Violet sodium polysulfide aluminosilicate with a SOD-type framework structure

Endpoint summary

Description of key information

Key Study: Sub-chronic repeated (90-day) toxicity study in rats. OECD 408, GLP study. The only test item-related effect found was minimal microscopic changes in thymus at the high dose of 1000 mg/kg bw/day in males (G4M). As this change was minimal in severity and was found to be reversible, it was considered as non-adverse effect. Thus, the NOAEL of the test item for both sexes was determined to be 1000 mg/kg bw/day.

Supporting Study: Dose Range Finding Study in rats, OECD 407, non GLP. The oral administration of test item by gavage to Sprague Dawley Rats for a period of 28 consecutive days did not produce test item-related effects in any of the parameters examined at the dose levels of 100, 300 and 1000 mg/kg bw/day under experimental conditions employed and the same are then considered appropriate for a subsequent 90-day repeated dose toxicity study in rats.

 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 2020 to 24 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

One deviation was recorded for administering 3.1 mL on Day 57 instead of 3.2 mL for Re7208 of G3M. This marginal variation in dose volume does not have any impact on the outcome of the study as variation was only 0.1 mL and also in mid dose group only.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: 152.08-153.74 g (Mean), Females: 139.62- 141.52 g (Mean)
- Fasting period before study: no
- Housing: Two animals of same sex were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7°C to 22.9°C
- Humidity (%): 46% to 65%
- Air changes (per hr): 12-15 Air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 14.08.2020 To: 16.12.2020

Route of administration:

oral: gavage

Details on route of administration:

The test formulations and vehicle was administered through oral gavage using oral intubation cannula attached to disposable syringe

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared before dose administration. The required quantity of test item was weighed separately and transferred, mixed, and triturated well in a mortar. A small quantity of vehicle was added and triturated until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred to measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure complete transfer of the contents to the measuring cylinder. Finally, the volume was adjusted to required mark in measuring cylinder with vehicle to get a desired concentration of 10, 30 and 100 mg/mL of test item for low, mid, and high dose groups, respectively.

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogenous suspension with test item
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): BCBN1690V (Sigma Aldrich)
- Expiry Date: 9 March 2024

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Verification of concentration and homogonetiy of the test item was conduced by BIONEEDS INDIA PRIVATE LIMITED DEVARAHOSAHALLY, SOMPURA HOBLI, NELAMANGALA TALUK, BANGALORE RURAL DISTRICT, PIN - 562 111, KARNATAKA, INDIA which hold the Good Laboratory Practice Certificate (GLP). 0.1 mL of sample was pipetted out into a digestion tube, a volume of 6 mL of concentrated nitric acid was added and placed in microwave sample preparation system and digested by the selected method. After digestion, the digestion tubes were cooled to room temperature and the contents were transferred to suitable volumetric flasks and diluted with Milli-Q water.


TECHNIQUE AND TEST METHOD: ICP-MS


TEST PARAMETERS: Timings Sweeps : 30 Seconds, Reading : 1, Replicates : 3, Analyte : Aluminium, Mass : 26.9815, Sample Flush : 35 seconds, Read Delay : 15 Seconds, Wash : 45 seconds, Mode : KED, Flow (Helium) : 4.0 mL, Vehicle: 0.5% Carboxymethyl Cellulose


ACCEPTANCE CRITERIA: Homogeneity and dose formulation analysis of prepared test formulations for the dose concentration verification were performed during week 1, month 2 and month 3 of the dosing period and the obtained mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1- control group

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1R- control recovery

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4R- high dose recovery

No. of animals per sex per dose:

10 males and 10 females per group for the main groups, 5 males and 5 females for the recovery groups (G1R and G4R).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 100, 300 and 1000 mg/kg/day were selected for test item based on the results obtained from the dose range finding study in which no adverse effects were found up to the dose of 1000 mg/kg/day.
- Fasting period before blood sampling for clinical biochemistry: overnight
- Post-exposure recovery period in satellite groups: 28 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity
- Cage side observations checked: clinical signs of toxicity, mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before initiation of the treatment and at weekly intervals thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: on the Day 1 before test item administration and weekly thereafter

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly.

FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during Week 12 for vehicle control and high dose main group animals (G1, and G4) and during Week 17 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1, G4, G1R and G4R

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on experimental day 91 and 119 for main and recovery groups respectively
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: Hemoglobin concentration, hematocrit, erythrocyte count, total leukocyte count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, mean platelet volume, reticulocyte count, absolute reticulocyte count, differential leucocytes count, absolute differential leucocytes count, prothrombin time and
activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on experimental day 91 and 119 for main and recovery groups respectively
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, cholinesterase, total protein, albumin, total bilirubin, glucose, total cholesterol, creatinine, urea, triglycerides, phosphorous, calcium, high density lipoprotein, low density lipoprotein, blood urea nitrogen, globulin, albumin/globulin ratio, sodium, potassium and chloride.

PLASMA/SERUM HORMONES/LIPIDS: Yes (Thyroid Hormones T3 (Triiodothyronine), T4 (Thyroxine) and Thyroid Stimulating Hormone (TSH))
- Time of blood sample collection: at termination.
- Animals fasted: Not specified
- How many animals: all of them

URINALYSIS: Yes
- Time schedule for collection of urine: on experimental day 91 and 119 for main and recovery groups respectively
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Blood, bilirubin, urobilinogen, ketones, protein, leuocytes, microalbumin, pH, nitrite and specific gravity.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during Week 12 for vehicle control and high dose main group animals (G1 and G4) and during Week 17 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1, G4, G1R and G4R
- Battery of functions tested: sensory activity / grip strength / motor activity / other: home cage observations, handling observations, open field observations, neuromuscular observations, physiological observation (rectal temperature)

IMMUNOLOGY: No

OTHER:
-Vaginal Smear Examination from all the females on the day of sacrifice.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On day 91, all the animals of groups G1, G2, G3 and G4 were subjected to necropsy and detailed gross pathological examination. All the animals from recovery groups (G1R and G4R) were subjected to necropsy and detailed gross pathological examination on day 119. 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 the fasted animals were recorded prior to exsanguination. The animals were euthanized using carbon dioxide asphyxiation exsanguination.
Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues. All the animals were sacrificed as per random numbers generated for the study and complete gross pathological examination was performed

TISSUE COLLECTION, ORGAN WEIGHTS, PRESERVATION AND HISTOPATHOLOGY: Yes (see table 1)
On the day of scheduled necropsy, a list of organs were collected from all animals and were trimmed of any adherent tissue and fat, as appropriate and weighed wet as soon as possible to avoid drying.
Organ weight ratio was calculated against fasting body weight. The organs and tissues were collected, trimmed off any adherent tissue, as appropriate. All the tissues mentioned below, from all animals, were preserved in 10% v/v Neutral Buffered Formalin and Modified Davidson’s fixative for eyes and testes.

HISTOPATHOLOGY: Yes
Histopathological examination was conducted on the tissues from the vehicle control (G1) and high dose (G4). Further, thymus from lower and recovery group males were processed and subjected for histopathological evaluation as it was found to be target organ of toxicity in high dose males.
All organs and tissue samples were processed, embedded in paraffin, cut at a thickness of 4 to 6 micrometers and stained with hematoxylin and eosin. The bone marrow smear was fixed in methanol and stained with Giemsa Stain. Testes were sectioned at 3-4 micrometers thickness and addition to hematoxylin and eosin stain, Periodic acid –Schiff and hematoxylin stain was 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 treatment 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

GROSS PATHOLOGY: Yes
All animals were fasted overnight (water allowed) and on the day of necropsy (day 91 for main groups, day 119 for recovery groups) all animals were weighed and then subjected to necropsy. Some organs (see Table 1) were collected, weighed and preserved for histopathology.
Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues.

HISTOPATHOLOGY: Yes
Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose (G4) group animals.

Other examinations:

BONE MARROW SMEAR EXAMINATION:
Bone marrow smear was prepared for all the animals at the time of necropsy. As there were no treatment related changes in haematology parameters or microscopic changes in organ like spleen, a cytologic evaluation of the bone marrow was not carried out. In thymus, minimal, decreased cellularity of lymphocytes was noted in high dose males (5/10) only and was found reversible and hence considered non-adverse finding.

Statistics:

The data was subjected to statistical analysis. The computer printout of the data was verified with the original raw data. After verification, the data was subjected to statistical analysis using SPSS software version 22. Body weight, percent change in body weight (with respect to Day 1 body weight), feed consumption, organ weights and ratios, hematological and clinical chemistry estimations and urine analysis parameters (pH, urine volume, specific gravity and urobilinogen), FOB parameters (Open field observations, Body temperature, Hind Limb Footsplay, Grip strength and Movement Counts) were subjected to statistical analysis. One way ANOVA followed by Dunnett’s post test was done for different treatment and recovery groups comparing with the respective vehicle control group data. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). Any other statistical tests used for the analysis of data was mentioned in the final report. Statistically significant changes obtained from the aforementioned tests was designated by the superscripts throughout the report as stated below:
*: Statistically significant (P<0.05)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant lower feed consumption was noted during week 13 in G4RF. The noted change is considered as incidental in the absence of any concomitant changes in body weights.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Increase in percent eosinophils (G4M), absolute neutrophils (G2F), absolute monocytes (G2F), decrease in MCHC (G3F, G4F), percent lymphocytes (G2F) were noted. The noted variation in differential counts is considered incidental in the absence of dose responsiveness. Decrease in MCHC without any variation in haemoglobin concentration is considered incidental. In recovery groups, increase in percent neutrophils (G4RM), decrease in percent lymphocytes (G4RM), absolute monocytes (G4RF), absolute basophils (G4RF) were noted. The observed changes in differential counts without any variation in total leucocyte count is considered incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Increase in glucose (G2M), calcium (G4M), sodium (G2M, G4M), phosphorus (G3F, G4F) was noted. The noted changes are considered incidental in the absence of dose responsiveness.
In recovery groups, increase in total cholesterol (G4RF), high density lipoprotein (G4RF), cholinesterase (G4RF), decrease in ALP (G4RF) was noted. The noted variations are considered incidental as similar variation was not noted in other sex nor at the end of treatment period.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistical significance decrease in T3 (G2M, G4F) and increase in TSH (G4F) was noted which is considered incidental in the absence of associated microscopic (or histopathological) or macroscopic changes in thyroid in the high dose group and dose responsiveness.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase in specific gravity (G2M), decrease in pH (G4RF) was noted. Increase in specific gravity is considered incidental in the absence of dose responsiveness. Decrease in pH is also considered incidental as no such variation was noted in other sex or at the end of treatment period.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Decreased movement counts (G4M, G4F); increased rearing (G4M), increase in hind limb footsplay (G4F) was noted. The noted variations are considered incidental in the absence of dose responsiveness and changes in other neuromuscular parameters. Also, no clinical signs were noted during the in-life phase of the study.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- Absolute weights: Decrease in weight of thymus (G2M, G4M), brain (G2M, G3M, G4M, G3F), adrenals (G2F, G3F), uterus with cervix (G2F, G3F), kidneys (G4RM); increase in weight of spleen (G3M), pituitary (G2M, G3M), uterus with cervix (G4F), heart (G4F), liver (G3F, G4F) was noted
- Relative weights (relative to body weight): Decrease in weight of thymus (G2M, G4M), brain (G2M, G3M, G3F), adrenals (G2F, G3F), ovaries with oviduct (G4F), uterus with cervix (G2F, G3F); increase in weight of spleen (G3M), uterus with cervix (G4F), heart (G4F), liver (G3F) was noted.

Variation in thymus was associated with microscopic changes like minimal, decreased cellularity of lymphocytes only in high dose male rats (G4M) and was characterized by paler staining of cortex and/or decreased cortico-medullary ratio with prominent stromal cells.

All other changes noted are considered incidental and not related to treatment in the absence of dose responsiveness and/or absence of microscopic changes in the high dose main groups.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In thymus, minimal, decreased cellularity of lymphocytes was observed only in high dose male rats and was characterized by, paler staining of cortex and/or decreased cortico-medullary ratio with prominent stromal cells. This change was found to be reversible at the end of 28-day treatment free recovery period as similar changes were not observed in recovery group. Further, as this change was minimal in severity and was found to be reversible, it can be considered as non-adverse finding. Few other microscopic findings observed in this study such as ultimobranchial cyst/s in thyroid gland, age related involution in thymus, dilatation of sinus in mandibular lymph node, dilatation of lumen in uterus in females and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats of this age.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Vaginal smear examination revealed normal stages of estrous cyclicity both in main and recovery females.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed up to highest dose tested.

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Table 2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. 

Group, Sex & Dose (mg/kg/day)	No. of Animals	Clinical Signs of Toxicity	Mortality (No. of Incidences/ No. of Animals)
G1, M & 0	10	N	0/10
G2, M & 100	10	N	0/10
G3, M & 300	10	N	0/10
G4, M & 1000	10	N	0/10
G1R, M & 0	5	N	0/5
G4R, M & 1000	5	N	0/5
G1, F & 0	10	N	0/10
G2, F & 100	10	N	0/10
G3, F & 300	10	N	0/10
G4, F & 1000	10	N	0/10
G1R, F & 0	5	N	0/5
G4R, F & 1000	5	N	0/5

M: Male; F: Female; R: Recovery; N: Normal

 

Table 3. Summary of body weights (g) record

Group, Sex & Dose (mg/kg/day)		Body Weight (g) on Days
		1	8	15	22	29	36	43	50	57	64	71	78	85	90	97	104	111	118
G1, M & 0	Mean	152.16	187.81	231.45	263.58	286.93	315.51	337.43	349.15	367.58	375.99	385.73	392.00	397.84	402.11	-	-	-	-
	±SD	9.25	17.98	17.17	19.73	24.86	29.52	33.07	35.85	42.06	45.52	49.18	49.77	50.53	51.40	-	-	-	-
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10	-	-	-	-
G2, M & 100	Mean	152.87	193.51	241.12	277.16	302.54	332.20	356.41	370.52	386.05	391.59	400.71	412.23	423.57	428.98	-	-	-	-
	±SD	6.43	14.04	18.87	22.26	25.16	30.10	34.67	37.20	41.79	44.10	42.62	40.09	34.69	36.14	-	-	-	-
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10	-	-	-	-
G3, M & 300	Mean	152.08	188.22	228.08	258.40	282.81	304.21	324.53	331.28	344.80	352.32	371.32	382.99	387.58	394.26	-	-	-	-
	±SD	4.57	6.46	7.72	13.00	16.24	22.73	26.03	29.22	27.77	32.92	36.59	41.37	44.46	44.33	-	-	-	-
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10	-	-	-	-
G4, M & 1000	Mean	152.98	188.39	223.67	248.07	274.56	301.28	316.91	326.15	329.98	346.73	359.82	369.54	378.32	382.55	-	-	-	-
	±SD	5.33	9.17	9.40	8.87	14.51	17.29	17.52	25.00	27.03	23.87	26.01	28.78	27.36	27.34	-	-	-	-
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10	-	-	-	-
G1R, M & 0	Mean	152.18	194.66	238.35	267.84	291.02	317.59	331.21	349.10	358.18	370.26	381.91	390.39	395.88	399.93	405.61	409.78	413.43	420.70
	±SD	6.19	9.83	15.88	18.54	27.85	23.94	24.55	19.28	25.20	19.68	17.43	27.49	31.00	30.17	31.60	30.53	30.96	30.04
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	153.74	190.61	223.75	255.61	279.31	299.54	321.26	348.73	355.75	371.42	379.62	379.68	390.13	397.15	401.61	406.95	410.49	417.01
	±SD	3.93	8.92	16.25	18.22	23.39	17.77	21.74	21.23	24.44	28.96	36.44	33.51	23.95	24.22	24.28	22.97	21.69	19.12
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Body Weight (g) on Days
		1	8	15	22	29	36	43	50	57	64	71	78	85	90	97	104	111	118
G1, F & 0	Mean	141.52	160.85	181.40	190.98	203.02	212.63	219.84	229.52	236.87	239.87	243.51	248.30	249.90	252.75	-	-	-	-
	±SD	6.19	6.59	5.87	6.56	6.89	6.51	8.50	13.70	12.18	13.80	13.21	13.35	12.21	12.06	-	-	-	-
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10	-	-	-	-
G2, F & 100	Mean	141.12	162.21	185.03	196.49	207.05	218.11	228.67	234.34	235.96	240.35	246.69	250.90	255.08	258.00	-	-	-	-
	±SD	7.58	9.36	11.05	12.51	12.35	13.60	15.45	15.41	19.92	22.29	23.35	21.05	19.83	19.03	-	-	-	-
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10	-	-	-	-
G3, F & 300	Mean	140.21	161.44	179.53	190.24	202.33	210.43	216.74	224.85	233.31	236.41	242.13	248.07	250.59	253.67	-	-	-	-
	±SD	6.96	8.01	9.29	11.59	12.08	13.93	12.16	13.55	13.27	11.92	12.59	13.94	13.24	13.44	-	-	-	-
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10	-	-	-	-
G4, F & 1000	Mean	140.93	163.88	185.30	198.32	211.24	217.91	226.25	238.18	242.60	245.33	249.75	254.92	256.56	263.73	-	-	-	-
	±SD	5.88	9.36	12.38	12.04	19.26	13.72	16.32	17.11	20.41	21.65	19.80	22.43	23.67	30.30	-	-	-	-
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10	-	-	-	-
G1R, F & 0	Mean	139.62	160.30	178.74	186.58	197.38	206.32	214.49	224.07	236.58	232.92	234.08	236.88	241.77	245.46	250.64	254.42	258.80	264.37
	±SD	6.24	6.38	5.53	6.63	9.21	9.07	9.90	9.82	12.75	12.75	14.46	13.66	16.53	15.96	15.13	13.51	13.69	12.39
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	140.20	165.41	185.44	201.64	212.04	213.72	225.32	235.60	242.69	246.06	250.82	255.23	254.56	264.07	267.85	272.42	276.82	282.60
	±SD	5.09	15.93	12.88	15.77	16.99	20.45	23.66	23.87	25.59	27.24	26.16	26.27	27.86	29.91	30.44	30.25	31.45	30.45
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

 

Table 4. Summary of feed consumption (g/rat/day) record

Group, Sex & Dose (mg/kg/day)		Week 1	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	18.27	19.62	24.04	25.20	25.54	25.44	25.28	25.01	25.27	25.42	24.68	24.56	25.07
	±SD	2.71	1.83	1.66	1.57	1.76	0.57	1.79	0.89	1.69	2.16	2.11	2.04	3.42
	n	5	5	5	5	5	5	5	5	5	5	5	5	5
G2, M & 100	Mean	20.09	21.19	24.69	25.45	25.82	24.52	24.76	25.40	24.75	25.92	26.18	26.51	23.95
	±SD	0.85	0.50	1.44	1.08	2.53	1.35	2.35	0.25	2.08	1.50	1.25	0.43	2.80
	n	5	5	5	5	5	5	5	5	5	5	5	5	5
G3, M & 300	Mean	18.78	21.38	22.61	24.93	25.70	25.60	24.79	25.88	24.40	26.48	25.96	26.39	23.11
	±SD	1.53	1.28	1.39	1.06	0.82	0.95	0.52	0.90	1.45	0.61	0.85	0.62	1.48
	n	5	5	5	5	5	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	19.09	21.43	23.72	25.51	25.45	25.68	25.38	25.52	24.69	25.97	25.84	25.69	24.61
	±SD	0.81	0.34	0.88	1.17	0.28	0.45	1.15	1.00	1.43	0.88	1.13	1.41	2.60
	n	5	5	5	5	5	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Week 1	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	16.68	17.05	17.63	18.49	18.96	19.89	18.40	17.17	17.90	19.22	17.92	18.24	19.41
	±SD	0.57	0.88	1.46	1.64	1.83	2.01	2.62	1.29	3.00	1.38	1.81	2.76	1.96
	n	5	5	5	5	5	5	5	5	5	5	5	5	5
G2, F & 100	Mean	15.87	17.40	17.77	18.28	19.15	21.22	17.07	17.43	17.62	17.80	18.14	18.51	19.13
	±SD	0.60	2.03	2.34	2.06	1.54	1.23	2.90	2.61	0.70	5.90	1.10	0.68	3.15
	n	5	5	5	5	5	5	5	5	5	5	5	5	5
G3, F & 300	Mean	15.85	17.07	17.13	16.83	19.67	18.31	16.37	17.61	17.42	18.93	16.31	16.52	15.38
	±SD	1.52	1.32	1.37	2.44	2.57	3.03	4.82	1.29	0.49	3.79	0.64	0.59	2.85
	n	5	5	5	5	5	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	15.67	17.77	18.25	18.54	19.40	20.32	17.18	18.22	18.55	19.15	19.09	19.38	17.29
	±SD	0.85	1.01	1.08	1.32	2.26	2.02	1.62	1.93	1.47	1.55	2.35	2.14	2.61
	n	5	5	5	5	5	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Week 9	Week 10
G1R, M & 0	Mean	20.58	20.95	25.45	26.34	27.08	26.12	25.02	25.63	25.27	24.85
	±SD	1.13	1.13	1.89	1.61	0.63	0.32	0.88	0.80	1.42	2.11
	n	3	3	3	3	3	3	3	3	3	3
G4R, M & 1000	Mean	18.74	21.75	24.84	26.41	26.75	25.82	25.65	25.59	27.37	26.17
	±SD	1.56	0.40	1.07	1.14	1.52	0.98	1.06	0.43	1.98	4.06
	n	3	3	3	3	3	3	3	3	3	3
G1R, F & 0	Mean	16.55	16.72	16.76	19.72	19.07	19.22	16.95	15.25	15.96	17.46
	±SD	0.99	1.65	1.65	2.66	1.36	1.16	0.67	1.84	1.42	0.40
	n	3	3	3	3	3	3	3	3	3	3
G4R, F & 1000	Mean	16.98	17.56	17.74	17.83	17.96	18.67	16.08	17.96	17.65	18.05
	±SD	0.72	2.06	0.36	0.37	0.88	5.08	3.53	3.66	2.97	2.75
	n	3	3	3	3	3	3	3	3	3	3

 

Group, Sex & Dose (mg/kg/day)		Week 11	Week 12	Week 13	Week 14	Week 15	Week 16	Week 17
G1R, M & 0	Mean	25.58	24.85	24.74	21.59	21.48	22.24	22.28
	±SD	1.26	1.51	2.85	2.17	1.78	2.00	1.64
	n	3	3	3	3	3	3	3
G4R, M & 1000	Mean	23.27	20.69	24.57	23.02	21.84	22.13	22.28
	±SD	3.79	4.99	1.33	3.35	2.17	1.96	1.93
	n	3	3	3	3	3	3	3
G1R, F & 0	Mean	16.35	19.60	18.03	17.70	16.21	16.30	16.70
	±SD	1.39	2.96	0.91	3.24	1.66	1.62	1.19
	n	3	3	3	3	3	3	3
G4R, F & 1000	Mean	16.52	18.42	15.60*	20.19	14.61	14.82	16.53
	±SD	1.04	1.37	0.71	1.57	0.48	0.73	1.52
	n	3	3	3	3	3	3	3

*: Statistically significant (P<0.05).

 

Table 5. Summary of neurological/functional observation battery (FOB) record

Week 12
Parameters↓	Group & Sex		G1 & M	G4 & M	G1 & F	G4 & F
	Dose (mg/kg/day)		0	1000	0	1000
	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	7.0	8.2*	6.9	7.4
		±SD	1.3	0.9	1.4	1.2
Numbers of Urination		Mean	1.5	1.2	1.5	1.1
		±SD	0.7	0.4	0.5	0.6
Number of Defecation		Mean	1.1	1.1	1.2	1.3
		±SD	0.6	0.6	0.4	0.5
Clonic involuntary movement			1	1	1	1
Tonic involuntary movement			1	1	1	1
Stereotype Behaviour			1	1	1	1
Excessive Grooming		Mean	4.5	3.8	4.2	4.6
		±SD	0.8	0.9	0.8	0.8
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	98.6	98.6	99.3	99.4
		±SD	0.2	0.2	0.3	0.2
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

*: Statistically significant (P<0.05).

 

Table 6. Summary of actimeter reading record

#Week 12/17
Group, Sex & Dose (mg/kg/day)		Actimeter Readings
		Movement Counts (no.)
G1, M & 0	Mean	2098.6
	±SD	53.0
	n	10
G4, M & 1000	Mean	2024.8*
	±SD	9.9
	n	10
G1, F & 0	Mean	2441.0
	±SD	94.2
	n	10
G4, F & 1000	Mean	2279.5*
	±SD	59.6
	n	10
G1R, M & 0	Mean	2208.6
	±SD	37.9
	n	5
G4R, M & 1000	Mean	2107.6
	±SD	94.5
	n	5
G1R, F & 0	Mean	2087.8
	±SD	53.6
	n	5
G4R, F & 1000	Mean	2090.6
	±SD	63.9
	n	5

*: Statistically significant (P<0.05)

#: Week 12 for main group and Week 17 for recovery group.

 

Table 7. Summary of haematology record

Group, Sex & Dose (mg/kg/day)		Total Leucocyte Count	Total Erythrocyte Count	Hemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Hemoglobin	Mean Corpuscular Hemoglobin 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	9.44	8.37	14.26	43.20	51.72	17.06	33.01
	±SD	1.77	0.53	0.69	2.10	2.32	0.80	0.81
	n	10	10	10	10	10	10	10
G2,M & 100	Mean	9.02	8.77	15.01	45.02	51.41	17.16	33.34
	±SD	2.38	0.59	0.98	2.75	1.92	0.80	0.59
	n	10	10	10	10	10	10	10
G3, M & 300	Mean	9.85	8.49	14.82	44.03	51.87	17.47	33.65
	±SD	4.22	0.74	1.45	4.01	1.80	0.75	0.46
	n	10	10	10	10	10	10	10
G4, M & 1000	Mean	8.33	8.44	14.59	44.04	52.32	17.31	33.11
	±SD	1.97	0.58	0.52	2.02	1.91	0.76	0.44
	n	10	10	10	10	10	10	10
G1R, M & 0	Mean	11.61	8.70	14.16	43.42	49.96	16.28	32.58
	±SD	3.10	0.76	1.26	3.19	0.92	0.34	0.66
	n	5	5	5	5	5	5	5
G4R, M & 1000	Mean	12.43	8.88	14.54	45.32	51.10	16.42	32.12
	±SD	5.12	0.92	1.18	3.92	1.81	0.38	0.69
	n	5	5	5	5	5	5	5

 

Group, Sex & Dose  (mg/kg/day)		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	749.40	6.33	2.26	24.09	68.41	4.60	1.70	0.35
	±SD	149.67	0.45	0.58	5.76	5.59	1.71	0.81	0.20
	n	10	10	10	10	10	10	10	10
G2, M & 100	Mean	844.70	6.90	1.96	33.13	58.74	4.89	1.88	0.36
	±SD	143.74	0.82	0.86	18.28	20.11	2.43	0.79	0.13
	n	10	10	10	10	10	10	10	10
G3, M & 300	Mean	699.10	6.39	1.84	22.59	70.29	4.09	1.67	0.35
	±SD	192.74	0.33	0.64	5.83	7.16	1.38	0.68	0.13
	n	10	10	10	10	10	10	10	10
G4, M & 1000	Mean	668.20	6.75	1.76	35.52	55.17	4.79	3.24*	0.25
	±SD	114.49	0.48	0.57	13.98	14.97	1.41	1.92	0.08
	n	10	10	10	10	10	10	10	10
G1R, M & 0	Mean	768.40	6.74	2.51	27.82	63.46	5.08	2.00	0.64
	±SD	82.34	0.46	1.07	2.53	4.90	2.51	0.37	0.27
	n	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	710.80	6.82	2.41	38.28*	52.12*	4.88	2.38	1.46
	±SD	114.11	0.26	0.74	7.21	7.50	0.74	1.37	0.75
	n	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		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, M & 0	Mean	187.71	2.32	6.44	0.42	0.15	0.03	18.11	27.45
	±SD	41.99	0.97	1.16	0.14	0.06	0.02	1.76	1.51
	n	10	10	10	10	10	10	10	10
G2, M & 100	Mean	168.40	2.76	5.55	0.42	0.18	0.03	18.87	25.77
	±SD	64.88	1.04	2.76	0.18	0.10	0.02	1.44	1.88
	n	10	10	10	10	10	10	10	10
G3, M & 300	Mean	155.56	2.06	7.11	0.40	0.15	0.04	18.02	26.21
	±SD	56.32	0.53	3.69	0.20	0.05	0.02	1.24	2.51
	n	10	10	10	10	10	10	10	10
G4, M & 1000	Mean	147.18	3.10	4.43	0.41	0.26	0.02	18.44	26.61
	±SD	43.50	2.08	1.38	0.21	0.17	0.01	1.89	5.77
	n	10	10	10	10	10	10	10	10
G1R, M & 0	Mean	213.10	3.26	7.34	0.60	0.24	0.07	16.36	23.24
	±SD	73.94	1.05	1.88	0.38	0.11	0.02	0.56	3.64
	n	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	209.40	4.91	6.39	0.60	0.27	0.17	16.30	23.68
	±SD	49.97	2.62	2.36	0.24	0.11	0.10	0.80	3.05
	n	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Total Leucocyte Count	Total Erythrocyte Count	Hemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Hemoglobin	Mean Corpuscular Hemoglobin 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	6.32	7.82	14.15	41.13	52.60	18.11	34.42
	±SD	1.81	0.36	0.87	2.39	1.80	0.52	0.73
	n	10	10	10	10	10	10	10
G2, F & 100	Mean	7.93	7.82	14.16	41.30	52.91	18.15	34.24
	±SD	1.65	0.38	0.57	1.42	1.90	0.74	0.45
	n	10	10	10	10	10	10	10
G3, F & 300	Mean	7.09	7.76	13.77	40.69	52.52	17.76	33.82*
	±SD	1.94	0.49	0.92	2.45	1.33	0.47	0.55
	n	10	10	10	10	10	10	10
G4, F & 1000	Mean	6.84	7.86	14.38	42.55	54.11	18.30	33.82*
	±SD	1.99	0.26	0.58	1.70	1.97	0.61	0.38
	n	10	10	10	10	10	10	10
G1R, F & 0	Mean	10.28	8.23	14.18	42.70	51.98	17.28	33.26
	±SD	4.28	0.83	0.89	2.93	2.23	0.80	0.40
	n	5	5	5	5	5	5	5
G4R, F & 1000	Mean	6.03	8.24	14.54	43.74	53.06	17.66	33.26
	±SD	1.22	0.66	1.26	3.92	1.34	0.38	0.39
	n	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		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	665.30	6.93	1.73	24.24	68.58	4.30	1.69	0.28
	±SD	200.24	0.68	0.44	6.40	6.87	1.39	0.52	0.09
	n	10	10	10	10	10	10	10	10
G2, F & 100	Mean	642.80	7.02	1.82	30.49	60.64*	5.07	2.40	0.34
	±SD	215.90	0.54	0.59	8.74	9.35	1.34	0.89	0.10
	n	10	10	10	10	10	10	10	10
G3, F & 300	Mean	712.10	6.75	1.88	23.22	68.76	4.41	2.41	0.29
	±SD	99.59	0.42	0.70	3.40	3.62	0.76	0.94	0.11
	n	10	10	10	10	10	10	10	10
G4, F & 1000	Mean	750.90	6.61	1.61	21.03	71.80	3.98	1.96	0.28
	±SD	182.92	0.53	0.56	5.68	6.05	1.67	0.45	0.08
	n	10	10	10	10	10	10	10	10
G1R, F & 0	Mean	815.80	6.48	1.87	25.62	66.14	4.48	1.88	0.56
	±SD	244.30	0.54	0.46	3.97	2.80	1.74	0.90	0.19
	n	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	785.20	6.72	2.13	27.92	65.50	3.96	1.56	0.38
	±SD	211.29	0.37	0.34	3.94	4.21	0.63	0.21	0.13
	n	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		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	135.83	1.49	4.38	0.27	0.11	0.02	19.38	26.55
	±SD	36.29	0.47	1.53	0.11	0.04	0.01	1.34	2.45
	n	10	10	10	10	10	10	10	10
G2, F & 100	Mean	141.99	2.42*	4.82	0.39*	0.18*	0.03	19.17	24.77
	±SD	45.52	0.93	1.37	0.10	0.06	0.01	2.53	2.10
	n	10	10	10	10	10	10	10	10
G3, F & 300	Mean	146.06	1.64	4.88	0.32	0.16	0.02	21.70	23.74
	±SD	54.79	0.54	1.34	0.09	0.06	0.01	4.28	1.87
	n	10	10	10	10	10	10	10	10
G4, F & 1000	Mean	126.36	1.39	4.95	0.28	0.14	0.02	19.90	26.27
	±SD	43.63	0.37	1.70	0.16	0.06	0.01	2.21	3.67
	n	10	10	10	10	10	10	10	10
G1R, F & 0	Mean	151.84	2.68	6.81	0.43	0.18	0.06	17.12	23.36
	±SD	29.59	1.35	2.80	0.15	0.10	0.02	1.03	2.24
	n	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	176.44	1.66	3.98	0.24*	0.10	0.02*	15.86	26.58
	±SD	34.63	0.27	0.97	0.05	0.03	0.01	2.03	6.69
	n	5	5	5	5	5	5	5	5

*: Statistically significant (P<0.05).

 

Table 8. Summary of clinical chemistry record

Group, Sex & Dose (mg/kg/day)		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	98.40	30.41	0.48	39.60	39.70	8.17	4.94	6.17	2.76	65.90	101.00
	±SD	11.07	3.55	0.03	6.29	11.25	1.28	1.15	0.33	0.14	9.09	12.42
	n	10	10	10	10	10	10	10	10	10	10	10
G2, M & 100	Mean	115.10*	32.91	0.46	41.30	33.20	8.54	4.62	6.19	2.85	59.70	95.20
	±SD	12.55	4.22	0.03	8.62	7.91	1.28	1.00	0.39	0.15	8.77	11.06
	n	10	10	10	10	10	10	10	10	10	10	10
G3, M & 300	Mean	111.40	33.57	0.49	37.20	31.20	7.65	4.82	6.36	2.82	64.50	114.40
	±SD	14.83	4.86	0.03	7.97	8.66	2.40	1.65	0.23	0.19	15.37	19.84
	n	10	10	10	10	10	10	10	10	10	10	10
G4, M & 1000	Mean	108.30	31.36	0.46	41.70	36.30	9.49	4.81	6.45	2.92	93.10	133.40
	±SD	11.56	5.51	0.03	10.20	9.68	2.01	1.15	0.26	0.14	102.51	95.73
	n	10	10	10	10	10	10	10	10	10	10	10
G1R, M & 0	Mean	101.20	36.28	0.52	32.00	35.00	8.16	4.72	6.84	2.90	63.40	115.60
	±SD	7.60	5.44	0.03	5.52	7.35	1.19	0.73	0.15	0.17	10.92	13.52
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	100.80	34.92	0.54	38.60	36.00	9.94	4.30	6.90	3.00	60.40	112.40
	±SD	12.64	4.31	0.03	11.87	5.70	1.90	0.70	0.35	0.12	13.63	17.81
	n	5	5	5	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		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	151.50	0.09	9.07	4.84	80.90	3.41	0.81	14.19	143.16	3.63	111.69
	±SD	30.16	0.03	0.41	0.27	11.97	0.30	0.08	1.66	1.01	0.22	1.71
	n	10	10	10	10	10	10	10	10	10	10	10
G2, M & 100	Mean	141.30	0.09	9.26	4.65	95.50	3.34	0.86	15.36	144.47*	3.72	113.48
	±SD	38.30	0.01	0.29	0.36	27.81	0.35	0.10	1.97	0.71	0.18	1.12
	n	10	10	10	10	10	10	10	10	10	10	10
G3, M & 300	Mean	157.50	0.09	9.41	4.96	87.80	3.54	0.80	15.67	143.86	3.53	112.33
	±SD	70.32	0.01	0.26	0.34	18.03	0.13	0.06	2.27	1.02	0.19	1.73
	n	10	10	10	10	10	10	10	10	10	10	10
G4, M & 1000	Mean	142.90	0.08	9.55*	4.99	84.90	3.53	0.84	14.64	144.95*	3.49	113.02
	±SD	45.17	0.01	0.28	0.46	15.93	0.35	0.12	2.57	1.26	0.16	2.46
	n	10	10	10	10	10	10	10	10	10	10	10
G1R, M & 0	Mean	108.20	0.05	9.06	4.84	128.80	3.94	0.74	16.93	142.92	3.31	111.22
	±SD	31.59	0.01	0.26	0.53	32.06	0.14	0.06	2.54	1.06	0.22	1.36
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	120.60	0.04	9.14	4.42	99.80	3.90	0.77	16.30	142.64	3.48	111.40
	±SD	17.34	0.02	0.30	0.72	30.26	0.26	0.04	2.01	0.94	0.15	1.36
	n	5	5	5	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		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	129.20	28.82	0.52	58.00	48.00	14.70	3.07	6.87	3.30	50.00	94.70
	±SD	16.65	4.29	0.03	6.85	24.38	1.91	0.41	0.32	0.15	14.01	14.29
	n	10	10	10	10	10	10	10	10	10	10	10
G2, F & 100	Mean	126.80	31.81	0.53	55.00	42.40	13.19	3.10	7.06	3.42	45.40	89.60
	±SD	8.80	4.25	0.04	10.30	10.25	2.64	0.81	0.48	0.33	6.62	8.54
	n	10	10	10	10	10	10	10	10	10	10	10
G3, F & 300	Mean	125.50	32.11	0.51	53.10	38.50	12.75	3.37	6.90	3.25	46.20	97.00
	±SD	15.10	3.26	0.04	11.95	10.32	2.62	1.04	0.30	0.24	13.31	18.68
	n	10	10	10	10	10	10	10	10	10	10	10
G4, F & 1000	Mean	135.70	28.50	0.51	51.60	37.50	12.45	3.13	7.12	3.29	39.30	91.00
	±SD	16.83	3.42	0.04	10.72	8.11	2.31	0.86	0.46	0.20	3.77	10.00
	n	10	10	10	10	10	10	10	10	10	10	10
G1R, F & 0	Mean	117.00	35.32	0.59	44.60	36.60	13.62	2.88	7.22	3.49	44.80	101.40
	±SD	12.39	5.31	0.03	8.53	12.88	1.91	0.75	0.26	0.21	9.04	13.32
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	118.20	32.08	0.58	66.80*	43.00	19.68*	3.68	7.38	3.77	41.00	113.40
	±SD	15.69	4.87	0.02	18.58	14.53	4.61	0.58	0.47	0.37	10.91	25.71
	n	5	5	5	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		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	114.90	0.07	9.95	3.69	634.40	3.57	0.93	13.45	141.55	3.28	110.48
	±SD	33.14	0.03	0.25	0.43	175.96	0.31	0.10	2.00	0.58	0.16	0.92
	n	10	10	10	10	10	10	10	10	10	10	10
G2, F & 100	Mean	90.80	0.09	9.94	3.77	611.40	3.64	0.96	14.85	141.48	3.52	110.36
	±SD	34.32	0.01	0.39	0.60	205.38	0.43	0.17	1.98	0.73	0.31	1.40
	n	10	10	10	10	10	10	10	10	10	10	10
G3, F & 300	Mean	103.80	0.09	10.07	4.88*	636.20	3.65	0.90	14.98	142.00	3.38	110.23
	±SD	77.06	0.02	0.30	0.34	278.72	0.30	0.12	1.52	0.85	0.23	1.38
	n	10	10	10	10	10	10	10	10	10	10	10
G4, F & 1000	Mean	82.10	0.07	9.97	4.59*	686.70	3.83	0.87	13.30	141.61	3.43	110.88
	±SD	19.28	0.03	0.32	0.77	251.66	0.33	0.07	1.60	0.95	0.55	1.38
	n	10	10	10	10	10	10	10	10	10	10	10
G1R, F & 0	Mean	83.80	0.03	9.48	3.42	612.40	3.73	0.94	16.48	142.20	3.41	112.38
	±SD	25.57	0.02	0.41	0.65	98.35	0.17	0.07	2.48	0.73	0.16	1.28
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	46.20*	0.05	9.50	3.62	995.20*	3.61	1.05	14.97	142.58	3.43	112.32
	±SD	9.73	0.01	0.45	0.43	232.70	0.25	0.12	2.27	0.93	0.22	2.21
	n	5	5	5	5	5	5	5	5	5	5	5

*: Statistically significant (P<0.05).

 

Table 9. Summary of urinalysis record

Examination	Group & Sex		G1 & M	G2 & M	G3 & M	G4 & M	G1R & M	G4R & M
	Dose (mg/kg/day)		0	100	300	1000	0	1000
	Number of Animals		10	10	10	10	5	5
Physical	Colour	Pale Yellow	8	10	8	8	5	5
		Yellow	2	-	2	2	-	-
	Appearance	Clear	9	8	9	9	4	5
		Turbid	1	2	1	1	1	-
	Volume (mL)	Mean	5.8	5.2	5.5	5.3	8.0	6.5
		±SD	1.5	1.7	0.8	1.1	2.3	0.9
Chemical	pH	Mean	8.6	8.5	8.6	8.5	7.3	7.4
		±SD	0.2	0.0	0.2	0.0	0.4	0.4
	Specific Gravity	Mean	1.007	1.009*	1.006	1.007	1.013	1.009
		±SD	0.002	0.002	0.002	0.002	0.004	0.002
	Urobilinogen (mg/dL)	Mean	1.3	1.4	1.3	1.1	0.2	0.2
		±SD	1.2	0.5	0.6	0.7	0.0	0.0
	Bilirubin (mg/dL)	Neg	-	-	-	-	5	5
		1	9	8	9	9	-	-
		3	1	2	1	1	-	-
	Ketones (mg/dL)	Neg	10	10	10	10	5	5
	Blood (Ery/µL)	Neg	10	10	10	9	-	-
		Ca10	-	-	-	1	2	1
		Ca25	-	-	-	-	1	3
		Ca80	-	-	-	-	2	1
	Proteins (mg/dL)	Neg	-	-	-	-	5	4
		Trace	-	-	-	-	-	1
		>=300	10	10	10	10	-	-
	Nitrite	Neg	10	10	10	10	3	5
		Pos	-	-	-	-	2	-
	Leucocytes (Leu/µL)	Ca70	7	6	6	6	-	-
		Ca15	1	-	-	1	5	5
		Ca125	1	4	4	3	-	-
		>=Ca500	1	-	-	-	-	-
	Glucose (mg/dL)	Neg	10	10	10	10	5	5
	Micro albumin (MALB) (mg/dL)	Neg	-	-	-	-	-	2
		15	-	-	-	-	5	2
		>15	10	10	10	10	-	1
Microscopic	Epithelial cells	0	6	6	7	7	3	2
		0-1	3	2	2	2	1	2
		1-2	1	2	1	1	1	1
	Casts	Absent	10	10	10	10	5	5
	Crystals	Present	10	10	10	10	5	5

 

Examination	Group & Sex		G1 & F	G2 & F	G3 & F	G4 & F	G1R & F	G4R & F
	Dose (mg/kg/day)		0	100	300	1000	0	1000
	Number of Animals		10	10	10	10	5	5
Physical	Colour	Pale Yellow	7	8	6	9	5	4
		Yellow	3	2	4	1	-	1
	Appearance	Clear	9	7	10	8	5	4
		Turbid	1	3	-	2	-	1
	Volume (mL)	Mean	5.9	5.4	5.2	5.5	9.2	7.8
		±SD	1.4	0.9	0.8	1.2	1.2	2.4
Chemical	pH	Mean	8.6	8.5	8.5	8.6	8.3	7.5*
		±SD	0.2	0.0	0.0	0.2	0.3	0.0
	Specific Gravity	Mean	1.007	1.008	1.008	1.009	1.006	1.010
		±SD	0.003	0.003	0.003	0.002	0.002	0.006
	Urobilinogen (mg/dL)	Mean	1.1	1.1	1.0	1.6	0.2	0.2
		±SD	0.7	0.8	0.6	1.0	0.0	0.0
	Bilirubin (mg/dL)	Neg	-	-	-	-	5	5
		1	8	7	8	5	-	-
		3	2	3	2	5	-	-
	Ketones (mg/dL)	5	-	-	-	-	-	3
		Neg	10	10	10	10	5	2
	Blood (Ery/µL)	Neg	10	10	10	10	-	-
		Ca10	-	-	-	-	1	-
		Ca80	-	-	-	-	2	3
		>=Ca200	-	-	-	-	2	2
	Proteins (mg/dL)	Neg	-	-	-	-	-	1
		Trace	-	-	-	-	5	3
		30	-	-	-	-	-	1
		>=300	10	10	10	10	-	-
	Nitrite	Neg	10	10	10	10	5	4
		Pos	-	-	-	-	-	1
	Leucocytes (Leu/µL)	Neg	-	-	-	-	2	-
		Ca70	5	3	6	3	-	4
		Ca15	-	1	-	-	3	1
		Ca125	4	6	4	6	-	-
		>=Ca500	1	-	-	1	-	-
	Glucose (mg/dL)	Neg	10	10	10	10	5	5
	Micro albumin (MALB) (mg/dL)	15	-	-	-	-	-	1
		>15	10	10	10	10	5	4
Microscopic	Epithelial cells	0	6	6	7	5	2	3
		0-1	3	2	3	4	2	1
		1-2	1	2	-	1	1	1
	Casts	Absent	10	10	10	10	5	5
	Crystals	Present	10	10	10	10	5	5

*: Statistically significant (P<0.05).

 

Table 10. Summary of absolute organ weights (g) record

Group, Sex & Dose (mg/kg/day)		Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys	Brain	Liver	Prostate+Seminal vesicles with coagulating glands (PSC)	Thyroid along with parathyroid	Pituitary
G1, M & 0	Mean	0.0607	0.3146	1.0518	3.4852	1.4990	1.3322	2.4524	2.2109	10.1183	3.4192	0.0311	0.0118
	±SD	0.0087	0.0286	0.0837	0.1792	0.0745	0.0992	0.1857	0.1872	0.7609	0.3992	0.0071	0.0013
	n	10	10	10	10	10	10	10	10	10	10	10	10
G2, M & 100	Mean	0.0725	0.2701*	1.0986	3.3317	1.5310	1.3420	2.6263	2.0286*	10.1493	3.3746	0.0301	0.0137*
	±SD	0.0150	0.0408	0.1116	0.1725	0.1082	0.0555	0.2540	0.0802	0.6137	0.4279	0.0058	0.0015
	n	10	10	10	10	10	10	10	10	10	10	10	10
G3, M & 300	Mean	0.0657	0.2874	1.4000*	3.4346	1.5825	1.3618	2.4372	1.9158*	9.6114	3.1373	0.0296	0.0135*
	±SD	0.0155	0.0511	0.2514	0.2645	0.1377	0.0573	0.1292	0.0736	0.2494	0.4327	0.0058	0.0013
	n	10	10	10	10	10	10	10	10	10	10	10	10
G4, M & 1000	Mean	0.0614	0.1822*	1.0159	3.4937	1.5284	1.3548	2.3200	2.0210*	10.0858	3.1556	0.0309	0.0126
	±SD	0.0056	0.0226	0.0657	0.1809	0.1050	0.0449	0.2220	0.0586	0.3796	0.4620	0.0073	0.0018
	n	10	10	10	10	10	10	10	10	10	10	10	10
G1R, M & 0	Mean	0.0879	0.4786	1.3579	3.5237	1.8048	1.4099	3.2460	2.1860	11.1301	3.3972	0.0320	0.0131
	±SD	0.0073	0.3408	0.4710	0.2732	0.3187	0.1364	0.0254	0.1077	0.9636	0.5847	0.0062	0.0009
	n	5	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	0.0821	0.2844	1.1994	3.5459	1.5579	1.6577	3.0017*	2.1412	10.6402	3.2913	0.0341	0.0124
	±SD	0.0161	0.0765	0.1641	0.2108	0.1186	0.3634	0.1140	0.2365	0.0840	0.3513	0.0088	0.0011
	n	5	5	5	5	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Adrenals	Thymus	Spleen	Ovaries With oviduct	Uterus with cervix	Heart	Kidneys	Brain	Liver	Thyroid along with parathyroid	Pituitary
G1, F & 0	Mean	0.0899	0.2970	0.7721	0.1834	0.7494	0.8778	1.5773	2.0595	6.6549	0.0269	0.0151
	±SD	0.0162	0.0343	0.0695	0.0188	0.1149	0.0673	0.1618	0.1884	0.2953	0.0041	0.0020
	n	10	10	10	10	10	10	10	10	10	10	10
G2, F & 100	Mean	0.0723*	0.3263	0.7774	0.1799	0.5693*	0.9394	1.6403	1.9483	7.1163	0.0250	0.0153
	±SD	0.0110	0.0500	0.1080	0.0296	0.0691	0.0671	0.1486	0.0834	0.5037	0.0042	0.0027
	n	10	10	10	10	10	10	10	10	10	10	10
G3, F & 300	Mean	0.0642*	0.3099	0.6830	0.1684	0.5218*	0.9030	1.6191	1.8763*	7.5492*	0.0251	0.0146
	±SD	0.0082	0.0525	0.0854	0.0169	0.0676	0.0764	0.1134	0.0461	0.4368	0.0042	0.0027
	n	10	10	10	10	10	10	10	10	10	10	10
G4, F & 1000	Mean	0.0814	0.3217	0.8393	0.1640	0.9336*	1.0134*	1.6846	1.9466	7.2948*	0.0288	0.0161
	±SD	0.0114	0.0749	0.0765	0.0155	0.1281	0.0682	0.1570	0.0859	0.7540	0.0048	0.0024
	n	10	10	10	10	10	10	10	10	10	10	10
G1R, F & 0	Mean	0.0791	0.2851	0.6121	0.1990	0.8530	1.0021	1.8805	1.9020	7.9569	0.0240	0.0146
	±SD	0.0149	0.0544	0.2953	0.0449	0.2254	0.0448	0.0744	0.0497	0.8616	0.0038	0.0020
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	0.0870	0.2979	0.7606	0.2072	0.9065	1.0576	2.0242	1.9721	8.5214	0.0271	0.0148
	±SD	0.0094	0.0570	0.1371	0.0731	0.2404	0.0867	0.3460	0.1723	1.2695	0.0041	0.0019
	n	5	5	5	5	5	5	5	5	5	5	5

*: Statistically significant (P<0.05).

 

Table 11. Summary of organ weight relative to fasting body weight (%) record

Group, Sex & Dose (mg/kg/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys	Brain	Liver	Prostate+Seminal vesicles with coagulating glands (PSC)	Thyroid along with parathyroid	Pituitary
G1, M & 0	Mean	373.20	0.0166	0.0852	0.2859	0.9494	0.4081	0.3611	0.6641	0.6007	2.7473	0.9309	0.0085	0.0032
	±SD	52.77	0.0036	0.0096	0.0398	0.1314	0.0542	0.0395	0.0650	0.0780	0.3336	0.1595	0.0023	0.0003
	n	10	10	10	10	10	10	10	10	10	10	10	10	10
G2, M & 100	Mean	400.65	0.0182	0.0678*	0.2757	0.8382	0.3845	0.3378	0.6582	0.5111*	2.5453	0.8478	0.0076	0.0034
	±SD	36.40	0.0037	0.0107	0.0309	0.0914	0.0390	0.0367	0.0628	0.0625	0.1836	0.1243	0.0018	0.0005
	n	10	10	10	10	10	10	10	10	10	10	10	10	10
G3, M & 300	Mean	367.64	0.0182	0.0788	0.3840*	0.9459	0.4348	0.3751	0.6728	0.5282*	2.6488	0.8609	0.0081	0.0037
	±SD	43.85	0.0050	0.0150	0.0759	0.1297	0.0560	0.0468	0.0974	0.0686	0.3334	0.1311	0.0012	0.0006
	n	10	10	10	10	10	10	10	10	10	10	10	10	10
G4, M & 1000	Mean	353.38	0.0175	0.0517*	0.2888	0.9967	0.4355	0.3852	0.6587	0.5749	2.8672	0.8989	0.0088	0.0036
	±SD	28.14	0.0023	0.0065	0.0258	0.1200	0.0503	0.0284	0.0638	0.0458	0.2068	0.1517	0.0024	0.0006
	n	10	10	10	10	10	10	10	10	10	10	10	10	10
G1R, M & 0	Mean	398.51	0.0222	0.1227	0.3461	0.8897	0.4542	0.3541	0.8177	0.5503	2.8181	0.8557	0.0081	0.0033
	±SD	29.21	0.0025	0.0951	0.1364	0.1160	0.0875	0.0265	0.0540	0.0402	0.4468	0.1614	0.0020	0.0004
	n	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	394.91	0.0210	0.0719	0.3041	0.9012	0.3943	0.4168	0.7614	0.5447	2.7003	0.8361	0.0086	0.0031
	±SD	21.49	0.0052	0.0181	0.0404	0.0920	0.0147	0.0715	0.0397	0.0761	0.1373	0.1055	0.0021	0.0004
	n	5	5	5	5	5	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries With oviduct	Uterus with cervix	Heart	Kidneys	Brain	Liver	Thyroid along with parathyroid	Pituitary
G1, F & 0	Mean	234.52	0.0385	0.1268	0.3304	0.0785	0.3212	0.3744	0.6737	0.8808	2.8411	0.0115	0.0064
	±SD	11.76	0.0074	0.0151	0.0383	0.0097	0.0578	0.0241	0.0739	0.0982	0.1359	0.0020	0.0010
	n	10	10	10	10	10	10	10	10	10	10	10	10
G2, F & 100	Mean	240.63	0.0301*	0.1370	0.3256	0.0749	0.2382*	0.3922	0.6833	0.8132	2.9690	0.0104	0.0064
	±SD	19.68	0.0044	0.0268	0.0565	0.0116	0.0379	0.0367	0.0594	0.0567	0.2456	0.0018	0.0015
	n	10	10	10	10	10	10	10	10	10	10	10	10
G3, F & 300	Mean	236.62	0.0273*	0.1312	0.2902	0.0713	0.2221*	0.3827	0.6859	0.7958*	3.2042*	0.0107	0.0061
	±SD	13.56	0.0044	0.0238	0.0449	0.0077	0.0378	0.0370	0.0583	0.0576	0.3139	0.0021	0.0009
	n	10	10	10	10	10	10	10	10	10	10	10	10
G4, F & 1000	Mean	246.80	0.0334	0.1307	0.3423	0.0671*	0.3836*	0.4144*	0.6883	0.7978	2.9773	0.0118	0.0066
	±SD	31.05	0.0059	0.0280	0.0297	0.0089	0.0732	0.0415	0.0789	0.0869	0.3381	0.0025	0.0013
	n	10	10	10	10	10	10	10	10	10	10	10	10
G1R, F & 0	Mean	245.78	0.0321	0.1164	0.2524	0.0810	0.3505	0.4087	0.7667	0.7758	3.2335	0.0097	0.0059
	±SD	12.20	0.0052	0.0244	0.1240	0.0186	0.1093	0.0312	0.0503	0.0517	0.2736	0.0014	0.0008
	n	5	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	263.62	0.0332	0.1130	0.2880	0.0792	0.3460	0.4033	0.7697	0.7558	3.2390	0.0104	0.0056
	±SD	30.13	0.0042	0.0180	0.0349	0.0280	0.0945	0.0345	0.1156	0.1024	0.3873	0.0018	0.0007
	n	5	5	5	5	5	5	5	5	5	5	5	5

*: Statistically significant (P<0.05).

 

Table 12. Incidences of test-item related changes in thymus in male rats.

Organ/Finding	G1	G2	G3	G4	G1R	G4R
Number examined	10	10	10	10	5	5
Thymus : Cellularity, decreased, lymphocytes	-	-	-	5	-	-

Key: - = No incidence, G = Group, R: Recovery

Conclusions:

In a 90-day repeated dose toxicity study conducted in Sprague Dawley Rats, the NOAEL of the test item for both sexes is 1000 mg/kg bw/day.

Executive summary:

A study was conducted to assess the toxic potential of the test item when administered for a period of 90 consecutive days repeatedly to Sprague Dawley rats, according to OECD 408 Guideline (GLP study). Four groups (G1, G2, G3 and G4) of 10 animals of each sex and two recovery groups (G1R and G4R) were tested, being G1 and G1R the vehicle control groups (0.5% w/v CMC). The treatment groups had the following concentrations: 100, 300 and 1000 mg/kg bw. The vehicle and test item formulations were administered orally by gavage to the animals at an equivolume of 10 mL/kg bw. 

No clinical signs of toxicity and no mortality/morbidity were noted, as well as no changes in body weight, feed consumption and gross pathology in all groups. Besides, no effects in the ophthalmological and neurological/functional examinations were observed. No treatment related changes were observed in hematology, coagulation, clinical chemistry and urinalysis parameters, as well as no treatment related changes were observed in thyroid hormones (T3, T4 and TSH) and in vaginal smear examination. No gross pathological changes were observed. Decrease in weight of thymus in high dose males (G4M) was noted which was correlated with microscopic changes like minimal, decreased cellularity of lymphocytes. This change was found to be reversible at the end of 28-day treatment free recovery period as similar changes were not observed in recovery group. Further, as this change was minimal in severity and was found to be reversible, it is considered as non-adverse finding.

Therefore, based on the obtained results from this 90-day repeated dose toxicity study conducted in Sprague Dawley Rats, the NOAEL is estimated as 1000 mg/kg bw/day under experimental conditions employed and the NOEL is 300 mg/kg bw/day for males considering the non-adverse reversible minimal microscopic changes in thymus at 1000 mg/kg/day dose males.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12 March 2020 to 19 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 166.04-168.65 g (Mean), Females: 147.46- 148.12 g (Mean)
- Fasting period before study: no
- Housing: Two animals of same sex were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the the acclimatization and experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0°C to 23.6°C
- Humidity (%): 46% to 66%
- Air changes (per hr): 12-15 Air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 12.03.2020 To: 29.04.2020

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered through oral route using stainless steel (gavage) cannula.

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared daily before dose administration. The required quantity of test item was weighed separately and transferred, mixed, and triturated well in a mortar. A small quantity of vehicle was added and triturated until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred to measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure complete transfer of the contents to the measuring cylinder. Finally, the volume was adjusted to required mark in measuring cylinder with vehicle to get a desired concentration of 10, 30 and 100 mg/mL of test item for low, mid, and high dose groups, respectively.

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogenous suspension with test item
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): BCBN1690V (Sigma Aldrich)
- Expiry Date: 9 March 2024

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1- control group

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1R- control recovery

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4R- high dose recovery

No. of animals per sex per dose:

5 males and 5 females for the main and recovery groups.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 100, 300 and 1000 mg/kg/day were selected based on available data.
- Fasting period before blood sampling for clinical biochemistry: overnight
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity
- Cage side observations checked: clinical signs, mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before initiation of the treatment and at weekly intervals thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: on the Day 1 before test item administration and weekly thereafter

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of treatment and during Week 4 for vehicle control and high dose main group animals (G1, and G4) and during Week 6 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1, G4, G1R and G4R

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on experimental day 29 and 43 for main and recovery groups respectively
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: Hemoglobin concentration, hematocrit, erythrocyte count, total leukocyte count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, mean platelet volume, reticulocyte count, absolute reticulocyte count, differential leucocytes count, absolute differential leucocytes count, prothrombin time and
activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on experimental day 29 and 43 for main and recovery groups respectively
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, cholinesterase, total protein, albumin, total bilirubin, glucose, total cholesterol, creatinine, urea, triglycerides, phosphorous, calcium, high density lipoprotein, low density lipoprotein, blood urea nitrogen, globulin, albumin/globulin ratio, sodium, potassium and chloride.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: on experimental day 29 and 43 for main and recovery groups respectively
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Blood, bilirubin, urobilinogen, ketones, protein, glucose, leuocytes, pH, nitrite and specific gravity.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during Week 4 for vehicle control and high dose main group animals (G1 and G4) and during Week 6 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1, G4, G1R and G4R
- Battery of functions tested: sensory activity / grip strength / motor activity / other: home cage observations, handling observations, open field observations, neuromuscular observations, physiological observation (rectal temperature)

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were fasted overnight (water allowed) and on the day of necropsy (day 29 for main groups, day 43 for recovery groups) all animals were weighed and then subjected to necropsy. Some organs (see Table 1) were collected, weighed and preserved for histopathology.
Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues.

HISTOPATHOLOGY: Yes
Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose (G4) group animals.

Statistics:

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

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A non-dose dependent statistically significant increase in mean body weight was observed in G3 females (Day 22) and considered as incidental due to lack of dose dependency. In G4R females, statistically significant decrease in percent change in body weight with respect to Day 1 (during Days 1-8) is considered incidental as the mean body weights of high dose groups are unaffected in either sex.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In main group males, non-dose dependent statistically significant increase in absolute and percent basophils in G2 and decrease in APTT in G3 were observed but are considered as incidental due to the absence of dose responsiveness.
In G4R males, statistically significant decrease in PT was noted which is considered incidental in the absence of similar change in other sex.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In G4R females, decrease in urea and BUN and increase in cholinesterase were noted. All these changes noted are considered as incidental in the absence of similar changes in other sex.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute Organ Weight: In main groups, statistically significant increase in thymus (G2M), increase in prostate+seminal vesicles and coagulating glands (G3M and G4M), increase in epididymis (G4M) and increase in brain weight (G3F) was observed.
Organ Weight Relative to body weight: In main groups, statistically significant increase in thymus (G2M), increase in prostate+seminal vesicles and coagulating glands (G3M and G4M) and increase in epididymis (G3M and G4M) was observed.
All these changes are considered incidental and not related to test item as there were no associated histopathological or gross pathological changes noted in high dose group animals.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Few microscopic findings were observed in this study such as ultimobranchial cyst in thyroid gland, cast(s) in the kidneys and all other findings. However, these effects noted 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 examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to the highest dose tested

Key result

Critical effects observed:

no

Table 2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. 

Group, Sex & Dose (mg/kg/day)	No. of Animals	Clinical Signs of Toxicity	Mortality                                (No. of Incidences/ No. of Animals)
G1, M & 0	5	N	0/5
G2, M & 100	5	N	0/5
G3, M & 300	5	N	0/5
G4, M & 1000	5	N	0/5
G1, F & 0	5	N	0/5
G2, F & 100	5	N	0/5
G3, F & 300	5	N	0/5
G4, F & 1000	5	N	0/5
G1R, M & 0	5	N	0/5
G4R, M & 1000	5	N	0/5
G1R, F & 0	5	N	0/5
G4R, F & 1000	5	N	0/5

M: Male; F: Female; R: Recovery; N: Normal

 

Table 3. Summary of body weights (g) record

Group, Sex & Dose (mg/kg/day)		Body Weight (g) on Days
		1	8	15	22	28
G1, M & 0	Mean	166.80	201.74	236.57	281.66	301.51
	±SD	10.86	12.52	18.92	23.04	26.55
	n	5	5	5	5	5
G2, M & 100	Mean	166.36	206.35	239.31	279.76	293.08
	±SD	9.80	7.43	12.68	18.50	20.72
	n	5	5	5	5	5
G3, M & 300	Mean	166.69	205.95	234.17	271.71	283.77
	±SD	11.12	14.88	16.17	16.46	18.39
	n	5	5	5	5	5
G4, M & 1000	Mean	166.04	203.02	238.30	281.55	292.37
	±SD	10.44	16.29	21.52	20.35	20.73
	n	5	5	5	5	5
G1, F & 0	Mean	147.46	169.25	181.46	204.32	213.73
	±SD	7.75	6.07	5.68	8.37	8.93
	n	5	5	5	5	5
G2, F & 100	Mean	147.92	164.33	177.30	205.17	212.26
	±SD	7.84	9.01	5.70	7.83	8.13
	n	5	5	5	5	5
G3, F & 300	Mean	148.12	170.99	190.39	217.20*	223.11
	±SD	6.58	7.16	6.88	4.53	5.73
	n	5	5	5	5	5
G4, F & 1000	Mean	147.52	166.86	176.27	205.05	210.28
	±SD	6.53	7.73	8.55	9.57	9.58
	n	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Body Weight (g) on Days
		1	8	15	22	28	35	42
G1R, M & 0	Mean	166.46	203.57	231.27	274.77	289.08	300.24	312.84
	±SD	8.27	10.92	11.50	15.46	17.72	17.05	16.92
	n	5	5	5	5	5	5	5
G4R, M & 1000	Mean	168.65	201.65	227.56	268.95	278.78	291.24	303.39
	±SD	7.77	11.01	13.43	19.37	20.40	20.70	21.25
	n	5	5	5	5	5	5	5
G1R, F & 0	Mean	146.98	172.87	188.75	215.04	222.92	230.84	238.60
	±SD	4.91	9.91	14.50	13.22	13.25	12.95	12.84
	n	5	5	5	5	5	5	5
G4R, F & 1000	Mean	147.97	165.96	179.73	203.68	210.24	219.45	227.09
	±SD	4.83	6.07	5.52	9.94	11.06	11.54	11.08
	n	5	5	5	5	5	5	5

*: Statistically significant (p<0.05).

 

Table 4. Summmary of percent change in body weight (%) with respect to day 1 record

Group, Sex & Dose (mg/kg/day)		Percent Change in Body Weight (%) during Days
		1 to 8	1 to 15	1 to 22	1 to 28
G1, M & 0	Mean	21.06	41.99	68.99	80.89
	±SD	5.08	9.95	10.78	12.75
	n	5	5	5	5
G2, M & 100	Mean	24.20	44.00	68.36	76.33
	±SD	3.94	6.45	10.25	10.80
	n	5	5	5	5
G3, M & 300	Mean	23.52	40.48	63.15	70.40
	±SD	1.01	2.51	6.09	7.60
	n	5	5	5	5
G4, M & 1000	Mean	22.17	43.39	69.51	76.02
	±SD	2.57	5.97	3.58	2.70
	n	5	5	5	5
G1, F & 0	Mean	147.46	169.25	181.46	204.32
	±SD	7.75	6.07	5.68	8.37
	n	5	5	5	5
G2, F & 100	Mean	147.92	164.33	177.30	205.17
	±SD	7.84	9.01	5.70	7.83
	n	5	5	5	5
G3, F & 300	Mean	148.12	170.99	190.39	217.20
	±SD	6.58	7.16	6.88	4.53
	n	5	5	5	5
G4, F & 1000	Mean	147.52	166.86	176.27	205.05
	±SD	6.53	7.73	8.55	9.57
	n	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Percent Change in Body Weight (%) during Days
		1 to 8	1 to 15	1 to 22	1 to 28	1 to 35	1 to 42
G1R, M & 0	Mean	22.32	39.06	65.36	73.97	80.74	88.34
	±SD	3.84	6.58	12.07	13.36	14.11	14.52
	n	5	5	5	5	5	5
G4R, M & 1000	Mean	19.55	34.92	59.42	65.25	72.66	79.87
	±SD	3.10	4.79	7.66	8.19	8.65	8.85
	n	5	5	5	5	5	5
G1R, F & 0	Mean	146.98	172.87	188.75	215.04	222.92	230.84
	±SD	4.91	9.91	14.50	13.22	13.25	12.95
	n	5	5	5	5	5	5
G4R, F & 1000	Mean	147.97*	165.96	179.73	203.68	210.24	219.45
	±SD	4.83	6.07	5.52	9.94	11.06	11.54
	n	5	5	5	5	5	5

*: Statistically significant (p<0.05).

 

Table 5. Summmary of haematology record

Group, Sex & Dose (mg/kg/day)		Total Leucocyte Count	Total Erythrocyte Count	Hemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Hemoglobin	Mean Corpuscular Hemoglobin Concentration	Platelet Count
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)
		(10^3 cells/µL)	(10^6 cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3 cells/µL)
G1, M & 0	Mean	10.85	7.23	15.02	46.20	64.08	20.86	32.54	847.80
	±SD	1.20	0.62	0.89	2.66	3.03	0.88	0.17	94.52
	n	5	5	5	5	5	5	5	5
G2, M & 100	Mean	13.28	7.27	14.94	45.92	63.56	20.70	32.56	877.80
	±SD	3.08	0.88	0.96	3.21	4.31	1.29	0.54	141.16
	n	5	5	5	5	5	5	5	5
G3, M & 300	Mean	10.67	7.00	14.54	44.52	63.62	20.78	32.66	842.40
	±SD	1.30	0.41	0.55	1.90	1.21	0.55	0.67	104.14
	n	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	9.22	7.36	14.98	45.88	62.30	20.32	32.62	917.80
	±SD	2.77	0.40	0.93	2.57	0.44	0.38	0.54	152.11
	n	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	10.34	7.68	14.50	44.34	58.08	18.94	32.64	741.80
	±SD	1.94	0.70	1.01	2.95	5.93	1.70	0.49	198.71
	n	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	8.61	7.80	14.86	45.44	58.44	19.12	32.72	752.60
	±SD	2.37	0.59	0.68	1.30	4.01	1.16	0.80	184.01
	n	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Mean Platelet Volume	Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Reticulocyte Count
		(MPV)	(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(Retic)
		(fL)	(%)	(%)	(%)	(%)	(%)	(%)	(10^9 cells/L)
G1, M & 0	Mean	10.24	3.56	19.80	74.68	2.96	1.18	0.50	251.02
	±SD	0.21	1.36	6.16	6.18	0.34	0.58	0.20	73.84
	n	5	5	5	5	5	5	5	5
G2, M & 100	Mean	10.20	4.21	18.18	75.84	3.32	0.94	0.80*	290.50
	±SD	0.16	2.83	4.69	4.96	0.89	0.66	0.22	153.34
	n	5	5	5	5	5	5	5	5
G3, M & 300	Mean	10.24	4.12	17.50	77.82	2.54	0.88	0.46	288.68
	±SD	0.55	1.22	5.44	6.32	0.74	0.55	0.09	86.81
	n	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	10.06	2.29	23.44	71.16	3.42	0.74	0.50	167.52
	±SD	0.54	0.29	2.77	2.46	0.58	0.30	0.14	15.42
	n	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	7.50	1.96	18.14	75.40	4.46	0.94	0.32	147.74
	±SD	0.43	0.63	4.34	4.55	0.81	0.40	0.13	41.46
	n	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	7.60	1.90	19.34	74.02	4.34	1.22	0.30	146.00
	±SD	0.56	0.68	10.77	10.97	0.98	0.45	0.07	47.02
	n	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute  Basophils	Prothrombin Time	Activated Prothrombin Time
		(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(Seconds)	(Seconds)
G1, M & 0	Mean	2.11	8.14	0.32	0.13	0.06	23.80	28.44
	±SD	0.58	1.39	0.06	0.05	0.01	1.61	1.37
	n	5	5	5	5	5	5	5
G2, M & 100	Mean	2.39	10.08	0.45	0.13	0.10*	23.26	28.52
	±SD	0.69	2.55	0.17	0.12	0.04	2.68	4.17
	n	5	5	5	5	5	5	5
G3, M & 300	Mean	1.92	8.25	0.27	0.10	0.05	23.00	24.16*
	±SD	0.85	0.50	0.11	0.06	0.01	0.96	1.28
	n	5	5	5	5	5	5	5
G4, M & 1000	Mean	2.22	6.51	0.30	0.07	0.04	21.38	24.48
	±SD	0.88	1.78	0.09	0.03	0.01	1.34	2.24
	n	5	5	5	5	5	5	5
G1R, M & 0	Mean	1.88	7.79	0.47	0.09	0.03	25.24	16.80
	±SD	0.63	1.45	0.16	0.03	0.02	2.53	4.72
	n	5	5	5	5	5	5	5
G4R, M & 1000	Mean	1.51	6.53	0.38	0.11	0.03	17.40*	21.66
	±SD	0.40	2.38	0.14	0.06	0.01	2.27	2.36
	n	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Total Leucocyte Count	Total Erythrocyte Count	Hemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Hemoglobin	Mean Corpuscular Hemoglobin Concentration	Platelet Count
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)
		(10^3 cells/µL)	(10^6 cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3 cells/µL)
G1, F & 0	Mean	8.52	7.20	14.86	44.48	61.82	20.66	33.40	920.40
	±SD	2.54	0.24	0.47	1.74	2.79	0.69	0.42	163.75
	n	5	5	5	5	5	5	5	5
G2, F & 100	Mean	10.67	6.27	13.18	41.52	66.64	20.82	31.48	808.60
	±SD	4.41	0.95	2.51	3.87	5.70	1.59	3.68	208.67
	n	5	5	5	5	5	5	5	5
G3, F & 300	Mean	8.86	6.68	13.90	42.96	65.38	20.98	32.36	906.00
	±SD	1.38	0.87	0.88	2.38	11.14	2.20	1.85	135.69
	n	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	8.79	6.82	13.52	42.80	63.50	19.84	31.54	1021.20
	±SD	3.56	0.82	1.53	0.92	7.51	1.12	3.18	90.67
	n	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	6.99	7.01	13.76	41.82	59.80	19.72	33.00	819.40
	±SD	2.56	0.55	0.53	2.10	3.55	1.13	0.48	69.72
	n	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	7.13	7.19	14.08	42.70	59.56	19.62	32.96	798.00
	±SD	1.94	0.51	0.51	1.25	3.29	0.96	0.71	86.47
	n	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Mean Platelet Volume	Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Reticulocyte Count
		(MPV)	(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(Retic)
		(fL)	(%)	(%)	(%)	(%)	(%)	(%)	(10^9 cells/L)
G1, F & 0	Mean	9.86	2.49	24.12	70.70	2.78	1.02	0.50	180.16
	±SD	0.44	0.79	14.34	13.84	0.65	0.40	0.16	61.01
	n	5	5	5	5	5	5	5	5
G2, F & 100	Mean	10.78	4.75	18.62	76.36	2.58	0.92	0.74	282.24
	±SD	0.99	2.61	3.95	4.32	0.47	0.28	0.66	133.39
	n	5	5	5	5	5	5	5	5
G3, F & 300	Mean	10.28	4.56	17.88	77.10	2.72	1.12	0.40	287.20
	±SD	0.54	2.75	5.70	5.77	0.29	0.24	0.19	139.86
	n	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	10.66	5.49	18.40	77.32	2.02	0.84	0.36	343.76
	±SD	0.54	5.08	3.40	3.29	0.58	0.32	0.09	262.77
	n	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	7.42	2.27	18.78	75.40	3.34	1.28	0.32	153.90
	±SD	0.68	1.63	6.44	7.63	1.18	0.83	0.11	97.40
	n	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	7.52	1.81	25.32	67.66	3.60	1.34	0.22	127.82
	±SD	0.68	0.68	4.51	2.35	0.97	0.64	0.04	38.55
	n	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute  Basophils	Prothrombin Time	Activated Prothrombin Time
		(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(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	1.97	6.10	0.24	0.09	0.04	25.00	24.92
	±SD	1.07	2.46	0.10	0.05	0.02	0.45	1.15
	n	5	5	5	5	5	5	5
G2, F & 100	Mean	1.91	8.19	0.27	0.09	0.10	26.76	28.12
	±SD	0.67	3.53	0.13	0.04	0.12	3.35	4.03
	n	5	5	5	5	5	5	5
G3, F & 300	Mean	1.57	6.85	0.24	0.10	0.04	24.72	25.28
	±SD	0.53	1.22	0.02	0.03	0.02	1.23	1.11
	n	5	5	5	5	5	5	5
G4, F & 1000	Mean	1.67	6.75	0.18	0.07	0.03	25.16	27.22
	±SD	0.97	2.54	0.07	0.03	0.01	0.60	2.90
	n	5	5	5	5	5	5	5
G1R, F & 0	Mean	1.26	5.33	0.25	0.08	0.02	22.04	19.74
	±SD	0.51	2.01	0.17	0.05	0.02	1.81	4.86
	n	5	5	5	5	5	5	5
G4R, F & 1000	Mean	1.77	4.83	0.25	0.10	0.02	22.68	19.72
	±SD	0.49	1.38	0.10	0.06	0.01	0.95	5.37
	n	5	5	5	5	5	5	5

*: Statistically significant (p<0.05).

 

Table 6. Summmary of clinical chemistry record

Group, Sex & Dose (mg/kg/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)	(ALB)	(ALT)	(AST)	(ALP)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)	(U/L)	(U/L)	(U/L)
G1, M & 0	Mean	76.40	37.88	0.41	49.00	56.60	6.22	3.23	64.20	114.00	180.60
	±SD	6.80	2.32	0.03	11.40	10.41	0.43	0.11	12.30	27.92	27.25
	n	5	5	5	5	5	5	5	5	5	5
G2, M & 100	Mean	79.00	34.68	0.40	55.20	53.80	6.08	3.24	58.00	100.80	193.00
	±SD	7.25	1.95	0.05	12.70	10.96	0.33	0.21	6.63	7.60	37.01
	n	5	5	5	5	5	5	5	5	5	5
G3, M & 300	Mean	93.60	36.14	0.39	49.80	39.20	5.88	3.17	60.00	106.20	178.00
	±SD	16.01	3.92	0.04	11.56	13.14	0.39	0.21	11.47	8.41	40.79
	n	5	5	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	89.40	37.10	0.42	53.60	57.80	6.30	3.25	57.20	95.20	162.40
	±SD	10.06	3.03	0.05	10.43	15.35	0.29	0.21	9.63	5.76	27.62
	n	5	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	96.60	33.90	0.49	43.00	47.20	7.50	3.09	63.00	126.00	180.60
	±SD	25.65	3.78	0.03	5.79	8.23	0.27	0.11	12.63	34.78	35.45
	n	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	84.00	34.80	0.51	47.80	61.20	7.60	3.27	60.60	129.40	159.40
	±SD	18.23	6.97	0.05	4.66	13.97	0.48	0.23	6.95	22.88	54.55
	n	5	5	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Total Bilirubin	Calcium	Phosphorous	Cholinesterase	Globulin	Albumin/ Globulin ratio	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(BIT)	(CAL)	(PHO)	(CHE)	(GLO)	(A/G Ratio)	(BUN)	(Na)	(K)	(CLO)
		(mg/dL)	(mg/dL)	(mg/dL)	(U/L)	(g/dL)		(mg/dL)	mmol/L)	(mmol/L)	(mmol/L)
G1, M & 0	Mean	0.00	9.70	6.94	116.40	2.99	1.09	17.68	145.46	3.05	107.46
	±SD	0.00	0.16	0.63	11.33	0.36	0.12	1.09	1.16	0.22	1.48
	n	5	5	5	5	5	5	5	5	5	5
G2, M & 100	Mean	0.02	9.76	6.86	118.00	2.84	1.14	16.19	145.50	3.22	108.76
	±SD	0.02	0.26	0.46	25.40	0.17	0.07	0.91	1.32	0.22	1.55
	n	5	5	5	5	5	5	5	5	5	5
G3, M & 300	Mean	0.01	9.46	6.54	116.40	2.71	1.18	16.87	145.72	3.20	109.14
	±SD	0.01	0.24	0.53	13.67	0.22	0.08	1.83	1.14	0.28	1.99
	n	5	5	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	0.01	9.82	6.68	135.40	3.05	1.07	17.31	146.72	3.27	109.84
	±SD	0.01	0.25	0.38	30.34	0.18	0.09	1.42	1.04	0.34	1.59
	n	5	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	0.00	9.02	6.66	110.40	4.41	0.70	15.82	143.62	3.47	106.38
	±SD	0.00	0.31	0.36	28.59	0.23	0.04	1.76	0.45	0.29	0.85
	n	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	0.01	9.04	6.54	124.40	4.33	0.77	16.24	143.58	3.46	106.30
	±SD	0.02	0.48	0.66	30.38	0.56	0.13	3.25	0.28	0.56	1.91
	n	5	5	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)	(ALB)	(ALT)	(AST)	(ALP)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)	(U/L)	(U/L)	(U/L)
G1, F & 0	Mean	107.60	36.22	0.45	58.20	35.20	6.58	3.39	45.00	91.00	98.80
	±SD	20.32	2.62	0.06	8.67	8.23	0.16	0.19	1.73	7.18	15.99
	n	5	5	5	5	5	5	5	5	5	5
G2, F & 100	Mean	107.00	40.02	0.43	54.20	31.60	6.52	3.31	50.20	93.80	94.00
	±SD	14.76	7.23	0.05	6.38	7.80	0.28	0.12	5.67	12.70	31.72
	n	5	5	5	5	5	5	5	5	5	5
G3, F & 300	Mean	105.00	34.14	0.44	58.60	24.40	6.52	3.28	95.00	88.40	103.00
	±SD	19.27	3.72	0.04	13.32	5.55	0.44	0.23	119.10	10.01	31.29
	n	5	5	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	97.40	39.04	0.42	52.40	26.00	6.62	3.20	41.80	91.40	92.20
	±SD	5.81	3.50	0.01	5.64	4.74	0.43	0.12	6.72	9.58	18.61
	n	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	115.40	46.34	0.61	52.80	45.80	7.62	3.37	38.00	99.80	74.00
	±SD	8.68	3.36	0.06	16.08	22.69	0.35	0.17	4.06	7.19	21.70
	n	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	104.20	37.30*	0.56	74.00	63.00	8.00	3.57	40.20	107.60	64.60
	±SD	15.69	2.89	0.02	15.78	34.69	0.49	0.12	4.66	13.11	10.81
	n	5	5	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Total Bilirubin	Calcium	Phosphorous	Cholinesterase	Globulin	Albumin/Globulin ratio	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(BIT)	(CAL)	(PHO)	(CHE)	(GLO)	(A/G Ratio)	(BUN)	(Na)	(K)	(CLO)
		(mg/dL)	(mg/dL)	(mg/dL)	(U/L)	(g/dL)		(mg/dL)	mmol/L)	(mmol/L)	(mmol/L)
G1, F & 0	Mean	0.01	9.50	5.74	339.20	3.19	1.07	16.90	146.80	3.26	109.98
	±SD	0.00	0.22	0.90	153.87	0.15	0.10	1.22	0.96	0.25	1.11
	n	5	5	5	5	5	5	5	5	5	5
G2, F & 100	Mean	0.04	9.64	6.08	339.20	3.21	1.04	18.68	145.80	3.25	110.44
	±SD	0.04	0.30	0.23	156.45	0.18	0.04	3.37	0.82	0.28	2.17
	n	5	5	5	5	5	5	5	5	5	5
G3, F & 300	Mean	0.03	9.52	6.18	392.80	3.24	1.02	15.93	146.08	3.25	109.42
	±SD	0.03	0.44	0.76	103.24	0.36	0.13	1.74	0.31	0.27	0.91
	n	5	5	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	0.02	9.58	5.82	295.60	3.42	0.95	18.22	146.80	3.35	112.30
	±SD	0.02	0.13	0.48	44.11	0.41	0.13	1.63	1.36	0.23	2.17
	n	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	0.00	9.24	4.96	363.80	4.25	0.79	21.63	143.12	3.13	107.76
	±SD	0.00	0.15	0.35	128.23	0.26	0.06	1.57	0.77	0.24	1.19
	n	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	0.00	9.36	4.82	522.00*	4.43	0.81	17.41*	143.14	3.13	106.60
	±SD	0.00	0.21	0.47	50.04	0.39	0.05	1.35	0.80	0.20	0.90
	n	5	5	5	5	5	5	5	5	5	5

*: Statistically significant (p<0.05).

 

Table 7. Summmary of absolute organ weights (g) record

Group, Sex & Dose (mg/kg/day)		Adrenals	Thymus	Spleen	Testes	Epididymes	Heart	Kidneys	Brain	Liver	Prostate+Seminal vesicles with coagulating glands (PSC)	Lungs	Thyroid along with parathyroid
G1, M & 0	Mean	0.0650	0.3827	1.1071	2.9619	0.9208	1.0555	2.2293	1.8932	9.4504	1.8162	2.2357	0.0265
	±SD	0.0054	0.0237	0.1226	0.1131	0.0658	0.0642	0.1133	0.0368	0.5226	0.0533	0.1654	0.0046
	n	5	5	5	5	5	5	5	5	5	5	5	5
G2, M & 100	Mean	0.0653	0.4345*	1.1480	3.1239	0.9164	1.0255	2.3303	1.9193	9.1267	1.8468	2.2892	0.0272
	±SD	0.0055	0.0241	0.1758	0.1064	0.0498	0.0535	0.1783	0.0600	0.2846	0.0482	0.2341	0.0030
	n	5	5	5	5	5	5	5	5	5	5	5	5
G3, M & 300	Mean	0.0640	0.3655	1.1745	3.0821	0.9945	1.1462	2.2259	1.9611	9.9354	2.0696*	2.0302	0.0257
	±SD	0.0052	0.0398	0.1221	0.2433	0.0593	0.1155	0.1601	0.1184	0.2256	0.1255	0.0603	0.0022
	n	5	5	5	5	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	0.0624	0.3582	1.0955	3.0368	1.0421*	1.0209	2.0452	1.9481	9.2955	1.9882*	2.1237	0.0268
	±SD	0.0034	0.0183	0.0852	0.1892	0.0658	0.0426	0.0693	0.0429	0.2726	0.0750	0.1233	0.0036
	n	5	5	5	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	0.0562	0.4038	1.0462	3.2560	1.0915	1.1330	2.9957	1.9890	10.7320	1.9709	2.2137	0.0261
	±SD	0.0042	0.0567	0.1133	0.1275	0.1171	0.1165	0.3451	0.0392	0.7802	0.0667	0.2298	0.0023
	n	5	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	0.0570	0.4199	1.0465	3.2101	1.0799	1.1174	2.8470	2.0440	11.0235	2.0310	2.2273	0.0278
	±SD	0.0035	0.0530	0.1095	0.1234	0.1159	0.0848	0.0877	0.0629	0.7546	0.0963	0.1983	0.0027
	n	5	5	5	5	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Adrenals	Thymus	Spleen	Ovaries	Uterus	Heart	Kidneys	Brain	Liver	Lungs	Thyroid along with parathyroid
G1, F & 0	Mean	0.0622	0.3508	1.0520	0.1481	0.4742	0.9289	1.7274	1.7529	7.0155	1.8666	0.0276
	±SD	0.0039	0.0347	0.0962	0.0138	0.0575	0.0411	0.0521	0.0789	0.3258	0.0876	0.0019
	n	5	5	5	5	5	5	5	5	5	5	5
G2, F & 100	Mean	0.0634	0.3558	1.1011	0.1561	0.4584	0.8925	1.6806	1.8289	7.0099	1.8620	0.0261
	±SD	0.0054	0.0186	0.1395	0.0144	0.0387	0.0083	0.1311	0.1068	0.1685	0.1193	0.0032
	n	5	5	5	5	5	5	5	5	5	5	5
G3, F & 300	Mean	0.0663	0.3936	1.1317	0.1510	0.4627	0.9646	1.8377	1.9906*	7.0515	1.9899	0.0269
	±SD	0.0037	0.0364	0.0792	0.0127	0.0401	0.0411	0.0692	0.1342	0.4025	0.0822	0.0022
	n	5	5	5	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	0.0642	0.3658	1.0344	0.1570	0.4702	0.8786	1.7861	1.8945	7.1033	1.9446	0.0264
	±SD	0.0043	0.0323	0.0956	0.0099	0.0442	0.0647	0.0829	0.0896	0.5884	0.0886	0.0037
	n	5	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	0.0576	0.3131	0.9075	0.1519	0.4456	0.9248	1.6678	1.9168	8.1132	1.9274	0.0271
	±SD	0.0044	0.0277	0.0520	0.0081	0.0508	0.0546	0.0420	0.0464	0.5800	0.0870	0.0027
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	0.0588	0.3065	0.8669	0.1479	0.4234	0.9631	1.6538	1.9619	8.2348	2.0209	0.0264
	±SD	0.0027	0.0271	0.0677	0.0129	0.0465	0.0408	0.0402	0.0415	0.5318	0.0818	0.0023
	n	5	5	5	5	5	5	5	5	5	5	5

*: Statistically significant (p<0.05).

 

Table 8. Summmary of organ weight relative to fasting body weight (%) record

Group, Sex & Dose (mg/kg/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymes	Heart	Kidneys	Brain	Liver	Prostate+Seminal vesicles with coagulating glands (PSC)	Lungs	Thyroid along with parathyroid
G1, M & 0	Mean	279.87	0.0234	0.1372	0.3983	1.0670	0.3304	0.3801	0.8010	0.6810	3.4091	0.6523	0.8004	0.0096
	±SD	25.26	0.0025	0.0086	0.0573	0.1271	0.0302	0.0475	0.0732	0.0657	0.4682	0.0491	0.0399	0.0022
	n	5	5	5	5	5	5	5	5	5	5	5	5	5
G2, M & 100	Mean	269.30	0.0243	0.1616*	0.4278	1.1635	0.3408	0.3822	0.8658	0.7152	3.4039	0.6893	0.8523	0.0102
	±SD	19.10	0.0025	0.0074	0.0710	0.0692	0.0145	0.0320	0.0397	0.0493	0.2852	0.0635	0.0948	0.0018
	n	5	5	5	5	5	5	5	5	5	5	5	5	5
G3, M & 300	Mean	259.43	0.0248	0.1410	0.4538	1.1933	0.3849*	0.4430	0.8596	0.7590	3.8403	0.7984*	0.7851	0.0099
	±SD	16.58	0.0031	0.0140	0.0516	0.1363	0.0382	0.0496	0.0674	0.0739	0.2224	0.0343	0.0560	0.0005
	n	5	5	5	5	5	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	268.67	0.0233	0.1343	0.4104	1.1339	0.3889*	0.3816	0.7661	0.7289	3.4819	0.7428*	0.7945	0.0100
	±SD	20.08	0.0023	0.0162	0.0520	0.0850	0.0267	0.0302	0.0822	0.0667	0.3720	0.0518	0.0794	0.0012
	n	5	5	5	5	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	287.50	0.0196	0.1415	0.3647	1.1388	0.3823	0.3970	1.0451	0.6941	3.7494	0.6891	0.7769	0.0091
	±SD	20.48	0.0003	0.0258	0.0386	0.1172	0.0563	0.0611	0.1263	0.0417	0.3818	0.0659	0.1303	0.0009
	n	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	277.44	0.0206	0.1528	0.3771	1.1625	0.3932	0.4037	1.0326	0.7416	3.9934	0.7380	0.8046	0.0101
	±SD	22.94	0.0010	0.0282	0.0213	0.0938	0.0675	0.0286	0.1030	0.0750	0.4191	0.0926	0.0691	0.0009
	n	5	5	5	5	5	5	5	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries	Uterus	Heart	Kidneys	Brain	Liver	Lungs	Thyroid along with parathyroid
G1, F & 0	Mean	196.99	0.0316	0.1780	0.5341	0.0756	0.2403	0.4719	0.8788	0.8904	3.5629	0.9479	0.0140
	±SD	9.45	0.0020	0.0133	0.0429	0.0105	0.0221	0.0195	0.0549	0.0335	0.1130	0.0285	0.0011
	n	5	5	5	5	5	5	5	5	5	5	5	5
G2, F & 100	Mean	194.16	0.0327	0.1833	0.5695	0.0804	0.2365	0.4603	0.8655	0.9435	3.6131	0.9595	0.0135
	±SD	8.42	0.0035	0.0072	0.0893	0.0072	0.0228	0.0184	0.0521	0.0724	0.0906	0.0573	0.0014
	n	5	5	5	5	5	5	5	5	5	5	5	5
G3, F & 300	Mean	206.72	0.0321	0.1903	0.5477	0.0731	0.2240	0.4669	0.8900	0.9639	3.4135	0.9633	0.0130
	±SD	6.14	0.0018	0.0154	0.0386	0.0058	0.0214	0.0221	0.0505	0.0761	0.2224	0.0488	0.0010
	n	5	5	5	5	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	193.26	0.0334	0.1892	0.5374	0.0813	0.2430	0.4550	0.9265	0.9822	3.6818	1.0104	0.0137
	±SD	10.49	0.0035	0.0120	0.0675	0.0038	0.0111	0.0296	0.0673	0.0624	0.3381	0.0969	0.0022
	n	5	5	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	222.25	0.0261	0.1416	0.4093	0.0686	0.2017	0.4183	0.7529	0.8654	3.6658	0.8710	0.0122
	±SD	13.52	0.0032	0.0181	0.0288	0.0060	0.0311	0.0484	0.0540	0.0630	0.3921	0.0829	0.0012
	n	5	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	208.00	0.0284	0.1474	0.4181	0.0714	0.2039	0.4644	0.7982	0.9465	3.9816	0.9740	0.0128
	±SD	12.28	0.0023	0.0108	0.0430	0.0083	0.0233	0.0342	0.0655	0.0718	0.4821	0.0650	0.0015
	n	5	5	5	5	5	5	5	5	5	5	5	5

*: Statistically significant (p<0.05).

 

Table 9. Summmary of gross pathology findings

Sex	Male						Female
Route of administration	Oral Gavage
Treatment	Vehicle Control	Ultramarine Violet					Vehicle Control	Ultramarine Violet
Group	G1	G2	G3	G4	G1R	G4R	G1	G2	G3	G4	G1R	G4R
Dose (mg/kg/day)	0	100	300	1000	0	1000	0	100	300	1000	0	1000
Number of animals	5	5	5	5	5	5	5	5	5	5	5	5
No. of dead rats during treatment	0	0	0	0	0	0	0	0	0	0	0	0
No. of moribund sacrificed rats	0	0	0	0	0	0	0	0	0	0	0	0
No. of terminally sacrificed rats	5	5	5	5	5	5	5	5	5	5	5	5
No of rats showing gross pathology	0	0	0	0	0	0	0	0	0	0	0	0

 

Table 10. Summmary of histopathology findings

Route of administration		Oral Gavage
Treatment		Vehicle Control		Ultramarine Violet
Dose (mg/kg/day)		0		1000
Group		G1		G4
Sex		M	F	M	F
Number of Animals		5	5	5	5
Adrenals	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Brain	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Cervix	Number examined	X	5	X	5
	Within normal limits	-	5	-	5
Coagulating glands	Number examined	5	X	5	X
	Within normal limits	5	-	5	-
Epididymides	Number examined	5	X	5	X
	Within normal limits	5	-	5	-
Eyes	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Heart	Number examined	5	5	5	5
	Within normal limits	4	5	5	5
Infiltrations, mononuclear cells	Minimal	1	-	-	-
Kidneys	Number examined	5	5	5	5
	Within normal limits	4	5	5	5
Cast(s)	Minimal	1	-	-	-
Liver	Number examined	5	5	5	5
	Within normal limits	3	5	4	5
Infiltrations, mononuclear cells	Minimal	2	-	1	-
Lungs	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Ovaries	Number examined	X	5	X	5
	Within normal limits	-	5	-	5
Parathyroid	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Prostate gland	Number examined	5	X	5	X
	Within normal limits	5	-	5	-
Seminal vesicles	Number examined	5	X	5	X
	Within normal limits	5	-	5	-
Skin	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Spinal cord	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Spleen	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Testes	Number examined	5	X	5	X
	Within normal limits	5	-	5	-
Thymus	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Thyroid	Number examined	5	5	5	5
	Within normal limits	5	5	5	3
Ultimobranchial cyst	Present	-	-	-	2
Uterus	Number examined	X	5	X	5
	Within normal limits	-	5	-	5
Vagina	Number examined	X	5	X	5
	Within normal limits	-	5	-	5

 -: No incidence; X: Not applicable.

 

Conclusions:

In a 28-day repeated dose toxicity study conducted in Sprague Dawley Rats, the NOAEL of the test item for both sexes is 1000 mg/kg bw/day.

Executive summary:

To establish doses for a subsequent sub-chronic toxicity study on the test substance, a dose range finding study was performed for a period of 28 consecutive days according to OECD 407 Guideline (GLP study) using Sprague Dawley rats. Four groups (G1, G2, G3 and G4) of 5 animals of each sex and two recovery groups (G1R and G4R) were tested, being G1 and G1R the vehicle control groups (0.5% w/v CMC). The treatment groups had the following concentrations: 100, 300 and 1000 mg/kg bw. The vehicle and test item formulations were administered orally by gavage to the animals at an equivolume of 10 mL/kg bw. 

No mortality and clinical signs were observed throughout the dosing and recovery period. No test item-related changes in body weight, percent change in body weight and feed consumption were observed throughout the experimental period. No test item-related neurological/functional abnormalities and no ocular changes were observed during examination. No test item related changes were noted in haematology, coagulation, clinical chemistry and urinalysis parameters. There were no test item-related effects observed in absolute and relative organ weights. No test item related gross and histopathological changes were observed up to the highest dose tested in both the sexes.

Therefore, based on the obtained results from this 28-day repeated dose toxicity study conducted in Sprague Dawley Rats, the NOAEL is estimated as 1000 mg/kg bw/day under experimental conditions employed. Therefore, the dose levels of 100, 300 and 1000 mg/kg/day can be selected for subsequent 90-day repeat dose toxicity study in rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

1 key study is available with a Klimisch score of 1.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the substance is not classified for specific target organ toxicity by repeated exposure (STOT-RE) according to CLP Regulation (EC) no 1272/2008.