Bis(trimethoxysilylpropyl)amine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Jun - 11 Oct 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2008)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Japanese guideline (MHLW No. 2, 5 and 7, 2011, 2012)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(trimethoxysilylpropyl)amine

EC Number:

280-084-5

EC Name:

Bis(trimethoxysilylpropyl)amine

Cas Number:

82985-35-1

Molecular formula:

C12H31NO6Si2

IUPAC Name:

3,3,11,11-tetramethoxy-2,12-dioxa-7-aza-3,11-disilatridecane

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Hino Breeding Center, Japan
- Age at study initiation: 5 weeks
- Weight at study initiation: 142.4 - 177 g (males), 123 - 143.5 g (females)
- Housing: animals were housed individually in hanging stainless steel cages with wire-mesh floor.
- Diet (e.g. ad libitum): pelleted diet (MF, Oriental Yeast), ad libitum
- Water (e.g. ad libitum): chlorinated water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.3 - 23.5
- Humidity (%): 51.4 - 65.2
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 04 Jun 2013 To: 26 Jul 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed and dissolved in olive oil to a prepare 50% (w/v) formulation. A part of the 50% (w/v) formulation was taken and diluted with olive oil to prepare formulations of 15 and 5% (w/v). Since the 50 and 15% (w/v) dosing formulation were confirmed to be stable for 168 h after preparation at a cold place, the formulations were prepared as the preservation period was within 168 h. The stability of the 5% (w/v) formulation was confirmed to be 4 h, therefore, the 5% (w/v) formulation was prepared each morning prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Because there is information that the test substance reacts with water, a vehicle for dosing formulation was investigated with olive oil. As a result, the test substance dissolved in olive oil at 20.0% (w/v). After one day of storage at room temperature, there were no colour changes in the test substance solution. In addition, olive oil has been generally used as a vehicle in general toxicity studies and historical control data based on olive oil as vehicle is available. Therefore, olive oil was selected as vehicle.
- Lot/batch no.: 242003
- Concentration in vehicle: 5, 15 and 50% (w/v)
- Amount of vehicle (if gavage): 2 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stabilities of the 50, 15 and 5% (w/v) formulations in the cold place were confirmed by gas chromatography (GC) in CERI Hita. The test substance in the 50 and 15% (w/v) formulations were judged as stable for 168 h under the storage conditions, because those concentrations after storage were within 100 ± 10% (101 and 101%, respectively) of those immediately after preparation. The test substance in the 5% (w/v) formulation was judged as stable for 4 h under the storage condition, because that concentration after 4 h storage was within 100 ± 10 (101%) of that immediately after preparation. However, the test substance in the 5% (w/v) formulation was judged as unstable for 24 h under the storage condition, because that concentration after storage was without 100 ± 10% (89.8%) of that immediately after preparation. Concentration analysis for the 50, 15 and 5% (w/v) formulations was performed by GC in CERI Hita immediately after the first preparation. The concentrations of the formulations were confirmed to be within 100 ± 10% of each nominal concentration (50% (w/v): 102%; 15% (w/v): 103% and 5% (w/v): 98.8%) and the preparation procedure was judged to be appropriate.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 (28 day main study)
5 (14 day recovery study)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose selection was based on the results of the 7-day range finding study. In this study groups of three Crl:CD(SD) rats of each sex and dose were administered the test material dissolved in olive oil at 0, 25, 250, 500 and 1000 mg/kg bw/day via oral gavage for 7 consecutive days. General clinical observations and body weight measurements were performed in the dosing period. Necropsy and organ weights measurements at the liver, kidney and spleen were performed on the next day after the last dosing. Since no toxic effects were observed in all treatment groups, the high dose of 1000 mg/kg bw/day which was the highest dose according to OECD TG 407 was selected for the 28-day study.
- Rationale for selecting satellite groups: Recovery groups were set for the 0 and 1000 mg/kg bw/day groups to investigate a reversibility of possible toxic effects for 14 days after the 28 day test material administration.
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the dosing period, all animals were observed their general clinical signs twice daily (before and after dosing). During the recovery period, observation was performed once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed once before dosing. Thereafter, animals were examined once a week during the dosing and recovery periods on a blind test basis.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded one day prior to dosing and on days 1, 3, 8, 12, 17, 21, 26 and 28 of the dosing period and on days 1, 5, 10 and 14 of the recovery period and on the necropsy days.

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from all animals on the last day of dosing for the main groups and on the last withdrawal day for the recovery groups.
- Anaesthetic used for blood collection: Yes (isoflurane anesthesia)
- Animals fasted: Yes (overnight fasting)
- How many animals: all animals
- Parameters examined: red blood cell count (RBC), haemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (Platelet), reticulocyte count ratio (Reticulo), white blood cell count (WBC), differential leucocyte counts (neutrophils (Neutro), lymphocytes (Lymph), basophils (Baso), eosinophils (Eosino), monocytes (Mono), large unstained cells (LUC)), prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from all animals on the last day of dosing for the main groups and on the last withdrawal day for the recovery groups.
- Animals fasted: Yes (overnight fasting)
- How many animals: all animals
- Parameters examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (gamma-GTP), total cholesterol (T-Cho), triglyceride (TG), blood urea nitrogen (BUN), creatinine, total protein (T-protein), albumin, A/G ratio, glucose, total bilirubin (T-bil), total bile acids (TBA), inorganic phosphorus (IP), calcium (Ca), sodium (Na), potassium (K), chloride (Cl)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected in individual metabolic cages from the afternoon of last day of the dosing period for the main groups and of the recovery period for the recovery groups to each next morning.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: urine volume, colour, turbidity, urine osmotic pressure, pH, protein, glucose, occult blood, urinary sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Animals were examined on week 4 (on days 27 and 28) of the dosing period. Reflex tests and measurements of grip strength were performed on a blind test basis. In the recovery period, the examinations were not performed since no abnormalities related to the test substance were observed in either sex of the high dose groups.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals were subjected to a detailed gross necropsy after blood sampling and bleeding from the ventral aorta on the next day after last administration for the main groups and on the next day after last withdrawal day for the recovery groups. External surface of the body, all orifices, subcutis, cranial, thoracic, abdominal and pelvic cavities and their content were observed. For females, vaginal smears were collected before gross necropsy and the stages of estrous cycle were determined.

HISTOPATHOLOGY: Yes
- Histopathological examinations included: trachea, lungs, submandibular gland, forestomach, glandular stomach, duodenum-ileum, cecum-rectum, pancreas, liver, heart, kidneys, urinary bladder, testes, epididymides, prostate, coagulating gland, seminal vesicle, ovaries, uterus, vagina, cerebrum, cerebellum, pons, spinal cord, sciatic nerve, bone marrow, axillar lymph nodes, mesenteric lymph nodes, spleen, thymus, pituitary gland, thyroid, parathyroid, adrenas, eye ball, skeletal muscle, bone, mammary gland

Other examinations:

ORGAN WEIGHTS
- The weights of the following organs were determined: liver, heart, kidneys, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, brain, spleen, thymus, thyroid, adrenals

Statistics:

Data regarding body weights, food consumption, grip strength and locomotor activity counts during the dosing period, and parameters of haematological and clinical chemistry examinations, urine volume, urine osmotic pressure, organ weights and body weights on the necropsy day of the main groups were analysed by the Bartlett´s test for homogeneity of variances. When the variances were homogeneous at a significant level of 5%, the Dunnett´s test was used. The frequencies of defecation and urination during the dosing period were analysed by the nonparametric Dunnett´s test. Data regarding body weights, food consumption during the recovery period and parameters of haematological and clinical chemistry examination, urine volume, urine osmotic pressure, organ weights and body weights on the necropsy day of the recovery groups were analysed by the F-test for variance ratio. When there were no significant differences at a significant level of 5% in this analysis, the Student´s t-test was used. When there was a significant difference, the Aspin-Welch t-test was used. The frequencies of defecation and urination during the recovery group were analysed by the Mann-Whitney U-test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

statistically significant decrease in males of the 1000 mg/kg bw/day dose group (recovery period) (non-adverse)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

statistically significant decrease on day 3 in females of the 1000 mg/kg bw/day dose group (non-adverse)

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

males: statistically significant increase in ratio of monocytes (1000 mg/kg bw/day), prolongation of PT (100 mg/kg bw/day); females: statistically significant shortenings of activated partial thromboplastin time (100 and 1000 mg/kg bw/day) (non-adverse)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

females: statistically significant decrease of AST (all dose groups), statistically significant increase of gamma-GTP (300 mg/kg bw/day); statistically significant decrease of total bilirubin (1000 mg/kg bw/day, recovery group) (non-adverse)

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

females: statistically significant increase of locomotor activity counts (100 mg/mg bw/day), statistically significant decrease of locomotor activity (300 mg/kg bw/day) (non-adverse)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

females: statistically significant decrease in relative weights of brain/adrenals (100 mg/kg bw/day) (non-adverse)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

edema (submucosal layer), slight hyperplasia of the surface epithelial cells (fundic mucosa (males) and pyloric mucosa (females)) and slight necrosis (pyloric mucosa (males)) in the 1000 mg/kg bw/day groups (including recovery) (adverse, local effects)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

edema (submucosal layer), slight hyperplasia of the surface epithelial cells (fundic mucosa (males) and pyloric mucosa (females)) and slight necrosis (pyloric mucosa (males)) in the 1000 mg/kg bw/day groups (including recovery) (adverse, local effects)

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
In males, no abnormalities were observed in the 1000 or 100 mg/kg bw/day dose groups. Loss of hair and exudate in the neck were observed in one male of the 300 mg/kg bw/day dose group. In females, no abnormalities were observed in the control or any treatment group. No abnormalities were observed in either sex of the control or the 1000 mg/kg bw/day recovery groups.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant changes were noted in either sex of any treatment groups. In males, statistically significant decrease or decreased tendency were observed throughout the recovery period in the 1000 mg/kg bw/day dose group.

FOOD CONSUMPTION AND COMPOUND INTAKE
In males, no statistically significant changes were noted in any treatment groups. In females, a statistically significant decrease was found on day 3 in the 1000 mg/kg bw/day dose group. No abnormalities were noted in the 100 and 300 mg/kg bw/day dose groups. No statistically significant changes were noted in either sex of the 1000 mg/kg bw/day dose group of the recovery group.

HAEMATOLOGY
In males, a statistically significant increase in ratio of monocytes was observed in the 1000 mg/kg bw/day dose group. A statistically significant prolongation of prothrombin time (PT) was observed in the 100 mg/kg bw/day dose group. No abnormalities were observed in any parameters in the 300 mg/kg bw/day dose group. The change of PT observed in the 100 mg/kg bw/day dose group was considered to be incidental, since no abnormalities were observed in the 1000 and 300 mg/kg bw/day dose groups and no dose-dependency were observed. In females, statistically significant shortenings of activated partial thromboplastin time were observed in the 1000 and 100 mg/kg bw/day dose groups. No abnormalities were observed in any parameters in the 300 mg/kg bw/day dose group. In the recovery group, a statistically significant increase of platelet count was found in males in the 1000 mg/kg bw/day dose group. In females, no significant changes were noted in the 1000 mg/kg bw/day dose group.

CLINICAL CHEMISTRY
In males, no significant changes were noted in any treatment groups. In females, statistically significant decreases of aspartate aminotransferase were observed in all treatment groups. A statistically increase of gamma-glutamyl transpeptidase was found in the 300 mg/kg bw/day dose group. This change was considered to be incidental since no abnormalities were observed in the 1000 mg/kg bw/day dose group and no dose-dependency was found. In the recovery group, no significant changes were noted in the 1000 mg/kg bw/day dose group in males. In females, a statistically significant decrease of total bilirubin was found in the 1000 mg/kg bw/day dose group.

URINALYSIS
No statistically significant changes in urine volume or urine osmotic pressure were noted in either sex or any treatment groups. No abnormalities were observed in the other examined parameters in either sex of the control or any dose groups. In the recovery group, no statistically significant changes in urine volume or urine osmotic pressure were noted in either sex of the 1000 mg/kg bw/day dose group. Moreover, no abnormalities were observed in the other examined parameters in either sex of the control or 1000 mg/kg bw/day groups.

NEUROBEHAVIOUR
In males, no statistically changes in grip strength or locomotor activity counts were noted in any treatment groups. No abnormalities were observed in the control or any treatment groups in the reflex test. In females, no statistically significant changes in locomotor activity counts were noted in the 1000 mg/kg bw/day. In the 100 mg/kg bw/day does group, locomotor activity counts of interval between 10 and 20 min and total score showed statistically significant increase. In the 300 mg/kg bw/day dose group, locomotor activity counts of interval between 40 and 50 min showed statistically significant decrease. These changes in the 100 and 300 mg/kg bw/day dose groups were considered to be incidental since these changes were not observed in the 1000 mg/kg bw/day dose group and dose-dependency was not observed. In grip strength, no significant changes were observed in any treatment groups. In reflex test, no abnormalities were observed in the control or any treatment groups. No neurobehavioural examinations were performed in animals of the recovery group since no effects of the test substance were noted in week 4 during the dosing period.

ORGAN WEIGHTS
In males, no significant changes were noted in any treatment groups. In females, no significant changes were noted in the 1000 and 300 mg/kg bw/day dose groups. In the 100 mg/kg bw/day dose group, statistically significant decreases in relative weights of the brain and adrenals were found. These changes were considered to be incidental since no abnormalities were observed in the 1000 and 300 mg/kg bw/day dose groups and no dose-dependency were found. In the recovery group, statistically significant decreases in relative and absolute weights of the spleen were found in the 1000 mg/kg bw/day dose group. In females, statistically significant increases in relative and absolute weights of the thyroid were found in the 1000 mg/kg bw/day dose group.

GROSS PATHOLOGY
In males, staining around nose and mouth, thickening of wall of the esophagus, roughening of the mucosa in the forestomach, bilateral enlargement of the mediastinal lymph node, enlargement of the hepatic lymph node, edematous change of the thymus, hydrothorax of the thoracic cavity, unilateral dark reddish change and bilateral enlargement at the adrenal were observed in one animal of the 1000 mg/kg bw/day dose group. Gritty and brownish substance in the lumen of the stomach was observed in two animals, gritty and brownish substance in the lumen of the cecum was recorded in another animal of the 1000 mg/kg bw/day dose group. In the 300 mg/kg bw/day dose group, erosion in the cervical skin and bilateral enlargement of the submandibular lymph node were observed in one animal in which loss of hair and exudate in the neck was observed in the general clinical signs observations. The erosion in the cervical skin observed in the 300 mg/kg bw/day dose group was considered to be incidental and occurred at the retention of the administration, and the bilateral enlargement of the submandibular lymph node was considered as the secondary change occurred from erosion in the cervical skin. In females, gritty and brownish substance in the lumen of the cecum was observed in two animals of the 1000 mg/kg bw/day dose group. Gritty and brownish substance in the lumen of the stomach was observed in one animal of the 1000 mg/kg bw/day dose group. In the 100 mg/kg bw/day dose group, cyst in the vagina of one animal was observed. No abnormalities were observed in the control or the 300 mg/kg bw/day dose groups. In the recovery group, no abnormalities were recorded in the control group. In males, blackish region of the mucosa and thickening of wall in the glandular stomach and solid and brownish substance in the lumen of the stomach were observed in one animal of the 1000 mg/kg bw/day dose group. In females, thickening of wall of the glandular stomach and solid and brownish substance in the lumen of the stomach were observed in one animal of the 1000 mg/kg bw/day dose group.

HISTOPATHOLOGY: NON-NEOPLASTIC
In males, hypertrophy of the acinar cells in the submandibular gland, severe ulcer related to the macroscopic findings in the esophagus, slight hyperplasia of the squamous epithelium in the limiting ridge and severe ulcer related to the macroscopic findings in the forestomach, slight edema in the submucosal layer, slight hyperplasia of the surface epithelial cells in the fundic mucosa and slight necrosis of the pyloric mucosa at the glandular stomach, single cell necrosis of the hepatocyte in the liver, focal myocardial necrosis in the heart, degeneration and necrosis of the tubular epithelium in the kidney, decreased hematopoietic cells in the bone marrow, germinal center development in the spleen, cell infiltration in the capsule and septa and phagocytosis of the degenerate lymphocytes related to the macroscopic findings in the thymus and unilateral hemorrhage and necrosis related to the macroscopic findings in the adrenals were observed in one animal of the 1000 mg/kg bw/day dose group. Solitary cyst in the medulla in the kidney, lymphocyte infiltration in the dorsolateral prostate, focal myocarditis in the heart and Rathke´s pouch remnant in the pituitary gland were observed in one animal each in the 1000 mg/kg bw/day dose group. In the 300 mg/kg bw/day dose group, unilateral pelvic dilatation and subcapsular solitary cyst in the medulla in the kidney, ulcer in the skin and germinal center development and increased plasmacytes in the submandibular lymph node related to the macroscopic findings in the skin and submandibular gland were observed in one animal. In the 100 mg/kg bw/day dose group, focal necrosis of the hepatocytes in the liver was observed in one animal. The changes in the kidney observed in the 300 mg/kg bw/day dose group and observed in the liver of the 100 mg/kg bw/day dose group were considered to be incidental and not related to the administration of the test substance, since those changes were historically observed in the control animals of the laboratory and no similar changes were observed in the 1000 mg/kg bw/day dose group. In females, degeneration and necrosis of the tubular epithelium and subcapsular solitary cyst in the kidney and decreased hematopoietic cells in the bone marrow were observed in one female of the 1000 mg/kg bw/day dose group. Slight hyperplasia of the surface epithelial cells in the pyloric mucosa in the glandular stomach was observed in one female. In the 100 mg/kg bw/darey dose group, mineralization of cortico-medullary junction in the kidney and squamous epithelial cyst in the vagina were observed in one animal. No abnormalities were observed in the 300 mg/kg bw/day dose group. The changes observed in the 100 mg/kg bw/day dose group were considered to be incidental and not related to the administration of the test substance, since the changes were historically observed in the control animals of the laboratory and no dose-dependency was found as no similar changes were observed in the 300 and 1000 mg/kg bw/day dose groups. In the control group, mineralization of the cortico-medullary junction in the kidney and focal necrosis of the hepatocytes in the liver were observed in one animal each. In the recovery group, slight hyperplasia of the surface of epithelial cells in the pyloric mucosa and slight focal necrosis of the fundic mucosa related to the macroscopic findings in the glandular stomach were observed in one male animal of the 1000 mg/kg bw/day dose group. No abnormalities were observed in the control group. In females, slight hyperplasia of the surface epithelial cells in the pyloric mucosa related to the macroscopic findings in the glandular stomach was observed in one animal. Subcapsular solitary cyst in the kidney was observed in one animal of the control group.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

(local)

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: necrosis of the pyloric/fundic mucosa, hyperplasia of the surface epithelial cells in the pyloric/fundic mucosa, edema in the submucosal layer (males), hyperplasia of the surface epithelial cells in the pyloric mucosa (females)

2

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed with respect to general/detailed clinical observations, haematological/clinical chemistry/neurobehaviour examination, body weights or urinalysis

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

no

Any other information on results incl. tables

Table 1: Summary of functional observations (motor activity)

Sex		Males				Females
Dose (mg/kg bw/day)		0	100	300	1000	0	100	300	1000
No. of animals		10	5	5	10	10	5	5	10
Motor activity (interval)	0-10 (mn)	213 ± 49	215 ± 44	224 ± 72	215 ± 49	217 ± 49	254 ± 30	265 ± 23	250 ± 52
	10-20 (min)	108 ± 46	101 ± 14	138 ± 64	104 ± 71	153 ± 65	237 ± 7*	186 ± 62	180 ± 51
	20-30 (min)	74 ± 42	59 ± 37	89 ± 39	51 ± 40	107 ± 52	164 ± 26	130 ± 74	103 ± 70
	30-40 (min)	77 ± 54	63 ± 41	68 ± 49	55 ± 41	97 ± 53	106 ± 57	91 ± 51	83 ± 67
	40-50 (min)	40 ± 42	39 ± 28	18 ± 13	31 ±45	74 ± 60	131 ± 78	4 ± 7*	56 ± 53
	50-60 (mIn)	34 ± 37	13 ± 17	15 ± 19	17 ± 25	54 ± 46	68 ± 41	57 ± 81	46 ± 54
	Total	546 ± 196	489 ± 118	552 ± 150	473 ± 200	702 ± 202	959 ± 99*	733 ± 241	718 ± 168

*: p<0.05

Table 2: Summary of haematological parameters (males)

Item	Sex	Male groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
Mono (%)		2.50 ± 0.27	2.64 ± 1.11	2.48 ± 0.69	3.74* ± 0.48	2.82 ± 0.63	3.02 ± 0.60
PT (sec)		15.4 ± 1.20	18.68* ± 0.98	15.48 ± 1.18	15.56 ± 1.15	17.26 ± 1.52	19.42 ± 4.56
Platelat (x10^4/µl)		137.34 ± 7.75	129.94 ± 9.41	141.18 ± 22.00	115.06 ± 15.69	109.44 ± 5.66	125.14** ± 3.49

*: p<0.05

**: p<0.01

Table 3: Summary of haematological parameters (females)

Item	Sex	Female groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
APTT (sec)		19.90 ± 0.62	16.98* ± 2.00	18.48 ± 1.50	17.18* ± 1.00	17.66 ± 2.71	17.44 ± 1.88

*: p<0.05

Table 4: Summary of clinical chemistry parameters (females)

Item	Sex	Female groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
AST (IU/l)		95.6 ± 6.5	76.2** ± 7.9	75.4** ± 5.9	72.4** ± 7.4	71.2 ± 5.9	74.8 ± 15.0
gamma-GTP (IU/l)		0.56 ± 0.17	0.72 ± 0.41	1.02* ± 0.16	0.82 ± 0.13	0.52 ± 0.27	0.64 ± 0.22
T-Bil (mg/dl)		0.064 ± 0.013	0.058 ± 0.008	0.052 ± 0.008	0.056 ± 0.011	0.076 ± 0.005	0.060** ± 0.007

*: p<0.05

**: p<0.01

Table 5: Summary of absolute organ weights (males)

Item	Sex	Male groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
Spleen (g)		0.722 ± 0.089	0.650 ± 0.157	0.712 ± 0.154	0.602 ± 0.147	0.888 ± 0.033	0.698* ± 0.102
Final body weight (g)		345.04 ± 45.59	335.56 ± 33.27	341.76 ± 40.21	329.42 ± 47.50	424.46 ± 20.98	391.42* ± 22.89

*: p<0.05

Table 6: Summary of absolute organ weights (females)

Item	Sex	Female groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
Thyroid (g)		14.22 ± 2.66	14.88 ± 1.70	13.36 ± 0.74	13.30 ± 1.68	13.12 ± 1.47	17.24* ± 2.64
Final body weight (g)		196.52 ± 21.66	210.92 ± 12.75	202.70 ± 11.87	202.44 ± 12.52	236.56 ± 14.91	230.58 ± 17.05

*: p<0.05

Table 7: Summary of relative organ weights (males)

Item	Sex	Female groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
Spleen (g/100 g)		0.208 ± 0.024	0.192 ± 0.026	0.210 ± 0.050	0.180 ± 0.035	0.208 ± 0.004	0.178* ± 0.020
Final body weight (g)		345.04 ± 45.59	335.56 ± 33.27	341.78 ± 40.21	329.42 ± 47.50	424.46 ± 20.98	391.42* ± 22.89

*: p<0.05

Table 8: Summary of relative organ weights (females)

Item	Sex	Female groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
Brain (g/100 g)		0.966 ± 0.057	0.888* ± 0.026	0.960 ± 0.055	0.920 ± 0.045	0.826 ± 0.047	0.828 ± 0.018
Thyroid (mg/100 g)		7.28 ± 1.28	7.06 ± 0.66	6.60 ± 0.66	6.60 ± 1.00	5.54 ± 0.54	7.54* ± 1.46
Adrenals (mg/100 g)		31.58 ± 4.16	25.18* ± 1.12	28.98 ± 4.81	29.96 ± 0.76	25.50 ± 2.90	26.74 ± 3.17
Final body weight (g)		196.52 ± 21.66	210.92 ± 12.75	202.70 ± 11.87	202.44 ± 12.52	236.56 ± 14.91	230.58 ± 17.05

*: p<0.05