD

6.10

6.43

4.62

5.83

5.73

7.17

9.54

13.28

13.40

13.46

13.05

11.44

10.20

10.65

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G5, F & 12000/4000

Mean

-11.24*

-5.29*

17.53

28.99

36.90

40.71

47.57

56.93

61.35

66.35

69.44

72.76

74.66

76.10

±SD

2.59

3.95

6.12

6.33

8.73

9.49

13.45

16.75

17.69

17.65

17.80

18.24

17.08

15.98

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

F: Female; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)  

SUMMARY OF FEED CONSUMPTION (g/rat/day) RECORD

Group, Sex & Dose

(ppm in diet)

Week 1-Day (1-3)

Week 1-Day (3-6)

Week 1-Day (3-8)

Week

2

Week

3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

G1, M & 0

Mean

17.70

-

23.72

24.49

24.51

24.45

24.53

24.82

24.86

25.66

26.53

26.86

26.55

25.93

25.67

±SD

0.86

-

1.65

0.72

1.39

0.59

1.17

1.08

1.44

1.33

1.37

0.96

0.89

0.79

1.02

n

5

-

5

5

5

5

5

5

5

5

5

5

5

5

5

G2, M & 750

Mean

17.93

--

20.41

24.17

24.19

24.22

24.26

24.69

24.96

25.35

25.89

26.27

26.14

25.60

25.32

±SD

0.51

-

1.61

0.72

0.51

0.71

1.10

0.92

1.68

1.42

1.36

0.86

0.94

0.75

0.79

n

5

-

5

5

5

5

5

5

5

5

5

5

5

5

5

G3, M & 2000

Mean

17.87

--

22.39

24.29

24.14

24.42

24.21

25.03

25.27

25.84

26.69

26.96

26.74

26.26

26.01

±SD

1.20

-

2.61

0.83

0.47

1.05

1.52

1.44

1.38

0.84

0.69

0.48

0.63

0.76

0.57

n

5

-

5

5

5

5

5

5

5

5

5

5

5

5

5

G4, M & 5000

Mean

12.84*

--

14.20*

17.48*

17.77*

17.91*

17.92*

18.99*

19.26*

20.54*

20.65*

20.72*

20.60*

20.31*

19.95*

±SD

0.68

-

2.75

4.28

1.24

0.52

1.36

1.11

1.16

1.46

1.27

1.20

1.22

1.08

1.02

n

5

-

5

5

5

5

5

5

5

5

5

5

5

5

5

G5, M & 12000/4000

Mean

9.63*

10.24

10.00*

18.01*

18.10*

18.52*

18.70*

19.51*

20.03*

20.97*

21.75*

22.51*

22.34*

22.08*

21.83*

±SD

1.13

0.81

1.19

3.12

0.79

1.17

1.18

1.11

0.99

1.33

1.24

1.40

1.31

1.24

1.13

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

M: Male, SD: Standard Deviation; n: Number of cages; -: Not applicable; *: Statistically significant (p<0.05)

SUMMARY OF FEED CONSUMPTION (g/rat/day) RECORD

Group, Sex & Dose

(ppm in diet)

Week 1-Day (1-3)

Week 1-Day (3-6)

Week 1-Day (3-8)

Week

2

Week

3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

G1, F & 0

Mean

16.01

-

20.63

22.43

22.62

22.67

22.90

23.42

23.55

24.08

23.61

23.75

23.67

23.09

23.41

±SD

0.61

-

2.09

1.62

1.53

0.69

0.92

0.71

1.11

0.96

1.02

1.33

1.55

1.18

1.54

n

5

-

5

5

5

5

5

5

5

5

5

5

5

5

5

G2, F & 750

Mean

15.53

--

20.47

22.84

22.45

22.50

22.93

23.35

23.82

24.63

23.86

24.07

23.91

23.54

23.03

±SD

0.89

-

3.47

0.93

0.61

0.70

1.30

0.68

1.48

1.00

1.23

1.33

1.41

1.25

1.07

n

5

-

5

5

5

5

5

5

5

5

5

5

5

5

5

G3, F & 2000

Mean

15.64

--

19.57

21.96

21.81

22.90

23.00

23.36

23.69

24.39

23.48

23.56

23.46

22.94

22.58

±SD

0.55

-

4.49

0.87

0.33

0.46

0.83

0.92

1.47

1.51

1.25

1.33

1.51

1.43

1.59

n

5

-

5

5

5

5

5

5

5

5

5

5

5

5

5

G4, F & 5000

Mean

11.51*

--

13.27*

15.53*

17.05*

17.98*

18.12*

19.14*

19.67*

20.83*

19.95*

20.05*

19.89*

19.34*

19.02*

±SD

0.97

-

5.96

1.71

0.81

1.05

1.57

0.82

1.41

1.08

0.83

0.63

0.88

0.57

0.55

n

5

-

5

5

5

5

5

5

5

5

5

5

5

5

5

G5, F & 12000/4000

Mean

9.25*

9.15

9.23*

18.92*

19.77*

20.13*

20.48*

21.04*

21.80

22.61

22.25

22.39

22.29

21.88

22.15

±SD

0.49

0.71

0.94

2.35

0.58

0.80

1.43

0.67

0.48

0.62

0.84

0.85

0.90

1.00

0.84

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

F: Female; SD: Standard Deviation; n: Number of cages; -: Not applicable; *: Statistically significant (p<0.05)

SUMMARY OF OPHTHALMOSCOPIC EXAMINATION RECORD

Week 13

Group, Sex & Dose

(ppm in diet)

G1, M & 0

G4, M & 5000

G1, F & 0

G4, F & 5000

Number of Animals

10

10

10

10

Observations

Eye

L

R

L

R

L

R

L

R

Eye Lids

N

N

N

N

N

N

N

N

Cornea

N

N

N

N

N

N

N

N

Iris

N

N

N

N

N

N

N

N

Aqueous Humour

N

N

N

N

N

N

N

N

Lens

N

N

N

N

N

N

N

N

Vitreous Humour

N

N

N

N

N

N

N

N

Retina/Optic disc

N

N

N

N

N

N

N

N

N: No Abnormality Detected; L: Left; R: Right; M: Male; F: Female

SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

Week 13

Parameters↓

Group & Sex

G1 & M

G4 & M

G1 & F

G4 & F

Dose(ppm in diet)

0

5000

0

5000

Number of Animals

10

10

10

10

Home cage observations

Home Cage posture

1

1

1

1

Respiratory pattern

1

1

1

1

Clonic involuntary movements

1

1

1

1

Tonic involuntary movements

1

1

1

1

Vocalization

1

1

1

1

Palpebral Closure

1

1

1

1

Handling observation 

Ease of removal from the cage

2

2

2

2

Ease of handling animal in hand

2

2

2

2

Red or crusty deposits

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

Lacrimation

1

1

1

1

Salivation

1

1

1

1

Fur Appearance

1

1

1

1

Piloerection

1

1

1

1

Eye Prominence

1

1

1

1

Muscle Tone

1

1

1

1

Home cage observations: Home cage posture - 1=Normal, Respiratory Pattern - 1=Normal, Clonic involuntary movements - 1=None/Normal, Tonic involuntary movements - 1=None/Normal, Vocalization - 1=Absent (Normal), Palpebral closure - 1=Eyelids wide open (Normal), Handling observation: Ease of removal from the cage - 2=Normal, Ease of handling animal in hand - 2=Normal, Red/Crusty deposits - 1=Absent, Lacrimation - 1=None, Salivation- 1=Absent (Normal), Fur Appearance - 1=Normal, Piloerection - 1=None, Eye Prominence - 1=Normal, Muscle tone - 1=Normal

M: Male; F: Female

SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

Week 13

Parameters↓

Group & Sex

G1 & M

G4 & M

G1 & F

G4 & F

Dose(ppm in diet)

0

5000

0

5000

Number of Animals

10

10

10

10

Open field Observation

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of Rearing

Mean

5.9

6.2

6.1

5.6

±SD

0.7

0.9

1.1

1.1

Numbers of Urination

Mean

1.1

1.0

1.2

1.1

±SD

0.6

0.5

0.4

0.3

Number of Defecation

Mean

0.9

1.1

1.2

0.8

±SD

0.6

0.3

0.6

0.6

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype Behaviour

1

1

1

1

Excessive Grooming

Mean

3.6

3.2

3.8

2.8*

±SD

0.7

0.8

0.8

0.6

Sensory Observations

Approach Response

2

2

2

2

Auditory Response

2

2

2

2

Touch Response  

2

2

2

2

Pupil Reflex

2

2

2

2

Tail Pinch Response

2

2

2

2

Righting Reflex

1

1

1

1

Physiological observation

 

Body temperature (°F)

Mean

96.8

96.6

96.8

96.8

±SD

0.2

0.2

0.3

0.4

Open field Observation: Mobility - 1=Normal, Gait - 1=Normal, Arousal - 3=Normal, Clonic involuntary movements - 1=None/Normal, Tonic involuntary movements - 1=None/Normal, Stereotype Behaviour - 1=Absent, Sensory Observations: Approach response - 2=Normal, Auditory Response - 2=Normal, Touch Response - 2=Normal, Pupil Reflex - 2=Normal, Tail Pinch Response - 2=Normal, Righting Reflex - 1=Present

M: Male;F: Female; *: Statistically significant (p<0.05)

SUMMARY OF LANDING FOOT SPLAY (cm) RECORD

Week 13

Group, Sex & Dose

(ppm in diet)

Hind Limb Foot Splay

(cm)

G1, M & 0

Mean

7.8

±SD

0.7

n

10

G4, M & 5000

Mean

7.9

±SD

0.4

n

10

G1, F & 0

Mean

7.0

±SD

0.6

n

10

G4, F & 5000

Mean

6.4*

±SD

0.6

n

10

M: Male; F: Female; SD: Standard Deviation; n: number of animals; *: Statistically significant (p<0.05)

SUMMARY OF ACTIMETER READING RECORD

Week 13

Group, Sex & Dose

(ppm in diet)

Movement Counts

(no.)

G1, M & 0

Mean

2522.2

±SD

186.8

n

10

G4, M & 5000

Mean

2533.4

±SD

187.3

n

10

G1, F & 0

Mean

2598.2

±SD

105.3

n

10

G4, F & 5000

Mean

2475.4

±SD

159.2

n

10

M: Male; F: Female; SD: Standard Deviation; n: number of animals

SUMMARY OF GRIP STRENGTH (kgf) MEASUREMENT RECORD

Week 13

Group, Sex &

Dose (ppm in diet)

Fore Limb

Hind Limb

G1, M & 0

Mean

1.491

0.495

±SD

0.043

0.033

n

10

10

G4, M & 5000

Mean

1.471

0.478

±SD

0.036

0.026

n

10

10

G1, F & 0

Mean

1.269

0.248

±SD

0.023

0.022

n

10

10

G4, F & 5000

Mean

1.255

0.229*

±SD

0.021

0.014

n

10

10

M: Male; F: Female; SD: Standard Deviation; n: number of animals; *: Statistically significant (p<0.05)

SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose

(ppm in diet)

Total Leucocyte Count

Total Erythrocyte Count

Haemoglobin

Haematocrit

Mean Corpuscular Volume

Mean Corpuscular Haemoglobin

Mean Corpuscular Haemoglobin Concentration

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(10^3 cells/µL)

(10^6 cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

G1, M & 0

Mean

11.36

7.75

13.78

42.75

55.23

17.86

32.31

±SD

2.94

0.59

0.86

2.53

1.40

1.66

2.36

n

10

10

10

10

10

10

10

G2, M & 750

Mean

11.51

7.79

13.91

44.06

56.63

17.85

31.51

±SD

3.34

0.42

0.81

1.84

1.68

0.65

0.61

n

10

10

10

10

10

10

10

G3, M & 2000

Mean

9.09

7.89

13.83

43.51

55.24

17.56

31.83

±SD

3.16

0.70

1.08

3.02

1.84

0.54

0.57

n

10

10

10

10

10

10

10

G4, M & 5000

Mean

9.81

7.99

13.89

43.50

54.53

17.42

31.94

±SD

2.79

0.78

1.07

3.33

1.83

0.66

0.45

n

10

10

10

10

10

10

10

G5, M & 12000/4000

Mean

12.07

7.67

13.63

42.16

55.00

17.75

32.28

±SD

3.58

0.39

0.63

2.02

2.06

0.52

0.59

n

10

10

10

10

10

10

10

M: Male; R: Recovery; SD: Standard Deviation; n: number of animals

SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose

(ppm in diet)

Platelet Count

Mean Platelet Volume

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

(PLT)

(MPV)

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(10^3 cells/µL)

(fL)

(%)

(%)

(%)

(%)

(%)

(%)

G1, M & 0

Mean

715.30

6.18

1.59

21.90

72.72

3.14

0.88

0.29

±SD

87.90

0.29

0.20

4.90

5.22

0.77

0.29

0.14

n

10

10

10

10

10

10

10

10

G2, M & 750

Mean

689.40

6.02

1.63

22.19

71.48

3.74

1.20

0.29

±SD

163.76

0.19

0.35

2.84

2.81

1.48

0.58

0.09

n

10

10

10

10

10

10

10

10

G3, M & 2000

Mean

691.10

6.23

1.46

28.24

65.84

2.94

1.77

0.21

±SD

152.53

0.44

0.60

11.05

10.58

0.96

2.48

0.10

n

10

10

10

10

10

10

10

10

G4, M & 5000

Mean

740.80

6.32

1.52

33.51*

60.77*

3.87

0.77

0.25

±SD

100.36

0.22

0.52

9.29

9.19

1.13

0.45

0.08

n

10

10

10

10

10

10

10

10

G5, M & 12000/4000

Mean

708.90

6.36

1.69

32.87

61.07

3.90

1.02

0.27

±SD

141.32

0.29

0.78

16.04

17.58

2.49

0.75

0.12

n

10

10

10

10

10

10

10

10

M: Male; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose

(ppm in diet)

Absolute Reticulocyte Count

Absolute Neutrophils

Absolute Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated Prothrombin Time

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(10^9 cells/L)

(10^9 cells/µL)

(10^3 cells/µL)

(10^3 cells/µL)

(10^3 cells/µL)

(10^3 cells/µL)

(Seconds)

(Seconds)

G1, M & 0

Mean

123.28

2.40

8.36

0.34

0.10

0.04

15.59

26.64

±SD

18.10

0.43

2.59

0.07

0.03

0.02

1.29

1.69

n

10

10

10

10

10

10

10

10

G2, M & 750

Mean

126.78

2.54

8.26

0.42

0.14

0.03

16.86

25.47

±SD

25.13

0.71

2.56

0.15

0.07

0.01

1.66

1.22

n

10

10

10

10

10

10

10

10

G3, M & 2000

Mean

112.77

2.40

6.11

0.27

0.18

0.02

19.21*

26.42

±SD

44.77

0.82

2.59

0.14

0.32

0.02

5.46

1.98

n

10

10

10

10

10

10

10

10

G4, M & 5000

Mean

118.43

3.12

6.13

0.37

0.08

0.03

16.05

27.49

±SD

32.52

0.55

2.48

0.13

0.06

0.02

1.99

5.85

n

10

10

10

10

10

10

10

10

G5, M & 12000/4000

Mean

128.32

3.60*

7.79

0.43

0.12

0.04

17.18

24.78

±SD

57.50

1.01

3.73

0.15

0.09

0.02

1.85

2.09

n

10

10

10

10

10

10

10

10

M: Male; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose

(ppm in diet)

Total Leucocyte Count

Total Erythrocyte Count

Haemoglobin

Haematocrit

Mean Corpuscular Volume

Mean Corpuscular Haemoglobin

Mean Corpuscular Haemoglobin Concentration

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(10^3 cells/µL)

(10^6 cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

G1, F & 0

Mean

8.26

7.21

13.24

40.85

56.69

18.38

32.42

±SD

2.50

0.46

0.72

2.21

1.33

0.39

0.23

n

10

10

10

10

10

10

10

G2, F & 750

Mean

8.52

6.97

12.89

40.57

58.36

18.47

31.73

±SD

2.53

0.61

1.11

2.17

2.98

0.38

1.45

n

10

10

10

10

10

10

10

G3, F & 2000

Mean

8.58

7.33

13.36

42.58

58.12

18.24

31.41

±SD

2.10

0.40

0.65

2.76

1.98

0.42

1.22

n

10

10

10

10

10

10

10

G4, F & 5000

Mean

7.94

6.80

12.42

39.14

57.64

18.24

31.64

±SD

2.65

0.78

1.52

3.81

1.79

0.64

1.01

n

10

10

10

10

10

10

10

G5, F & 12000/4000

Mean

9.84

6.87

12.85

40.47

59.02

18.73

31.74

±SD

3.22

0.67

0.99

2.89

2.11

0.61

0.44

n

10

10

10

10

10

10

10

F: Female; SD: Standard Deviation; n: Number of animals.

 

SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose

(ppm in diet)

Platelet Count

Mean Platelet Volume

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

(PLT)

(MPV)

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(10^3 cells/µL)

(fL)

(%)

(%)

(%)

(%)

(%)

(%)

G1, F & 0

Mean

739.20

6.26

1.71

22.65

71.36

3.38

1.29

0.20

±SD

121.61

0.30

0.33

4.42

4.92

0.95

0.41

0.07

n

10

10

10

10

10

10

10

10

G2, F & 750

Mean

778.70

6.34

2.57

23.92

69.71

3.79

1.20

0.20

±SD

69.29

0.47

2.14

6.72

7.51

0.88

0.42

0.09

n

10

10

10

10

10

10

10

10

G3, F & 2000

Mean

742.40

6.26

2.01

25.00

69.60

3.31

0.81*

0.22

±SD

145.51

0.24

0.81

3.59

4.19

1.12

0.41

0.09

n

10

10

10

10

10

10

10

10

G4, F & 5000

Mean

720.80

6.57

2.30

31.81*

62.22*

3.91

0.96

0.20

±SD

94.85

0.46

0.73

12.29

12.40

1.42

0.49

0.11

n

10

10

10

10

10

10

10

10

G5, F & 12000/4000

Mean

751.00

6.55

1.55

24.20

70.20

3.40

1.00

0.24

±SD

178.51

0.37

0.35

4.21

3.96

0.66

0.34

0.08

n

10

10

10

10

10

10

10

10

F: Female; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose

(ppm in diet)

Absolute Reticulocyte Count

Absolute Neutrophils

Absolute Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated Prothrombin Time

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(10^9 cells/L)

(10^3 cells/µL)

(10^3 cells/µL)

(10^3 cells/µL)

(10^3 cells/µL)

(10^3 cells/µL)

(Seconds)

(Seconds)

G1, F & 0

Mean

122.53

1.82

5.94

0.28

0.11

0.02

17.22

24.96

±SD

19.82

0.47

2.03

0.12

0.05

0.01

1.71

4.14

n

10

10

10

10

10

10

10

10

G2, F & 750

Mean

170.82

1.95

6.04

0.31

0.10

0.02

15.20*

24.47

±SD

118.46

0.56

2.28

0.09

0.05

0.01

0.96

3.23

n

10

10

10

10

10

10

10

10

G3, F & 2000

Mean

146.96

2.14

5.97

0.29

0.07

0.02

16.36

25.85

±SD

59.24

0.62

1.55

0.14

0.04

0.01

1.79

6.15

n

10

10

10

10

10

10

10

10

G4, F & 5000

Mean

155.92

2.46

5.00

0.31

0.08

0.02

15.62*

23.21

±SD

49.93

1.06

2.22

0.14

0.05

0.01

1.17

1.99

n

10

10

10

10

10

10

10

10

G5, F & 12000/4000

Mean

105.54

2.34

6.94

0.34

0.10

0.02

15.17*

25.64

±SD

22.23

0.75

2.35

0.15

0.06

0.02

0.89

3.04

n

10

10

10

10

10

10

10

10

F: Female; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose

(ppm in diet)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

High-density lipoprotein

Low-density lipoprotein

Total Protein

Albumin

Alanine aminotransferase

Aspartate aminotransferase

(GLU)

(CRE)

(CHO)

(TRI)

(HDL)

(LDL)

(TPR)

(ALB)

(ALT)

(AST)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

(U/L)

(U/L)

G1, M & 0

Mean

109.60

33.16

0.52

47.60

60.10

10.66

3.93

6.54

3.14

56.20

94.10

±SD

18.51

7.63

0.03

9.00

17.20

1.88

2.11

0.37

0.21

9.95

12.27

n

10

10

10

10

10

10

10

10

10

10

10

G2, M & 750

Mean

98.00

31.16

0.51

45.60

63.70

9.87

3.27

6.45

3.21

47.50

85.50

±SD

8.45

2.89

0.03

6.82

17.90

1.97

1.10

0.32

0.14

5.68

9.47

n

10

10

10

10

10

10

10

10

10

10

10

G3, M & 2000

Mean

103.20

34.78

0.54

40.00

46.60

8.87

3.15

6.22

3.20

66.70

111.00

±SD

15.58

9.50

0.06

7.45

14.84

1.94

0.66

0.25

0.15

42.58

44.58

n

10

10

10

10

10

10

10

10

10

10

10

G4, M & 5000

Mean

109.90

47.98*

0.52

48.10

40.10*

11.39

5.01

6.51

3.24

59.30

213.40*

±SD

7.14

13.67

0.05

6.97

17.70

1.51

1.89

0.37

0.20

19.00

144.41

n

10

10

10

10

10

10

10

10

10

10

10

G5, M & 12000/4000

Mean

109.90

34.00

0.58*

49.90

51.30

11.57

4.75

6.51

3.22

81.40

176.40

±SD

17.09

9.11

0.05

15.60

18.30

3.18

1.39

0.28

0.21

94.33

154.89

n

10

10

10

10

10

10

10

10

10

10

10

M: Male; SD: Standard Deviation; n: number of animals; *: Statistically significant (p<0.05)

SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose

(ppm in diet)

Alkaline phosphatase

Total Bilirubin

Calcium

Phosphorous

Cholinesterase

Globulin

Albumin/Globulin ratio

Blood Urea Nitrogen

Sodium

Potassium

Chloride

(ALP)

(BIT)

(CAL)

(PHO)

(CHE)

(GLO)

(A/G Ratio)

(BUN)

(Na)

(K)

(CLO)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

(U/L)

(g/dL)

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1, M & 0

Mean

130.50

0.04

9.57

5.51

189.60

3.40

0.93

15.48

143.25

3.65

110.31

±SD

44.53

0.02

0.40

0.42

109.32

0.34

0.11

3.56

0.66

0.17

1.09

n

10

10

10

10

10

10

10

10

10

10

10

G2, M & 750

Mean

139.60

0.04

9.72

5.30

154.10

3.24

1.00

14.54

143.32

3.58

109.87

±SD

35.41

0.02

0.30

0.52

31.59

0.20

0.04

1.35

0.83

0.17

1.94

n

10

10

10

10

10

10

10

10

10

10

10

G3, M & 2000

Mean

123.10

0.06*

9.55

4.99

159.80

3.02*

1.06*

16.23

143.66

3.65

110.37

±SD

30.49

0.01

0.27

0.44

35.40

0.15

0.06

4.44

0.92

0.27

1.43

n

10

10

10

10

10

10

10

10

10

10

10

G4, M & 5000

Mean

190.80*

0.07*

9.52

5.44

149.20

3.27

0.99

22.39*

143.54

3.45

109.54

±SD

52.16

0.02

0.37

0.68

24.12

0.29

0.10

6.38

0.92

0.11

1.35

n

10

10

10

10

10

10

10

10

10

10

10

G5, M & 12000/4000

Mean

151.10

0.06*

8.84*

5.23

169.60

3.29

0.98

15.87

144.20

3.32

111.16

±SD

51.78

0.02

0.30

0.56

59.58

0.22

0.10

4.25

1.19

0.64

1.59

n

10

10

10

10

10

10

10

10

10

10

10

M: Male; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose

(ppm in diet)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

High-density lipoprotein

Low-density lipoprotein

Total Protein

Albumin

Alanine aminotransferase

Aspartate aminotransferase

(GLU)

(CRE)

(CHO)

(TRI)

(HDL)

(LDL)

(TPR)

(ALB)

(ALT)

(AST)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

(U/L)

(U/L)

G1, F & 0

Mean

115.20

34.73

0.65

53.50

38.10

15.05

2.23

6.85

3.59

51.10

91.10

±SD

11.80

4.84

0.05

15.62

11.09

3.94

0.56

0.32

0.22

12.56

16.20

n

10

10

10

10

10

10

10

10

10

10

10

G2, F & 750

Mean

121.00

37.33

0.68

46.00

35.20

13.07

2.00

6.75

3.51

43.00

85.80

±SD

13.23

4.13

0.06

8.25

6.46

2.62

0.39

0.32

0.16

7.54

7.41

n

10

10

10

10

10

10

10

10

10

10

10

G3, F & 2000

Mean

120.30

44.19

0.71

49.60

39.40

13.09

2.70

6.75

3.45

44.00

109.90

±SD

10.40

7.84

0.06

12.64

11.50

3.46

0.79

0.38

0.21

10.11

43.48

n

10

10

10

10

10

10

10

10

10

10

10

G4, F & 5000

Mean

149.60*

58.00*

0.70

50.30

32.60

12.88

3.19*

6.36*

3.27*

55.80

302.60*

±SD

35.78

23.81

0.08

18.87

13.24

5.21

1.06

0.71

0.31

14.79

237.06

n

10

10

10

10

10

10

10

10

10

10

10

G5, F & 12000/4000

Mean

132.30

39.92

0.69

50.20

34.50

12.92

2.81

6.67

3.41

53.40

110.20

±SD

21.44

4.66

0.04

9.52

7.46

3.17

0.53

0.26

0.23

25.19

50.21

n

10

10

10

10

10

10

10

10

10

10

10

F: Female; SD: Standard Deviation; n: number of animals; *: Statistically significant (p<0.05)

 

SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose

(ppm in diet)

Alkaline phosphatase

Total Bilirubin

Calcium

Phosphorous

Cholinesterase

Globulin

Albumin/Globulin ratio

Blood Urea Nitrogen

Sodium

Potassium

Chloride

(ALP)

(BIT)

(CAL)

(PHO)

(CHE)

(GLO)

(A/G Ratio)

(BUN)

(Na)

(K)

(CLO)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

(U/L)

(g/dL)

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1, F & 0

Mean

64.30

0.06

9.35

4.12

886.90

3.26

1.10

16.21

143.52

3.42

110.90

±SD

10.51

0.02

0.28

0.60

166.81

0.17

0.07

2.26

0.83

0.17

1.41

n

10

10

10

10

10

10

10

10

10

10

10

G2, F & 750

Mean

75.50

0.08

9.31

4.32

852.60

3.24

1.09

17.42

143.47

3.34

110.88

±SD

28.47

0.04

0.24

0.77

230.07

0.32

0.12

1.92

1.14

0.26

1.71

n

10

10

10

10

10

10

10

10

10

10

10

G3, F & 2000

Mean

114.50

0.07

9.10

4.59

635.00*

3.30

1.05

20.62

144.35

3.28

112.46

±SD

70.39

0.02

0.42

2.31

208.45

0.23

0.07

3.66

2.19

0.29

2.14

n

10

10

10

10

10

10

10

10

10

10

10

G4, F & 5000

Mean

130.70*

0.08

8.87*

5.23

440.20*

3.10

1.07

27.07*

146.35*

2.86*

114.47*

±SD

50.50

0.01

0.64

1.19

250.03

0.46

0.12

11.11

3.95

0.48

4.08

n

10

10

10

10

10

10

10

10

10

10

10

G5, F & 12000/4000

Mean

94.30

0.05

9.12

4.78

588.80*

3.26

1.05

18.63

144.39

3.29

111.84

±SD

56.74

0.03

0.37

0.73

189.92

0.22

0.12

2.17

1.20

0.35

2.12

n

10

10

10

10

10

10

10

10

10

10

10

F: Female; SD: Standard Deviation; n: number of animals; *: Statistically significant (p<0.05)

SUMMARY OF URINALYSIS RECORD

Examination

Group & Sex

G1 & M

G2 & M

G3 & M

G4 & M

G5 &

M

Dose (ppm in diet)

0

750

2000

5000

12000/4000

Number of Animals

10

10

10

10

10

Physical

Colour

Pale Yellow

10

10

9

10

9

Yellow

-

-

1

-

1

Appearance

Clear

10

7

4

8

8

Turbid

-

3

6

2

2

Volume (mL)

Mean

7.0

6.1

4.7*

5.8*

5.1*

±SD

0.7

1.2

0.9

1.6

0.6

Chemical

pH

Mean

8.4

8.5

8.5

8.5

8.4

±SD

0.3

0.0

0.0

0.0

0.3

Specific Gravity

Mean

1.005

1.005

1.005

1.005

1.006

±SD

0.000

0.000

0.000

0.000

0.002

Urobilinogen (mg/dL)

Mean

0.2

0.4

1.0*

0.2

0.3

±SD

0.0

0.3

0.0

0.0

0.3

Bilirubin (mg/dL)

Neg

10

9

6

10

9

1

-

1

4

-

1

Ketones (mg/dL)

Neg

10

10

10

10

10

Blood (Ery/µL)

Neg

10

10

10

7

8

Ca10

-

-

-

2

2

Ca25

-

-

-

1

-

 

Proteins (mg/dL)

Neg

7

-

-

-

2

Trace

3

-

-

3

-

30

-

-

-

1

4

100

-

-

-

1

3

>=300

-

10

10

5

1

Nitrite

Neg

8

10

10

10

7

Pos

2

-

-

-

3

Leucocytes (Leu/µL)

Neg

8

3

-

7

9

Ca15

2

7

9

3

-

Ca70

-

-

1

-

1

Glucose (mg/dL)

Neg

10

10

10

10

10

Microscopic

Epithelial cells

0

8

6

6

8

7

0-1

2

2

3

1

2

1-2

-

2

1

1

1

Casts

Absent

10

10

10

10

10

Crystals

Present

10

10

10

10

10

M: Male; Neg: Negative; Ca: Calculated Approximately; Pos: Positive; SD: Standard Deviation; >=: more than equal to; *: Statistically significant (p<0.05)

SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g) RECORD

Group, Sex & Dose

(ppm in diet)

Adrenals

Thymus

Spleen

Testes

Epididymis

Heart

Kidneys

Brain

Liver

Prostate + Seminal Vesicles with Coagulating Glands

Lungs

Thyroid along with Parathyroid

Pituitary Gland

G1, M & 0

Mean

0.0726

0.3702

0.7062

3.2673

1.4095

1.3495

3.1178

2.1629

12.0135

3.1544

3.0560

0.0292

0.0179

±SD

0.0092

0.0734

0.1125

0.3328

0.1271

0.1307

0.3156

0.1413

1.0085

0.4980

0.5329

0.0056

0.0020

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, M & 750

Mean

0.0689

0.3308

0.7163

2.7693

1.2676

1.4902

2.7329

2.0890

12.1128

3.1716

2.3502*

0.0305

0.0183

±SD

0.0096

0.0664

0.1106

0.7481

0.2305

0.1042

0.3412

0.0915

1.0680

0.4097

0.2272

0.0025

0.0026

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, M & 2000

Mean

0.0730

0.4437*

0.6486

3.0819

1.2850

1.2536

3.0511

2.2111

11.9603

3.4712

2.2573*

0.0284

0.0174

±SD

0.0143

0.0617

0.1128

0.5410

0.1617

0.1386

0.5866

0.1125

1.3146

0.7617

0.2853

0.0036

0.0019

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, M & 5000

Mean

0.0640

0.2661*

0.5118*

3.1188

1.2820

1.2431

3.4232

2.0499

10.1786*

2.3242*

2.6039

0.0288

0.0175

±SD

0.0095

0.0613

0.0976

0.2395

0.2333

0.1643

0.4287

0.2106

2.1961

0.4964

0.5157

0.0032

0.0027

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G5, M & 12000/4000

Mean

0.0627

0.2624*

0.5635

3.3056

1.3758

1.3834

3.4573

2.0777

11.6877

2.8438

2.5776*

0.0290

0.0168

±SD

0.0114

0.0563

0.2099

0.2580

0.2385

0.2681

0.3865

0.1230

1.5498

0.4145

0.4228

0.0026

0.0023

n

10

10

10

10

10

10

10

10

10

10

10

10

10

M: Male; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g) RECORD

Group, Sex & Dose

(ppm in diet)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

Thyroid along with Parathyroid

Pituitary Gland

G1, F & 0

Mean

0.0743

0.3433

0.4162

0.1342

0.4998

1.0723

2.0470

2.0595

8.1811

1.8623

0.0274

0.0183

±SD

0.0077

0.0718

0.0451

0.0383

0.1241

0.1421

0.1889

0.0794

0.9870

0.2851

0.0054

0.0025

n

10

10

10

10

10

10

10

10

10

10

10

10

G2, F & 750

Mean

0.0704

0.3702

0.4890

0.1509

1.0058*

1.0442

2.0001

2.0156

8.8584

1.9139

0.0274

0.0176

±SD

0.0104

0.0637

0.1437

0.0198

0.2683

0.1220

0.2446

0.1174

0.7790

0.2731

0.0038

0.0013

n

10

10

10

10

10

10

10

10

10

10

10

10

G3, F & 2000

Mean

0.0796

0.3178

0.4855

0.1452

0.6756

1.0003

2.0201

1.9891

7.3375*

1.6913

0.0275

0.0180

±SD

0.0066

0.0625

0.0892

0.0237

0.1744

0.0467

0.2359

0.1423

0.4605

0.1451

0.0024

0.0022

n

10

10

10

10

10

10

10

10

10

10

10

10

G4, F & 5000

Mean

0.0727

0.2841

0.3469

0.1092

0.6163

0.9968

2.1388

1.8903*

7.1107*

1.7549

0.0278

0.0180

±SD

0.0111

0.0524

0.0583

0.0248

0.1940

0.1335

0.2487

0.1542

0.8868

0.2075

0.0049

0.0019

n

10

10

10

10

10

10

10

10

10

10

10

10

G5, F & 12000/4000

Mean

0.0765

0.3459

0.5265*

0.1621

0.5842

1.0567

2.1191

2.0072

8.6751

1.9781

0.0278

0.0176

±SD

0.0127

0.0588

0.0741

0.0434

0.1513

0.1210

0.2301

0.1590

0.4184

0.1631

0.0015

0.0015

n

10

10

10

10

10

10

10

10

10

10

10

10

F: Female; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

SUMMARY OF ORGAN WEIGHT RELATIVE TO FASTING BODY WEIGHT (%) RECORD

Group, Sex & Dose

(ppm in diet)

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Testes

Epididymis

Heart

Kidneys

Brain

Liver

Prostate + Seminal Vesicles with Coagulating Glands

Lungs

Thyroid along with Parathyroid

Pituitary Gland

G1, M & 0

Mean

371.25

0.0199

0.1015

0.1935

0.8911

0.3860

0.3693

0.8482

0.5921

3.2856

0.8590

0.8343

0.0080

0.0049

±SD

46.28

0.0041

0.0261

0.0427

0.1271

0.0674

0.0633

0.1117

0.0909

0.5174

0.1585

0.1757

0.0020

0.0008

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, M & 750

Mean

375.00

0.0189

0.0902

0.1953

0.7539

0.3485

0.4062

0.7420

0.5691

3.2927

0.8603

0.6388*

0.0083

0.0050

±SD

59.28

0.0050

0.0241

0.0459

0.2101

0.0938

0.0719

0.1328

0.0895

0.5349

0.1482

0.1044

0.0016

0.0012

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, M & 2000

Mean

384.62

0.0192

0.1159

0.1708

0.8011

0.3360

0.3295

0.8007

0.5809

3.1382

0.9059

0.5903*

0.0075

0.0046

±SD

44.56

0.0046

0.0136

0.0374

0.1242

0.0432

0.0486

0.1609

0.0624

0.4198

0.1774

0.0695

0.0013

0.0008

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, M & 5000

Mean

310.18*

0.0212

0.0883

0.1660

1.0279

0.4178

0.4105

1.1247*

0.6732

3.3093

0.7699

0.8449

0.0095

0.0059

±SD

56.37

0.0043

0.0278

0.0222

0.1477

0.0695

0.0787

0.1765

0.0937

0.6336

0.2131

0.1509

0.0017

0.0020

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G5, M & 12000/4000

Mean

351.84

0.0178

0.0752*

0.1571

0.9439

0.3917

0.3941

0.9911

0.5957

3.3298

0.8092

0.7361

0.0084

0.0048

±SD

35.24

0.0025

0.0175

0.0565

0.0726

0.0617

0.0706

0.1429

0.0650

0.3781

0.0951

0.1239

0.0015

0.0008

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

M: Male; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

SUMMARY OF ORGAN WEIGHT RELATIVE TO FASTING BODY WEIGHT (%) RECORD

Group, Sex & Dose

(ppm in diet)

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

Thyroid along with Parathyroid

Pituitary Gland

G1, F & 0

Mean

260.82

0.0286

0.1313

0.1594

0.0516

0.1922

0.4112

0.7846

0.7920

3.1411

0.7152

0.0105

0.0070

±SD

12.28

0.0037

0.0248

0.0138

0.0151

0.0488

0.0526

0.0602

0.0606

0.4036

0.1140

0.0020

0.0011

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, F & 750

Mean

249.66

0.0282

0.1481

0.1952

0.0606

0.4032*

0.4185

0.8014

0.8080

3.5548

0.7678

0.0110

0.0070

±SD

9.98

0.0043

0.0235

0.0540

0.0089

0.1072

0.0489

0.0952

0.0478

0.3555

0.1127

0.0016

0.0006

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, F & 2000

Mean

256.77

0.0312

0.1241

0.1902

0.0568

0.2649

0.3926

0.7923

0.7804

2.8729

0.6650

0.0108

0.0070

±SD

22.40

0.0038

0.0230

0.0365

0.0092

0.0702

0.0436

0.1142

0.0932

0.2604

0.0973

0.0014

0.0007

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, F & 5000

Mean

196.96*

0.0371*

0.1456

0.1765

0.0552

0.3156*

0.5082*

1.0884*

0.9619*

3.6210*

0.8947*

0.0141*

0.0092*

±SD

11.87

0.0060

0.0331

0.0297

0.0111

0.1139

0.0794

0.1297

0.0856

0.4895

0.1261

0.0023

0.0012

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G5, F & 12000/4000

Mean

250.92

0.0309

0.1387

0.2101*

0.0654

0.2311

0.4238

0.8478

0.8031

3.4803

0.7920

0.0111

0.0071

±SD

23.04

0.0066

0.0261

0.0263

0.0199

0.0427

0.0604

0.0922

0.0661

0.3264

0.0735

0.0007

0.0006

n

10

10

10

10

10

10

10

10

10

10

10

10

10

F: Female; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

SUMMARY OF SERUM TRIIODOTHYRONINE (T3) LEVELS (ng/mL) RECORD

Group, Sex & Dose

(ppm in diet)

Serum T3 Levels

(ng/mL)

G1, M & 0

Mean

3.774

±SD

0.308

n

10

G2, M & 750

Mean

3.253*

±SD

0.114

n

10

G3, M & 2000

Mean

3.202*

±SD

0.212

n

10

G4, M & 5000

Mean

3.151*

±SD

0.209

n

10

G5, M & 12000/4000

Mean

3.755

±SD

0.188

n

10

G1, F & 0

Mean

3.606

±SD

0.439

n

10

G2, F & 750

Mean

3.000*

±SD

0.095

n

10

G3, F & 2000

Mean

3.460

±SD

0.406

n

10

G4, F & 5000

Mean

3.230*

±SD

0.205

n

10

G5, F & 12000/4000

Mean

3.730

±SD

0.203

n

10

M: Male, F: Female; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05)

 

SUMMARY OF SERUM THYROXINE (T4) LEVELS (ng/mL) RECORD

Group, Sex & Dose

(ppm in diet)

Serum T4 Levels

(ng/mL)

G1, M & 0

Mean

77.976

±SD

6.918

n

10

G2, M & 750

Mean

77.191

±SD

5.672

n

10

G3, M & 2000

Mean

115.150*

±SD

8.375

n

10

G4, M & 5000

Mean

119.186*

±SD

21.605

n

10

G5, M & 12000/4000

Mean

88.656

±SD

8.597

n

10

G1, F & 0

Mean

75.662

±SD

4.865

n

10

G2, F & 750

Mean

84.023

±SD

10.332

n

10

G3, F & 2000

Mean

122.576*

±SD

13.300

n

10

G4, F & 5000

Mean

122.331*

±SD

32.736

n

10

G5, F & 12000/4000

Mean

91.223

±SD

10.769

n

10

M: Male, F: Female; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05)

 

SUMMARY OF SERUM THYROID STIMULATING HORMONE (TSH) LEVELS (µIU/mL) RECORD

Group, Sex & Dose

(ppm in diet)

Serum TSH Levels

(µIU/mL)

G1, M & 0

Mean

5.380

±SD

4.926

n

10

G2, M & 750

Mean

4.201

±SD

6.245

n

10

G3, M & 2000

Mean

4.220

±SD

4.371

n

10

G4, M & 5000

Mean

1.662

±SD

1.294

n

10

G5, M & 12000/4000

Mean

1.076

±SD

1.389

n

10

G1, F & 0

Mean

2.185

±SD

2.257

n

10

G2, F & 750

Mean

3.803

±SD

6.944

n

10

G3, F & 2000

Mean

3.242

±SD

6.372

n

10

G4, F & 5000

Mean

1.435

±SD

1.030

n

10

G5, F & 12000/4000

Mean

1.264

±SD

1.188

n

10

M: Male, F: Female; SD: Standard Deviation, n: Number of animals.

HISTORICAL CONTROL DATA – 90-DAYS REPEATED ORAL DOSE TOXICITY STUDIES IN SPRAGUE DAWLEY RATS

 

SUMMARY OF SERUM RECORD – MALES

Serum

Mean

±SD

N

Min. Value

Max. Value

Serum Triiodothyronine (T3) Levels (ng/mL)

2.439

0.286

54

2.076

3.205

Serum Thyroxine (T4) levels (ng/mL)

95.248

31.263

54

68.641

182.155

Serum Thyroid Stimulating Hormone (TSH) Levels (μIU/mL)

4.744

5.167

54

0.740

21.244

SD: Standard Deviation; n: Number

 

SUMMARY OF SERUM RECORD – FEMALES

Serum

Mean

±SD

N

Min. Value

Max. Value

Serum Triiodothyronine (T3) Levels (ng/mL)

2.493

0.451

54

2.084

4.147

Serum Thyroxine (T4) levels (ng/mL)

94.862

33.972

54

68.422

185.804

Serum Thyroid Stimulating Hormone (TSH) Levels (μIU/mL)

4.063

3.576

53

0.385

20.797

SD: Standard Deviation; n: Number

Conclusions:
Under the conditions of the study, based on the observed results and considering the decrease in body weight, feed consumption at 5 000 (G4) and 12 000/4 000 ppm (G5) and microscopic findings observed in brain and thymus which included neuronal necrosis in piriform cortex and/or CA1&2 of the brain and depletion of lymphocytes in thymus at 5 000 (G4) and at 12 000/4 000 ppm (G5) in both sexes, the No Observed Adverse Effect Level (NOAEL) of the test material is 2 000 ppm in the diet (163.12 mg/kg bw/day males; 199.52 mg/kg bw/day females) when administered for a period of 91 consecutive days by oral (dietary) route to Sprague Dawley rats.
Executive summary:

The toxic potential of the test material was assessed according to OECD Test Guideline 408 and in compliance with GLP.

The objective of this study was to assess the toxic potential of the test material, when administered for a period of 91 consecutive days by oral (dietary) route to Sprague Dawley rats. This study provides information on toxic effects, target organs and an estimate of the No Observed Adverse Effect Level (NOAEL).

A total of 100 Sprague Dawley rats (50 males + 50 females) were distributed to 5 groups. Each group consisted of 10 animals/sex/group. The animals allocated to groups G2, G3, G4 and G5 were administered with doses of 750, 2 000, 5 000 and 12 000/4 000 ppm in diet, respectively. The animals allocated to group G1 received only the basal diet without any fortification. The dose of 12 000 ppm was administered to G5 group initially till Day 5. From Day 6 onwards, the dose level was reduced to 4 000 ppm because of reduction in body weights and feed consumption and associated clinical signs.

The homogeneity and dose concentration verification was carried out during week 1, month 2 and month 3 of the treatment period. The obtained results were considered acceptable as the mean results were within the range of 80 to 120 % of the nominal concentrations with a Relative Standard Deviation (RSD) of < 20 %.

All the animals were observed once daily for clinical signs and twice daily for mortality; detailed clinical examination, body weight and feed consumption measurements were performed weekly (additionally body weight and feed consumption were measured on Day 3 for all groups and feed consumption on Day 6 for the G5 group); ophthalmoscopic examination was performed during acclimatisation on all the animals and during week 13 on the G1 and G4 groups. Functional observational battery was performed during week 13 for G1 and G4 group animals.

At the end of treatment period, blood (haematology, coagulation, clinical chemistry) and urine samples were collected and analysed from all animals on Day 92. Vaginal smear examination was carried out on Day 92 for all the females. Subsequently, all animals were sacrificed and subjected to gross pathological examination. All the organs specified in the study plan were collected and preserved. All collected organs/tissues from the control (G1) and high dose (G4) groups were subjected to histopathological examination. In addition, the brain and thymus were evaluated in lower dose groups as they were identified as target organs in this study.

No clinical signs of toxicity and mortality were observed in all tested dose groups up to 5 000 ppm (G4) in either sex. Clinical signs were observed in a few rats of either sex at 12 000 ppm (G5). These consisted of dehydration from Day 4 onwards (G5M: 6/10, and up to 7/10; G5F: 6/10, and up to 9/10) and piloerection from Day 5 onwards (G5M: 5/10, and up to 6/10; G5F: 4/10, and up to 5/10). Hence, the dose level of G5 group was reduced to 4 000 ppm on Day 6 and post dose reduction, only piloerection was observed on Day 7 in two rats (G5M:1/10; G5F:1/10). All animals appeared normal from Day 8 onwards up to the completion of treatment period.

No treatment related changes in body weight or feed consumption were noted up to 2 000 ppm (G3). All animals of G5 lost weight during the first 3 days of treatment at 12 000 ppm (8 to 21 % for individual males and 7 to 15 % for individual females). Decrease in mean body weights were noted during the initial weeks of treatment in the G5 (12 000/40 00) group and recovered from week 3 once the dose level was reduced. Mean body weights at termination were 16 % and 24 % lower than controls in males and females, and treatment related decreased body weight gains of up to 28 % and 51 % over the entire treatment period were noted in G4 males and females (5 000 ppm), respectively. Treatment related decreased feed consumption in G4 groups (5 000 ppm) was also noted.

No treatment related changes were observed in control and high dose group animals during ophthalmological examination, functional observation battery tests and motor activity assessment.

No adverse treatment related changes were noted in haematology, clinical chemistry, coagulation and urinalysis parameters. No adverse treatment related changes were observed in thyroid hormone levels (T3, T4 and TSH).

Significant decrease in fasting body weight was noted in high dose group (G4) animals of either sex. Decrease in weight of thymus and brainin the G4 and G5 groups was associated to microscopic changes.

Notreatmentrelated gross pathological changeswere observed at any of the tested dose group animals.

During histopathology, test material-related changes were observed in the brain and thymus of animals of both sexes at 5 000 and at 12 000/4 000 ppm. In the brain, minimal to mild neuronal necrosis characterised by cytoplasmic shrinkage with intense eosinophilia (red neuron) accompanied by karyorrhexis or karyolysis of nucleus in the piriform cortex and/or cornu ammonis (CA1&2) was observed. In the thymus, minimal to moderate depletion of lymphocytes was observed at 5 000 ppm and at 12 000/4 000 ppm in both sexes.

Under the conditions of the study, based on the observed results and considering the decrease in body weight, feed consumption at 5 000 (G4) and 12 000/4 000 ppm (G5) and microscopic findings observed in brain and thymus which included neuronal necrosis in piriform cortex and/or CA1&2 of the brain and depletion of lymphocytes in thymus at 5 000 (G4) and at 12 000/4 000 ppm (G5) in both sexes, the No Observed Adverse Effect Level (NOAEL) of the test material is 2 000 ppm in the diet (163.12 mg/kg bw/day males; 199.52 mg/kg bw/day females) when administered for a period of 91 consecutive days by oral (dietary) route to Sprague Dawley rats.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 March 2019 to 09 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
GLP compliance:
no
Remarks:
Dose range-finding study only.
Limit test:
no
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 and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: Yes, females used were nulliparous and non-pregnant.
- Age at study initiation: 8 weeks
- Weight at study initiation: Weight at receipt: 132.07 g to 146.75 g - male; 121.14 g to 138.41 g - female.
- Housing: Maximum of two animals of same sex (two rats in two cages + one rat in another cage) were housed in standard polypropylene cages (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilised paddy husk was provided as bedding material.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: The animals were acclimatised for a period of five days to experimental room conditions and were observed for clinical signs once daily. Veterinary examination and body weight recording of all animals were performed on the day of receipt and on the day of randomisation. Ophthalmoscopic examination of all animals were performed on the day of grouping.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 °C to 23.1 °C.
- Humidity (%): 47 % 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.
Route of administration:
oral: feed
Details on route of administration:
The test material was administered orally as admixture with the diet. Doses were expressed as parts per million (ppm) in diet. The oral route was chosen because this route is a possible route of human exposure.
Vehicle:
unchanged (no vehicle)
Remarks:
The test material was admixtured with powder diet.
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: Fortified diets were prepared once a week and used.
- Mixing appropriate amounts with: The following procedure was followed when 3.5 kg of fortified diet was prepared.
The required quantity of test material was weighed and initially mixed with a small volume of acetone. This was then transferred into a stainless steel container with the total required quantity of feed and hand mixed using a stainless steel spoon for at least 2 minutes. The fortified diet was prepared using a blender, the mixing was carried out for 10 minutes after initial pre-mixing in a drum for 2 minutes.
- Storage temperature of food: The fortified diets were stored in polyethylene bags which were kept in stainless steel drums in the study room at a temperature of 22 ± 3 ºC and used. The quantity of experimental diet prepared and the amount of test material weighed was not varied. The left out formulated diet was sent for disposal.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
Daily
Dose / conc.:
500 ppm
Dose / conc.:
1 500 ppm
Dose / conc.:
3 000 ppm
No. of animals per sex per dose:
Five per sex per dose.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses of 0, 500, 1 500 and 3 000 ppm in the diet were selected for vehicle control, low dose, mid dose and high dose as recommended by the Sponsor.
- 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 the experimental groups using a 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: +5.69 % to -7.68 % and female: +6.71 % to -9.96 %). The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analysed statistically for mean body weight to rule out any statistically significant difference between groups within each sex.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity throughout the experimental period.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weight was recorded on the day of randomisation, prior to start of treatment (Day 1) and weekly thereafter. Fasting body weights of all animals were recorded at their scheduled terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Cage wise feed consumption was measured at weekly intervals. 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. Test material intake (in mg/kg bw/day) was calculated and presented.

FOOD EFFICIENCY: No.

WATER CONSUMPTION AND COMPOUND INTAKE: No.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from all animals on Day 29. Blood samples were collected from the animals separately into tubes containing K2-EDTA and 3.2 % Sodium citrate tubes for Prothrombin time and activated partial thromboplastin time parameters. Blood samples were collected by retro-orbital plexus puncture method with the help of a fine capillary tube.
- Anaesthetic used for blood collection: Yes, mild Isoflurane anesthesia.
- Animals fasted: Yes. The animals were fasted overnight before blood collection. Water was provided ad libitum during the fasting period.
- How many animals: All animals.
- Parameters checked: The following haematology parameters were estimated using Advia 2120 Haematology System (Siemens Limited).
Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC).
Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by coagulation analyser.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from all animals on Day 29. Blood samples were collected from the animals separately into tubes containing sodium heparin. Blood samples were collected by retro-orbital plexus puncture method with the help of a fine capillary tube.
- Animals fasted: Yes. The animals were fasted overnight before blood collection. Water was provided ad libitum during the fasting period.
- How many animals: All animals.
- Parameters checked: The following clinical chemistry parameters were analysed using a clinical chemistry analyser.
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Triglycerides, Phosphorous, Calcium, Blood Urea Nitrogen, Globulin, Albumin/Globulin ratio.
Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using a Prolyte Na/K/Cl analyser (Diamond Diagnostics). The plasma was separated by centrifuging the blood samples at 5 000 rpm for 10 minutes for determining the clinical chemistry parameters and 1 500 rpm for 15 minutes for determining Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT).


URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected and analysed from all rats on the day of necropsy (day 29).
- Metabolism cages used for collection of urine: Yes. The animals were kept in urine collection cages overnight on day 28.
- Animals fasted: Yes fasted but water was provided ad libitum during this period.
- Parameters checked:
The overnight urine volume (mL) collected from these animals was measured.
The volume of urine collected (mL), appearance and the colour was recorded by physical evaluation.
The urine was analysed using Urine Analyser for the following parameters:
Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Leucocytes.
In addition pH, nitrite and specific gravity were also analysed. After analysis of the above parameters, the urine was subjected to centrifugation at 1 500 rpm for 3 minutes. Then the urine was subjected to microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION: No.

IMMUNOLOGY: No.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
- On day 29, all animals of Group G1, G2, G3, and G4 were subjected to necropsy and detailed gross pathological examination. The animals were fasted overnight (water was provided ad libitum) prior to scheduled necropsy. On the day of terminal sacrifice, the body weight of all fasted animals was recorded prior to exsanguination. The animals were euthanised using CO2 asphyxiation followed by exsanguination.
- Gross pathological changes were recorded for each rat. Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, with organs and tissues of each animal examined.
- Organ Weights: The following organs from all animals at the scheduled sacrifice were trimmed of any adherent tissue and fat, as appropriate, and weighed wet as soon as possible to avoid drying.
List of the organs weighed: Kidneys*, Adrenals*, Spleen, Heart, Liver, Thymus, Brain, Lungs, Testes*/Ovaries*, Epididymides*/Uterus*.
*Paired organs were weighed together.
- Relative Organ weights were calculated against fasting body weight.
- Tissue Collection and Preservation: The tissues collected from all animals were preserved in 10 % v/v Neutral Buffered Formalin except testes which were preserved in Modified Davidson’s fluid.
Statistics:
The raw data were subjected to statistical analysis. The computer printout of the data was verified with the original raw data. After verification, the data were subjected to statistical analysis using SPSS software version 22. Body weight, percent change in body weight with respect to day 1, feed consumption, haematological and clinical chemistry parameters, urinalysis, absolute organ weights and relative organ weights data were subjected to statistical analysis. One way ANOVA followed by Dunnett’s post test were done for comparing different treatment groups with the respective 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 or morbidity were observed in any of the test material administered animals of either sex.
Mortality:
no mortality observed
Description (incidence):
No mortality/morbidity were observed in any of the test material administered animals of either sex.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment related changes in mean body weight and percent change in body weight with respect to day 1 were observed in any of the treated group animals of either sex.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment related changes were observed in feed consumption in any of the treated group animals of either sex.

The test material intake was as follows:
Males: 48.38, 141.79 and 297.01 mg/kg/day for low dose, mid dose and high dose groups, respectively.
Females: 56.93, 164.98 and 320.44 mg/kg/day for low dose, mid dose and high dose groups, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematological parameters of test material administered animals (G2, G3 and G4) were comparable with those of the vehicle control.

In males, statistically significant changes included a decrease in relative neutrophils (G2, G3), and increases in relative lymphocytes (G2, G3), absolute monocytes (G4), absolute basophils (G4), APTT (G3). In females, a statistically significant decrease in APTT (G2, G3) was noted.
All the variations noted are considered incidental due to lack of dose relationship and/or because these were observed in only one sex.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males, a statistically significant increase in triglycerides (G4) and a decrease in calcium (G2, G3, G4) were noted. In females, statisticaly significant increases in total protein (G2), albumin (G2) and a decrease in phosphorous (G2, G4) were noted. The decrease in calcium in test material treated males is considered incidental as all the values are within historical range of the species (except Rd5261-G2M-10.6 mg/dL, which is higher than historical range; historical range: 8.0 to 10.4 mg/dL). All other variations noted are considered incidental and not related to treatment due to lack of dose responsiveness.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant increase in urine pH in G3 males and a decrease in urine pH in G4 females were noted, considered incidental due to lack of dose responsiveness.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant changes in fasting body weight of treated animals when compared to the control group.
Absolute organ weight: In males, statistically significant increases in adrenals (G2), thymus (G4), spleen (G3), heart (G2, G4) and liver (G3); and a decrease in kidneys (G4) were noted. In females, statistically significant increases in heart (G3) and lungs (G3); and a decrease in uterus (G3, G4) were noted.
Relative organ weight: In males, statistically significant increases in thymus (G4) and heart (G4); and a decrease in kidneys (G4) were noted. In females, a statistically significant decrease in uterus (G3, G4); and an increase in heart (G3) were noted.
Although some individual values were outside Historical control data, all the above changes were considered incidental due to lack of dose responsiveness and/or because not supported by any other finding.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related gross pathological lesions were observed in any of the treated group animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
NOAEL
Remarks on result:
not measured/tested
Critical effects observed:
no
Conclusions:
Under the conditions of the study the test material was well tolerated up to 3 000 ppm in diet (males: 297.01 mg/kg/day and females: 320.44 mg/kg/day) and did not reveal any adverse changes when administered for a period of 28 consecutive days by oral (diet) route to Sprague Dawley rats. For the subsequent toxicity studies, dose levels up to 3 000 ppm in diet (297.01 mg/kg/day for males/320.44 mg/kg/day for females) will be considered.
Executive summary:

The objective of this repeated oral (dietary) toxicity study was to generate information on health hazards likely to arise from repeated exposure to the test material for a period of at least three weeks (actual: four weeks) to aid selecting doses for subsequent toxicity studies and was conducted according to OECD Test Guideline 407.

A total of 40 (20 males + 20 females) Sprague Dawley rats were distributed to four groups viz., Vehicle control (G1), Low dose (G2), Mid dose (G3) and High dose (G4). Each group consisted of 5 males and 5 females. The test material was admixture with powder diet and fed to rats at the doses of 500, 1 500 and 3 000 ppm. Control group rats were fed with basal diet throughout the treatment period without any test material.

All animals were observed once daily for clinical signs and twice daily for mortality. Body weight and feed consumption were measured at weekly intervals. At the end of the treatment period, blood and urine samples were collected and analysed. Subsequently, the animals were sacrificed and subjected to gross pathological examination where organs were collected and preserved.

No mortality and clinical signs were observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight with respect to day 1, feed consumption, haematology, clinical chemistry, urinalysis, fasting body weights and organ weights were observed.

There were no treatment related gross pathological changes observed at any of the tested doses in either sex during gross pathological examination.

Based on the results observed, it is concluded that the test material was well tolerated up to 3 000 ppm in diet (males: 297.01 mg/kg/day and females: 320.44 mg/kg/day) and did not reveal any adverse changes when administered for a period of 28 consecutive days by oral (diet) route to Sprague Dawley rats under the experimental conditions employed. Hence, for the subsequent toxicity studies, dose levels up to 3 000 ppm in diet (297.01 mg/kg/day for males/320.44 mg/kg/day for females) will be considered.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
163.12 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A subchronic guideline and GLP compliant key study is available on the test material. The quality of the database is therefore considered to be good.
System:
nervous system
Organ:
brain
thymus

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Remarks:
Doses / Concentrations:

Basis:

Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Remarks:
Doses / Concentrations:

Basis:

Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Remarks:
Doses / Concentrations:

Basis:

Critical effects observed:
not specified