Fructofuranosidase, β-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August to 28 or 29 November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted: 21 September 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

August 1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Directive 87/302/EEC Part B:Methods for Determination of Toxicity: Subchronic Oral Toxicity Test: 90 day Repeated Oral Dose using Rodent species

Version / remarks:

18 November 1987

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministry of Health Welfare and Sports, Inspectorate for Health Protection, Commodities and Veterinary Public Health, GLP Compliance Monitoring unit, The Netherlands

Limit test:

no

Test material

Test material information

1

Reference substance name:

Xylanase, endo-1,4-

EC Number:

232-800-2

EC Name:

Xylanase, endo-1,4-

Cas Number:

9025-57-4

Molecular formula:

Not applicable, see remarks.

IUPAC Name:

endo-1,4-beta-xylanase

Test animals

Species:

rat

Strain:

other: Hsd Cpb WU rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxicology Department, Rallis Research Center, Rallis India Limited, Bangalore, India
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks
- Weight at study initiation: 208 ± 9.9 - 210 ± 9.7 (males), 153 ± 6.9 - 154 ± 7.3 (females)
- Housing: two rats per cage in in a sterilized suspended standard polypropylene rat cage (size: L 410 x W 282 x H 150 mm) with stainless steel mesh bottom and stainless steel top grill.
- Diet: rats/mice pellet food (Ssniff Spezialdiaten GmbH, Söest, Germany), ad libitum
- Water: deep borewell water passed through activated charcoal filter and exposed to UV rays in aquaguard on-line water filter-cum-purifier, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 -24
- Humidity (%): 30 -70
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24.08. 2001 To: 28./29.11.2001

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: double distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in double distilled water and was prepared daily. The quantities of test substance was not weighed instead volume was measured based on specific gravity (specific gravity 1 06). Approximate quantities of 2000 mg (1.89 mL), 8000 mg (7.55 mL) and 32000 mg (30.19 mL) of test substance were separately mixed and volume of each was made up to 50 mL with double distilled water to get the test substance concentrations of 40, 160 and 640 mg.
- Dose volume: 10 mL/kg bw/day

VEHICLE
- Amount of vehicle: varied, depending on the body weights of the rats recorded during different intervals of treatment period

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance preparations were analysed for protein content by digesting the sample of the test item with concentrated sulphuric acid and total nitrogen content was estimated by Micro-Kjeldahl method on day 1, months 2 and 3 of the treatment period. The total protein content in the test item was calculated by multiplying total nitrogen content by a factor 6.25. The results showed mean concentrations of 34.40 ± 1.01, 135.50 ± 4.48 and 543.18 ± 17.33 mg of the test substance/mL as against the nominal concentrations of 40, 160 and 640 mg/mL.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

1 600 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

6 400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: Grouping was done based on body weight stratification and distribution. The rats procured for the study were weighed and grouped in to body weight ranges (selected ranges: 191 - 200 g, 201 - 210 g, 211 - 220 g for males and 141 - 150 g, 151 - 160 g for females). These body weight stratified rats were distributed to all the study groups in equal numbers. The rats with extreme body weights were not used for the study. Grouping was done on last day of acclimatization.
- Fasting period before blood sampling for clinical biochemistry: overnight

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations: clinical signs of toxicity (once a day), pre-terminal deaths (twice a day)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to initiation of treatment and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: shortly before test substance 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

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: one day before start of the treatment and at the end of the treatment period prior to sacrifice
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: two days prior to sacrifice (blood smear: differential leucocyte count), at the end of the treatment period (blood collection)
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the treatment period (12/13th week of the treatment period)
- Dose groups that were examined: all dose groups
- Battery of functions tested: grip strength / motor activity / sensory activity / other: visual response, auditory response, proprioceptive response, righting reflex

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (liver, adrenals, kidneys, testes, ovaries, epididymides, uterus, thymus, spleen, brain, heart were collected and weighed)

HISTOPATHOLOGY: Yes (please refer to table 2 in the "Any other information on materials and methods incl. tables" section). All the tissues from control and high dose group rats and gross lesions from low and mid dose group rats were subjected to detailed histopathological examination. The lungs of animals in the low and mid dose groups were subjected to histopathological examination for evidence of infection to provide an assessment of the health status of the animals.

Statistics:

Body weights, cumulative net body weight gains, food consumption, laboratory investigations (haematology and clinical chemistry), organ weights and organ weight ratio data were compared by Bartlett's test for homogeneity of intra group variances. When the variances proved to be heterogeneous, the data were transformed using appropriate transformation. The data with homogeneous intra group variances were subjected to one-way analysis of variance (ANOVA - Snedecor and Cochran, 1987). Following ANOVA, When 'F' was found to be significant, Dunnett's pairwise comparison (Scheffe 1953) of means of treated groups with the mean of.the control group was done individually. Following a significant difference of a test group with the control group, the dose-response correlation was estimated by including the control and all the treated groups and tested by , t' test.
All analyses and comparisons are evaluated at 5% (P ≤ 0.05) level. Statistically significant differences (P ≤ 0.05) indicated by the a forementioned tests are designated by the superscripts as stated below:
+/- : Significantly higher(+)/lower(-) than the control group
d : Significant dose correlation

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

control and 400, 1600, 6400 mg/kg bw/day: An incidence of alopecia in a female at control group and incidence of hair thinning with hair regrowth in a female each at low and mid doses and in two females at high dose group were observed (incidental finding). For details please refer to table 6 in the " Any other information on results incl. tables" section.

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):

1600 mg/kg bw: in males and females higher food intake was observed in week 8, but was considered an incidental finding. In week 9, a lower food intake was observed in females, but considered an incidental finding.
6400 mg/kg bw/day: lower food intake was observed in week 9 in both sexes and in week 10 in males only. Since the food intake was not consistently affected the effect on food consumption was considered an incidental finding.
For details please refer to table 3 in the " Any other information on results incl. tables" section.

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):

400 mg/kg bw/day: a higher level of mean corpuscular volume (MCV) was observed in females, but considered incidental as the changes were isolated incidences.

1600 mg/kg bw/day: in males isolated incidences of higher level of MCH, platelets, prothrombin time, neutrophils, and lower level of lymphocytes were observed. Since there were no effects observed at high dose level, the observed effects were considered incidental. In males significantly higher level of MCHC was observed, but considered incidental as the corresponding changes were not observed in RBC and Hb. In females, an incidence of higher level of mean corpuscular volume and a higher level of haematocrit were observed, but considered incidental as the changes were isolated incidences.

6400 mg/kg bw/day: in males a significantly higher level of MCHC was observed, but considered incidental as the corresponding changes were not observed in RBC and Hb. In females, higher neutrophil percentage with lower lymphocyte percentage were observed compared to control, but considered incidental since they were within the range of biological variation.

For details please refer to table 4 in the " Any other information on results incl. tables" section.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

1600 mg/kg bw/day: the creatinine level was significantly higher in males. Although the increase was dose-correlated it was considered incidental as there were no corresponding histopathological changes in the kidneys.

6400 mg/kg bw/day: sodium, chloride and creatinine levels were significantly higher in males. Since the higher levels of chloride and sodium were within the normal range of variation, they were not considered to be treatment-related. Although the creatinine increase was dose-correlated it was considered incidental as there were no corresponding histopathological changes in the kidneys.

For details please refer to table 5 in the " Any other information on results incl. tables" section.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

1600 mg/kg bw/day: a significant decrease in the absolute and relative weight of ovaries was observed in females, but was not considered treatment-related as it was not observed at high dose and there were no corresponding gross and histopathological changes.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

control/1600 mg/kg bw/day:dilatation of pelvis in kidneys were considered incidental.

400 mg/kg bw/day: enlarged testes and heart were condisered incidental.

control, 400, 1600 and 6400 mg/kg bw/day: a few incidences of skin-alopecia were considered incidental.

6400 mg/kg bw/day: one male showed a white liver focus, this isolated finding was considered incidental.

For details please refer to table 6 in the " Any other information on results incl. tables" section.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

400 mg/kg bw/day: in males lower grip strength values were observed in forelimbs, but considered incidental as these changes were isolated incidences.

1600 and 6400 mg/kg bw/day: significantly higher incidence of landing foot splay was observed in males and females, but considered incidental as there was no dose-dependency and no change in gait observed in these animals. In mid dose males higher grip strength values were observed in hindlimbs, but considered incidental as these changes were isolated incidences. In females, significantly higher grip strength values were observed in forelimbs at mid and high dose and hindlimbs at high dose, but were also considered incidental findings since dose correlation was not evident.

For details please refer to table 7 in the " Any other information on results incl. tables" section.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

control, 400, 1600 and 6400 mgkg bw/day: perivascular lymphocytic infiltration in the lung (minimal to mild) was observed at all doses in females and in males at all doses with exception of the low dose. The incidence of perivascular lymphocytic infiltration in the lungs was not considered treatment-related as there was no intergroup statistical significance. Blood vessel mineralisation (minimal) was observed in males and females at all doses, except in males of the control group. Since the highest indidence was observed in females of the control group this finding was considered incidental.

400 and 6400 mg/kg bw/day: mineralisation in kidneys was observed in one male of the low dose group and one female of the high dose group.

control, 1600 and 6400 mg/kg bw: basophilic tubules (minimal) were observed in kidneys of males.

control and 6400 mg/kg bw/day: hyaline droplets and proteinaceous material were observed in tubular epithelium (minimal to moderate) of the kidneys in males.

The changes in kidneys and other tissues were considered incidental as the incidences were similar or higher in control.

For a detailed overview of all findings and details please refer to table 8 in the " Any other information on results incl. tables" section.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

6 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed at this dose level

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 3: Cagewise average food intake (g/rat/day)

Group	weeks
		1	2	3	4	5	6	7	8	9	10	11	12	13
males
control	mean	25.9	27.0	27.4	27.6	27.2	26.0	26.2	25.9	29.1	28.6	25.8	25.2	25.7
	SD	1.22	1.03	0.98	0.84	1.58	1.91	1.39	0.59	1.59	1.53	0.67	0.96	1.35
400	mean	26.4	27. 1	27.4	27.9	26.7	26.4	26.0	26.2	27.5	27.5	25.8	25.8	26.6
	SD	0.50	0.69	0.98	1.49	0.94	1.07	0.76	0.31	0.99	1.52	0.57	0.91	0.64
1600	mean	27.2	27.6	28.1	28.2	28.0	25.9	26. 9	27.6+	29.1	2 7.8	2 7.1	26.6	27.1
	SD	1.46	1.41	0.96	1.22	1.21	2.54	1.57	1.09	1.81	1.45	1.59	1.58	1.45
6400	mean	26.9	27.5	27.7	28.0	27.8	26.0	24.7	25.1	24.2-	25.9-	25.9	25.4	25.7
	SD	0.83	1.12	0.75	1.30	1.01	1.19	1.26	1.19	0.78	0.95	1.34	1.64	1.05
females
control	mean	17.9	18.0	18.3	19.2	19.7	19.9	18.8	17.8	22.3	20.6	17.4	17.6	17.6
	SD	0.71	0.63	1.00	0.87	0.90	1.57	1.21	1.25	1.34	1.38	0.98	1.69	0.79
400	mean	17.3	17.8	18.1	18.6	19.3	19.0	18.2	18.0	21.3	20.0	16.7	18.0	18.1
	SD	0.58	0.74	0.73	0.98	1.22	1.08	0.78	0.73	1.48	1.28	0.78	0.90	0.85
1600	mean	17.7	18.3	18.5	18.7	19.2	19.7	18.2	20.0+	18.1-	19.1	17.8	18.5	18.7
	SD	1.07	1.08	0.79	0.89	1.21	1.51	1.15	1.63	0.62	0.86	1.36	1.33	1.47
6400	mean	18.0	18.4	19.3	19.0	19.6	18.1	17.4	20.5	18.6-	19.4	16.6	17.4	17.9
	SD	0.60	0.56	0.78	0.68	0.57	0.78	0.41	0,45	1.02	0.82	0.75	0.36	0.93

SD Standard deviation

+ significantly higher than the control group

- significantly lower than the control group

 

Table 4: Summary of haematological results

males
		WBC g/L	RBC L/L	Hb g/L	Hct L/L	MCV fl	MCH pg	MCHC g/L	Plat g/L	P.T. S	Neut %	Lymp%	Eosi%	Mono%	Baso%
control	mean	5.7	8.76	162	0.432	49.4	18.5	375	957	18.4	14.6	82.2	2.4	0.8	0.0
	SD	1.76	0.31	3.88	0.016	1.46	0.44	9.25	139.14	1.34	5.50	5.71	2.27	1.03	0.0
400	mean	7.4	8.55	161	0.426	49.8	18.8	378	1002	19.5	1 6.0	78.3	4. 4	1.3	0.0
	SD	2.56	0. 39	6.53	0.021	1.61	0.34	11.89	99.08	2.08	5.01	6. l7	2.41	1.57	0.0
1600	mean	8.6	8.40	163	0.417	49.7	19.4+	391+	1081+	20.5+	26.3+	71.1-	2.1	0.5	0.0
	SD	2.64	0.39	8.09	0.022	1.02	0.49	11.73	71.41	1.85	9.93	10.54	1.91	0.53	0.0
6400	mean	7.2	8.76	167	0.428	48.9	19.0	390+	943	18.2	19.7	75.8	4.0	0.5	0.0
	SD	2.21	0.46	5.74	0.025	1.17	0.74	13.42	82.13	1.71	7.30	7.76	2.75	0.53	0.0
females
		WBC g/L	RBC L/L	Hb g/L	Hct L/L	MCV fl	MCH pg	MCHC g/L	Plat g/L	P.T. S	Neut %	Lymp%	Eosi%	Mono%	Baso%
control	mean	4.9	7.91	159	0.397	50.2	20.1	400	1034	15.9	11.3	85.8	1.8	1.1	0.0
	SD	1.03	0.29	4.57	0.014	1.11	0.65	9.60	82.49	1.60	6.04	7.13	1.62	1.45	0.0
400	mean	5.9	7.88	163	0.411	52.2+	20.7	397	974	15.0	18.2	78.8	1.7	1.3	0.0
	SD	1.45	0.31	5.10	0.018	1.64	0.80	12.83	116.11	1.03	8.72	8.72	1.34	0.67	0.0
1600	mean	5.9	7.95	164	0.415+	52.2+	20.6	395	1018	15.3	15.5	80.2	2.5	1.8	0.0
	SD	1.88	0.26	5.25	0.013	1.37	0.59	11.74	88.66	1.29	8.54	9.75	1.84	1.62	0.0
6400	mean	5.9	7.70	159	0.395	51.3	20.6	402	1006	16.1	22.3+	73.8-	2.7	1.2	0.0
	SD	1.58	0.24	4.26	0.014	1.39	0.55	8.14	89.83	1.09	8.03	8.95	1.83	1.48	0.0

SD Standard deviation

+ Significantly higher than the control group

- Significantly lower than the control group

 

Table 5: Summary of clinical chemistry results

	males
		Glu mmol/L	BUN mmol/L	Urea mmol/L	Tot. Pro g/L	AST U/L	ALT U/L	GGT U/L	Tot.Bil µmol/L	Creat µmol/L	Alb g/L	Pi mmol/l	Ca mmol/l	Chol mmol/l	Cl mmol/l	Na mEq/L	K mEq/L
control	mean	8.28	2.45	5.25	58.4	81	38	3	3.60	77	32.43	1.97	2.67	2.23	108	143.2	4.12
	SD	0.72	0.20	0.42	1.62	11.83	4.88	2.07	0.62	3.74	1.05	0.17	0.05	0.42	0.92	1.64	0.24
400	mean	8.79	2.73	5.85	59.7	88	36	4	5.19	82	33.22	2.13	2.72	2.29	109	143.9	4.10
	SD	0.90	0.33	0.70	3.00	35.36	4.58	5.09	2.46	7.07	1.97	0.18	0.06	0.46	1.51	1.84	0.36
1600	mean	8.52	2.49	5.32	58.3	81	34	3	4.70	85+	31.49	2.04	2.69	2.22	108	143.5	4.24
	SD	0.71	0.38	0.82	2.37	11.29	8.91	4.80	2.05	7.02	1.06	0.23	0.04	0.36	1.75	1.45	0.24
6400	mean	8.74	2.55	5.45	58.7	86	40	3	5.25	88d+	32.67	2.10	2.68	2.13	111+	145.5+	4.22
	SD	0.69	0.30	0.64	2.66	10.91	6.00	1.71	2.11	4.65	1.89	0.15	0.06	0.35	2.22	1.68	0.35
	females
		Glu mmol/L	BUN mmol/L	Urea mmol/L	Tot. Pro g/L	AST U/L		GGT U/L	Tot.Bil µmol/L	Creat µmol/L	Alb g/L	Pi mmol/l	Ca mmol/l	Chol mmol/l	Cl mmol/l	Na mEq/L	K mEq/L
control	mean	6.99	2.64	5.64	61.7	93	35	3	4.08	75	34.23	1.79	2.68	1.92	108	144.1	4.04
	SD	0.42	0.26	0.56	2.87	15.77	8.71	2.13	0.82	6.00	2.15	0.32	0.06	0.22	2.01	2.73	0.37
400	mean	7.12	3.01	6.45	63.0	100	34	4	4.59	80	34.36	1.81	2.68	1.91	108	146.1	3.95
	SD	0.79	0.80	1.72	3.08	18.06	6.84	4.03	1.99	8.14	1.90	0.34	0.08	0.20	2.42	2.43	0.39
1600	mean	7.08	2.77	5.92	64.3	112	36	5	4.54	79	35.65	1.67	2.76	2.19	109	145.6	4.10
	SD	1.06	0.32	0.68	3.06	32.9	5.14	2.67	1.65	10.55	2.09	0.29	0.08	0.33	1.89	2.96	0,30
6400	mean	7.15	2.95	6.31	63.8	101	39	3	3.66	83	34.69	1.82	2.71	2.05	109	145.5	3.98
	SD	0.56	0.56	1.20	2.21	14.70	7.95	0.74	0.44	4.20	2.35	0.22	0.06	0.38	2.04	3.40	0.46

SD Standard deviation

+ Significantly higher than the control group

- Significantly lower than the control group

d Dose-response

 

Table 6: Summary of necropsy findings

	males				females
Dose (mg/kg bw/day)	control	400	1600	64000	control	400	1600	64000
Number of animals examined	10	10	10	10	10	10	10	10
Number of animals showing gross pathology	1	2	1	1	1	3	2	2
Number of animals showing external pathology	0	0	0	0	1	1	1	2
Skin alopecia focal	0	0	0	0	1	0	0	0
Skin-hair thinning and regrowth focal	0	0	0	0	0	1	1	2
Number of animals showing visceral organ pathology	1	2	1	1	0	2	1	0
Liver-focus white	0	0	0	1	0	0	0	0
Kidney unilateral/bilateral pelvis dilated	1	0	1	0	0	2	1	0
Testes unilateral enlarged, abcess	0	1	0	0	0	0	0	0
Heart enlarged	0	1	0	0	0	0	0	0

 

Table 7: Neurological examination results

Incidences - males
Dose(mg/kg bw/day)			control			400			1600			6400
Number of rats			10			10			10			10
1. Visual response
Normal (present)			10			10			10			10
Absent			0			0			0			0
2. Auditory response
Normal			10			10			10			10
No (absent)			0			0			0			0
Exaggerated			0			0			0			0
3. Gait
Normal			10			10			10			10
Abnormal			0			0			0			0
Ataxic			0			0			0			0
4. Landing foot splay(cm)
Mean±			6.5			7.0			7.8+			8.8+
SD			0.74			1.15			1.26			0.94
5. Righting reflex
on back
- Present	10					10			10			10
- Slow	0					0			0			0
- Absent	0					0			0			0
dropped
- Present	10					10			10			10
- Slow	0					0			0			0
- Absent	0					0			0			0
6. Motor activity (Score)
Mean ±			719			666			658			744
SD			122.26			92.56			191.67			201.92
7. Grip strength (g)
Forelimb
Mean±.		1054		984 -			1031			1089
SD		90.94		115.48			97.17			68.78
Hindlimb
Mean±		636				638			712+			670
SD		128.38				91 .43			79.40			140.45
Incidences - females
Dose(mg/kg bw/day)		control			400			1600			6400
Number of rats		10			10			10			10
1. Visual response
Normal (present)		10			10			10			10
Absent		0			0			0			0
2. Auditory response
Normal		10			10			10			10
No (absent)		0			0			0			0
Exaggerated		0			0			0			0
3. Gait
Normal		10			10			10			10
Abnormal		0			0			0			0
Ataxic		0			0			0			0
4. Landing foot splay (cm)
Mean±		6.1			6.1			7.8+			1.9+
SD		0.85			1.10			0.95			1.53
5. Righting reflex
on back
- Present		10			10			10			10
- Slow		0			0			0			0
- Absent		0			0			0			0
dropped
- Present		10			10			10			10
- Slow		0			0			0			0
- Absent		0			0			0			0
6. Motor activity (Score)
Mean ±		703			847			757			825
SD		200.42			107.87			149.62			139.75
7. Grip strength (g)
Forelimb
Mean ±		936		978			1067+			1034+
SD		127.15		135.58			89.40			96.54
Hindlimb
Mean±		575				611			619			664+
SD		85.55				98.63			97.57			102. 05

SD Standard deviation

+ Significantly higher than the control group

- Significantly lower than the control group

 

Table 8: Summary of selected Histopathological findings

	males				females
Dose (mg/kg bw/day)	control	400	1600	64000	control	400	1600	64000
Number of animals examined	10	10/-	10/-	10	10	10/-	10/-	10
Stomach- cystic glands	0	-	-	1	0	-	-	0
Colon- parasites	0	-	-	1	0	-	-	0
Pancreas						-	-
- Atrophy	1	-	-	0	0	-	-	0
- Adipocytes	1	-	-	0	0	-	-	0
- Increased cytoplasmic eosinopholia	0	-	-	0	1	-	-	0
Liver
- Haemorrhage	0	-	-	1	0	-	-	0
- Lymphocytic infiltration	1	-	-	0	0	-	-	0
- Necrosis	0	-	-	2	0	-	-	0
- Necrotic foci	0	-	-	1	0	-	-	0
Lungs
- Perivascular lymphocytic infiltration	1	0	1	3	1	1	4	3
- Granulocytic infiltration	1	0	0	0	0	0	0	0
- Mineralisation blood vessels	0	1	1	2	3	2	1	2
- Chronic pneumonia	1	0	0	1	0	0	0	0
- Chronic pneumonic foci	0	0	0	0	0	0	1	0
- Pneumonic foci	0	0	1	1	0	0	1	2
- Osseous metaplasia	0	1	0	0	0	0	0	0
Heart- dilatation	0	1	-	0	0	-	-	0
Kidneys
- Mineralisation	0	-	1	0	0	0	0	1
- Dilatation of pelvis	2	-	1	0	0	2	1	0
- Basophilic tubules	4	-	1	2	0	0	0	0
- Proteinaceous material in tubules	2	-	0	2	0	0	0	0
- Hyaline droplets-tubular epithelium	7	-	0	7	0	0	0	0
Testes
- Atrophy-seminiferous	0	1	-	0	/	/	/	/
- Spermatic granuloma	0	1	-	0	/	/	/	/
Epididymis- Lymphocytic infiltration	1	-	-	0	/	/	/	/
Ovaries- Follicular cyst(s)	/	/	/	/	0	-	-	1
Uterus- Dilatation	/	/	/	/	3	-	-	2
Thyroids
- Ultimobronchial cyst	1	-	-	1	0	-	-	0
- Ectopic thymus	1	-	-	1	0	-	-	1
- Accessory parathyroid	0	-	-	1	0	-	-	0
Parathyroids– Connective tissue proliferation	1	-	-	0	0	-	-	0
Pituitary
- Cysts(s)	1	-	-	1	0	-	-	0
- Dilated Rathke’s cleft	4	-	-	5	2	-	-	2
- Eosinopohilic vacuoles	0	-	-	1	0	-	-	0
Adrenals– Accessory adrenal	0	-	-	0	1	-	-	1
Skin
- Epidermal hyperplasia	0	-	-	1	1	0	0	0
- Necrotising dermatitis	0	-	-	0	0	0	0	1
- Parakeratosis	0	-	-	0	1	0	0	1
- Epithelial hyperplasia	1	-	-	0	0	0	0	0
Thymus
- Hemorrhage	2	-	-	0	2	-	-	1
- Epithelial hyperplasia	0	-	-	0	0	-	-	1
Mammary gland
- Hyperplasia	/	/	/	/	0	-	-	1
- Lymphocytic infiltration/	/	/	/	/	0	-	-	1
Rectum- Parasites	1	-	-	1	1	-	-	0