D-Glucitol, 1-deoxy-1-(dimethylamino)-

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:

26 November 2016 to 21 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline No. 407, “Repeated Dose 28-Day Oral Toxicity Study in Rodents” adopted on 03 October 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

k.A.

IUPAC Name:

k.A.

Test material form:

solid: flakes

Details on test material:

- Name of test material (as cited in study report): LICOCARE RBW 106 FL TP

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Clariant Produkte (Deutschland) GmbH and RAK-KRS-00022

- Expiration date of the lot/batch: 01.04.2018
- Purity test date: 30.09.2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: Stable at 6 hours at room temperature with a concentration 100 mg/mL
- Solubility and stability of the test substance in the solvent/vehicle: Clearly miscible (at a concentration of 140 mg/mL) with Distilled water evidenced by the in-house miscibility test results and Stable at 6 hours at room temperature with a concentration 100 mg/mL

FORM AS APPLIED IN THE TEST: Liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the recommended species by regulatory agencies for conducting preclinical toxicological studies among rodent species

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 100 to 160 g
- Housing:Maximum of three animals of same sex and group 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 water bottle. Clean sterilized paddy husk was provided as bedding material.
- Diet :ad libitum
- Water: ad libitum
- Acclimation period: 05 Days

DETAILS OF FOOD AND WATER QUALITY: Teklad Certified (2014SC) Global 14 % Protein Rodent Maintenance Diet - Pellet (Manufactured by Envigo); Water: eep bore-well water passed through an activated charcoal filter and exposed to ultraviolet rays in an aquaguard water filter with purifier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1°C to 22.8°C
- Humidity (%): 46% to 67%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 2 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 26 November 2016 To: 12 January 2017

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered through oral route using stainless steel gavage cannula attached to disposable syringe

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared by mixing to the required quantity with the vehicle to get desired concentrations 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): Distilled water was used as vehicle for formulation preparation as the test item was clearly miscible (at a concentration of 140 mg/mL) with Distilled water evidenced by the in-house miscibility test results.
- Concentration in vehicle: G2: 10 mg/mL; G3: 30 mg/mL; G4: 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For active ingredient (a.i.) concentration analysis, the prepared formulations were sampled in duplicate sets (5 mL for each set from middle layer) from each dose level during week 1 and week 4 of the treatment period.
The results were considered acceptable, as the mean results are within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is equal to or less than 10.0%.

Duration of treatment / exposure:

28 Days

Frequency of treatment:

Daily once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 100, 300 and 1000 mg/kg body weight were selected as low (G2),mid (G3) and high dose (G4/G4R) levels based on the NOAEL)of Genamin DMG 75 (N, N-Dimethyl-D-glucamine) was 1000 mg/kg/day when administered for a period of 14 consecutive days by oral (gavage) to Sprague Dawley rats obtained from the dose range finding study.
- Rationale for animal assignment: Randamization and grouping based on the body weight
- Rationale for selecting satellite groups: based on the OECD 407 guidelines
- Post-exposure recovery period in satellite groups: 14 days

The dose applied refers to the amount of N, N-Dimethyl-D-glucamine.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily once
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly once.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly once

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Week 4 for main group animals and week 6 for recovry group animals
- Dose groups that were examined: Vehicle control and high dose group animals of both sex.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination (Day 29 for amin group animals and Day 43 for recovery group animals)
- Anaesthetic used for blood collection: Yes (identity): isolflurane mild
- Animals fasted: Yes
- How many animals: all nimals one day prior to sacrifice
- Parameters checked in table [No.10] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination (Day 29 for amin group animals and Day 43 for recovery group animals)
- Animals fasted: Yes
- How many animals: all nimals one day prior to sacrifice
- Parameters checked in table [No.11] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at termination (Day 29 for amin group animals and Day 43 for recovery group animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.12] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 4 for main group animals and week 6 for recovery group animals
- Dose groups that were examined: Vehicle control and high dose group animals of both sex.
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 15)

HISTOPATHOLOGY: Yes (see table 16)

Other examinations:

Not applicable

Statistics:

SPSS software version 22. One way ANOVA followed by Dunnett’s post-test for main group parameters and independant sample t-test for recovery groups comparing with the control group data at the 95% level of confidence (P<0.05).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity and mortality/morbidity observed at any of the doses tested in both the sex.

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any of the doses tested in both the sex.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

statistical significant increase in mean body weight on day 22 with respect to day 1 in G2 females; statistical significant decrease in percent change in body weight during week 1 in G3 and G4 males; statistical significant increase in percent change in body weight during week 1 and 3 in G4R females was observed when compared with respective vehicle control groups. These changes can be considered as incidental, but not treatment related as the mean body weights and feed consumption during this period was unaffected and there were no clinical signs were observed.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment related changes noted in feed consumption at any of the tested doses in both the sex.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed during the ophthalmological examination carried out during week 4 for vehicle control and high dose group animals and during week 6 for vehicle control recovery and high dose recovery group animals.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main group: Statistical significant increase in mean platelet volume in G2 and G4 males; statistical significant decrease in hemoglobin in G4 females; statistical significant decrease in mean corpuscular volume in G4 females; statistical significant decrease in mean corpuscular hemoglobin in G3 and G4 females; statistical significant decrease in mean corpuscular haemoglobin concentration in G4 females; statistical significant increase in percent eosinophills in G2 females; statistical significant increase in absolute eosinophills in G4 females when compared with vehicle control group animals were noted.

Recovery group: Statistical significant decrease in percent monocytes in G4R males, statistical significant decrease in absolute reticulocytes in G4R females when compared with vehicle control recovery group animals were noted.

All the observed changes are considered incidental and not treatment related because of the lack of dose dependency and/or due to random biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decrease in total cholesterol and statistically significant increase in sodium levels in G4R males; statistically significant decrease in glucose in G4R females; statistically significant increase in total bilurubin in G4R females were noted when compared with vehicle control recovery group.

These observed changes are considered as incidental and not treatment related as there were no changes were noted in main group animals of the same group and these changes can be due to random biological variation.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment related changes observed in the urinalysis parameters at any of the doses tested in either sex

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no treatment related changes observed in neurological/functional examination battery carried out during week 4 for vehicle control and high dose group animals and during week 6 for vehicle control recovery and high dose recovery group animals

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase in absolute spleen weight and decrease in relative thymus weight in G2 females; statistically significant increase in relative kidneys weight in G4R males; statistically significant decrease in absolute kidneys weight in G4R females; statistically significant decrease in relative thymus, kidneys and liver weight in G4R females were noted.
All the observed changes are considered incidental and not treatment related because of the lack of dose dependency and/or there were no associated macroscopic and microscopic abnormalities noted at the high dose main groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment related changes observed on histopathological examination of high dose group animals.
All observed lesions were considered as spontaneous and incidental to Sprague Dawley rats.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Details on results:

The active ingredient (a.i.) concentration analysis of the prepared dose formulations of Genamin DMG 75 (N, N-Dimethyl-D-Glucamine) was performed on week 1 and week 4 of the experiment as per the validated method (BIO-ANM 374). The results of the analysed formulations were within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is equal to or less than 10.0%.

All animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight and feed consumption. Ophthalmological examination and neurological/functional examination was carried out during week 4 for main groups (G1 and G4 group animals) and during week 6 for recovery groups (G1R and G4R group animals). Haematology, clinical chemistry analysis, urinalysis, gross pathology and organ weighing were performed on day 29 for main groups and on day 43 for recovery groups. Histopathological examination was conducted on the tissues from the vehicle control (G1) and high dose (G4) group animals.

The animals did not reveal any clinical signs of toxicity throughout the treatment and recovery period. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, ophthalmoscopic examination, neurological/functional examination, haematology, clinical chemistry, urinalysis, organ weights (both absolute and relative) were observed.

There were no gross pathological changes observed at any of the doses tested in either sex. No treatment related histopathological findings were noticed.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

TABLE 2. SUMMARY OF BODY WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight)		Body Weight on Days
		1	8	15	22	28
G1, M & 0	Mean	180.39	218.05	262.18	285.29	314.09
	±SD	14.38	14.68	17.88	16.52	20.15
	n	5	5	5	5	5
G2, M & 100	Mean	181.26	218.46	268.26	296.23	330.72
	±SD	9.54	10.58	9.66	10.36	13.09
	n	5	5	5	5	5
G3, M & 300	Mean	182.53	213.85	259.27	292.11	322.75
	±SD	13.00	14.32	20.46	21.16	22.16
	n	5	5	5	5	5
G4, M & 1000	Mean	178.28	207.94	255.99	284.52	307.84
	±SD	9.94	8.40	16.36	14.83	24.04
	n	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Body Weight on Days
		1	8	15	22	28
G1, F & 0	Mean	154.65	171.95	195.12	211.85	227.03
	±SD	9.72	11.49	13.22	14.98	17.61
	n	5	5	5	5	5
G2, F & 100	Mean	152.53	172.77	198.01	220.45	232.59
	±SD	8.28	8.25	8.54	6.66	9.39
	n	5	5	5	5	5
G3, F & 300	Mean	152.87	169.42	193.68	217.50	230.59
	±SD	7.20	7.03	11.25	9.30	7.68
	n	5	5	5	5	5
G4, F & 1000	Mean	155.74	172.60	199.21	219.72	232.71
	±SD	9.05	12.29	11.15	14.71	15.13
	n	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Days
		1	8	15	22	28		36	42
G1R, M & 0	Mean	178.54	211.73	250.07	276.32	302.69		329.01	353.91
	±SD	9.08	8.43	21.25	19.61	18.03		18.41	18.96
	n	5	5	5	5	5		5	5
G4R, M & 1000	Mean	179.48	212.43	255.13	282.25	310.70		335.70	360.55
	±SD	9.01	9.99	11.41	10.56	12.26		14.76	15.51
	n	5	5	5	5	5		5	5
G1R, F & 0	Mean	154.90	174.67	199.31	214.97	232.80		247.30	264.98
	±SD	6.48	8.93	10.63	10.58	13.11		15.16	14.22
	n	5	5	5	5	5		5	5
G4R, F & 1000	Mean	156.04	182.14	204.64	227.30	241.57		254.50	271.34
	±SD	6.62	11.49	14.82	15.80	16.74		15.94	16.42
	n	5	5	5	5	5		5	5

TABLE 3. SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose (mg/kg body weight)		Days
		1 to 8	1 to 15	1 to 22	1 to 28
G1, M & 0	Mean	20.98	45.46	58.43	74.30
	±SD	1.73	2.91	5.88	3.27
	n	5	5	5	5
G2, M & 100	Mean	20.54	48.16	63.65	82.70
	±SD	0.77	5.63	7.15	8.70
	n	5	5	5	5
G3, M & 300	Mean	17.19	42.00	60.02	76.87
	±SD	1.35	3.24	1.93	3.83
	n	5	5	5	5
G4, M & 1000	Mean	16.76	43.64	59.69	72.62
	±SD	3.79	6.06	5.37	7.97
	n	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Days
		1 to 8	1 to 15	1 to 22	1 to 28
G1, F & 0	Mean	11.17	26.14	36.93	46.71
	±SD	1.41	1.57	1.65	3.78
	n	5	5	5	5
G2, F & 100	Mean	13.31	30.04	44.76	52.69
	±SD	1.05	7.64	6.57	6.75
	n	5	5	5	5
G3, F & 300	Mean	10.85	26.67	42.35	50.96
	±SD	1.44	3.44	4.43	4.46
	n	5	5	5	5
G4, F & 1000	Mean	10.76	27.96	41.04	49.39
	±SD	1.64	3.59	3.05	3.07
	n	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Days
		1 to 8	1 to 15	1 to 22	1 to 28		1 to 36	1 to 42
G1R, M & 0	Mean	18.67	39.96	54.72	69.62		84.35	98.31
	±SD	2.99	7.15	6.34	7.62		6.70	7.17
	n	5	5	5	5		5	5
G4R, M & 1000	Mean	18.37	42.24	57.41	73.26		87.16	101.01
	±SD	0.87	4.78	5.59	6.05		5.70	5.89
	n	5	5	5	5		5	5
G1R, F & 0	Mean	12.74	28.68	38.79	50.30		59.61	71.04
	±SD	2.27	4.52	4.09	6.01		5.83	4.60
	n	5	5	5	5		5	5
G4R, F & 1000	Mean	16.65	31.02	45.54	54.68		63.00	73.79
	±SD	2.89	4.39	4.13	4.38		3.65	3.77
	n	5	5	5	5		5	5

TABLE 3. SUMMARY OF AVERAGE FEED CONSUMPTION (g/rat/day) RECORD

Group, Sex & Dose (mg/kg body weight)		Week
		Week 1	Week 2	Week 3	Week 4
G1, M & 0	Mean	21.1	23.0	24.4	26.8
	±SD	1.2	0.9	2.1	1.2
	n	5	5	5	5
G2, M & 100	Mean	21.7	23.3	23.9	27.4
	±SD	1.2	2.1	2.6	1.7
	n	5	5	5	5
G3, M & 300	Mean	20.9	22.6	23.6	26.9
	±SD	0.5	0.7	1.1	1.8
	n	5	5	5	5
G4, M & 1000	Mean	20.6	21.3	23.8	26.2
	±SD	1.7	2.1	2.7	2.0
	n	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Week
		Week 1	Week 2	Week 3	Week 4
G1, F & 0	Mean	18.4	19.4	20.7	22.8
	±SD	0.1	1.0	2.2	3.1
	n	5	5	5	5
G2, F & 100	Mean	18.5	19.6	20.8	22.8
	±SD	0.1	0.5	0.9	0.8
	n	5	5	5	5
G3, F & 300	Mean	18.8	20.1	21.0	22.8
	±SD	0.3	1.1	1.4	1.5
	n	5	5	5	5
G4, F & 1000	Mean	18.6	19.6	20.9	22.2
	±SD	0.3	0.1	0.6	0.3
	n	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Week
		Week 1	Week 2	Week 3	Week 4		Week 5	Week 6
G1R, M & 0	Mean	20.4	21.9	24.0	25.7		24.6	27.0
	±SD	1.3	2.6	2.6	1.6		2.0	0.3
	n	5	5	5	5		5	5
G4R, M & 1000	Mean	20.2	21.5	23.6	25.8		25.2	25.7
	±SD	1.4	2.1	2.3	1.7		2.4	0.7
	n	5	5	5	5		5	5
G1R, F & 0	Mean	18.6	19.7	20.3	21.8		21.6	25.5
	±SD	0.1	0.3	0.6	0.1		0.6	0.4
	n	5	5	5	5		5	5
G4R, F & 1000	Mean	18.3	19.0	20.3	21.2		21.9	24.2
	±SD	0.7	0.5	0.9	0.8		0.3	0.8
	n	5	5	5	5		5	5

TABLE 5. SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

			Week 4		Week 6
Parameters↓	Group & Sex		G1 & M	G4 & M	G1R & M	G4R & M
	Dose (mg/kg body weight)		0	1000	300	1000
	Number of Animals		5	5	5	5
Home cage observations
a. Convulsions			1	1	1	1
b. Tremors			1	1	1	1
c. Palpebral closure			1	1	1	1
Handling observation
a. Ease of removal from the cage			2	2	2	2
b. Ease of handling animal in hand			2	2	2	2
c. Lacrimation			1	1	1	1
d. Red deposits around		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
e. Crusty deposits around		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
f. Salivation			1	1	1	1
g. Fur appearance			1	1	1	1
h. Piloerection			1	1	1	1
i. Palpebral closure			1	1	1	1
j. Respiratory character			1	1	1	1
k. Eye prominence			1	1	1	1
l. Muscle tone			1	1	1	1
Home cage observations: a. Convulsions - 1=Absent, 2=Present, b. Tremors- 1=Absent, 2=Present, c. Palpebral closure - 1=Normal, Handling observation : a. Ease of removal from the cage - 2=Normal, b. Ease of handling animal in hand - 2=Normal, c. Lacrimation - 1=None, 2=Slight, 3=Severe, d. Red deposits around - 1=Absent, 2=Present, e. Crusty deposits around - 1=Absent, 2=Present , f. Salivation- 1=Normal , g. Fur Appearance - 1=Normal, h. Piloerection - 1=None, 2=Slight, 3=Severe, i. Palpebral Closure - 1=Normal, j. Respiratory character 1=Normal, k. Eye Prominence - 1=Normal, l. Muscle tone - 1=Normal

			Week 4		Week 6
Parameters↓	Group & Sex		G1 & M	G4 & M	G1R & M	G4R & M
	Dose (mg/kg body weight)		0	1000	300	1000
	Number of Animals		5	5	5	5
Open field Observation
a. Mobility			1	1	1	1
b. Gait			1	1	1	1
c. Arousal			3	3	3	3
d. Rearing		Mean	3.2	3.6	2.0	2.8
		±SD	0.8	1.1	0.7	0.8
e. Urination		Mean	1.8	1.6	2.6	2.2
Numbers of pools of urine		±SD	0.8	1.3	0.5	0.8
f. Defecation		Mean	1.4	2.0	2.0	1.6
Number of fecal pellets		±SD	0.5	0.7	0.7	0.5
g. Stereotypies- repetitive circling			1	1	1	1
h. Excessive grooming		Mean	2.6	3.0	2.8	2.8
		±SD	0.9	0.0	0.8	0.8
Sensory observations
a. Startle response			2	2	2	2
b. Touch response			2	2	2	2
c. Pupil response			2	2	2	2
d. Response to nociceptive stimuli			2	2	2	2
e. Righting reflex			1	1	1	1
Physiological observation		Mean	98.2	98.9	98.8	98.4
Body temperature (°F)		±SD	0.9	0.6	0.4	0.6
Open field Observation: a. Mobility - 1=Normal, b. Gait - 1=Normal, c. Arousal - 3=Normal, g. Stereotypies - repetitive circling- 1= Absent, 2=Present,Sensory Observations: a.Startle Response -2=Normal,b. Touch Response -2=Normal,c. Pupil Response 2=Normal,d. Response to Nociceptive stimuli - 2=Normal,e. Righting Reflex -1=Present, 2=Slow, 3=Absent

		Week 4		Week 6
Parameters↓	Group & Sex	G1 & F	G4 & F	G1R & F	G4R & F
	Dose (mg/kg body weight)	0	1000	300	1000
	Number of Animals	5	5	5	5
Home cage observations
a. Convulsions		1	1	1	1
b. Tremors		1	1	1	1
c. Palpebral closure		1	1	1	1
Handling observation
a. Ease of removal from the cage		2	2	2	2
b. Ease of handling animal in hand		2	2	2	2
c. Lacrimation		1	1	1	1
d. Red deposits around	Eyes	1	1	1	1
	Nose	1	1	1	1
	Mouth	1	1	1	1
e. Crusty deposits around	Eyes	1	1	1	1
	Nose	1	1	1	1
	Mouth	1	1	1	1
f. Salivation		1	1	1	1
g. Fur appearance		1	1	1	1
h. Piloerection		1	1	1	1
i. Palpebral closure		1	1	1	1
j. Respiratory character		1	1	1	1
k. Eye prominence		1	1	1	1
l. Muscle tone		1	1	1	1
Week 4 Week 6 Parameters↓ Group & Sex G1 & F G4 & F G1R & F G4R & F Dose (mg/kg body weight) 0 1000 300 1000 Number of Animals 5 5 5 5 Open field Observation   a. Mobility 1 1 1 1 b. Gait 1 1 1 1 c. Arousal 3 3 3 3 d. Rearing Mean 2.4 3.0 2.8 2.8 ±SD 1.1 1.0 0.8 0.8 e. Urination Mean 1.8 2.4 2.0 2.2 Numbers of pools of urine ±SD 0.8 1.1 1.0 0.8 f. Defecation Mean 1.8 1.8 1.8 1.2 Number of fecal pellets ±SD 0.8 0.8 0.8 0.5 g. Stereotypies- repetitive circling 1 1 1 1 h. Excessive grooming Mean 3.2 2.4 2.6 2.8 ±SD 1.3 1.1 0.5 0.8 Sensory observations   a. Startle response 2 2 2 2 b. Touch response 2 2 2 2 c. Pupil response 2 2 2 2 d. Response to nociceptive stimuli 2 2 2 2 e. Righting reflex 1 1 1 1 Physiological observation Mean 98.8 98.6 98.5 98.7 Body temperature (°F) ±SD 1.2 0.6 0.5 0.8 Open field Observation: a. Mobility - 1=Normal, b. Gait - 1=Normal, c. Arousal - 3=Normal, g. Stereotypies - repetitive circling- 1= Absent, 2=Present,Sensory Observations: a.Startle Response -2=Normal,b. Touch Response -2=Normal,c. Pupil Response 2=Normal,d. Response to Nociceptive stimuli - 2=Normal,e. Righting Reflex -1=Present, 2=Slow, 3=Absent Home cage observations: a. Convulsions - 1=Absent, 2=Present, b. Tremors- 1=Absent, 2=Present, c. Palpebral closure - 1=Normal, Handling observation : a. Ease of removal from the cage - 2=Normal, b. Ease of handling animal in hand - 2=Normal, c. Lacrimation - 1=None, 2=Slight, 3=Severe, d. Red deposits around - 1=Absent, 2=Present, e. Crusty deposits around - 1=Absent, 2=Present , f. Salivation- 1=Normal , g. Fur Appearance - 1=Normal, h. Piloerection - 1=None, 2=Slight, 3=Severe, i. Palpebral Closure - 1=Normal, j. Respiratory character 1=Normal, k. Eye Prominence - 1=Normal, l. Muscle tone - 1=Normal

TABLE 6. LANDING FOOT SPLAY (cm) RECORD

Group, Sex & Dose                            (mg/kg body weight)		Landing Foot Splay (cm)
G1, M & 0	Mean	8.0
	±SD	1.0
	n	5
G4, M & 1000	Mean	8.6
	±SD	1.5
	n	5
G1R, M & 0	Mean	8.5
	±SD	1.2
	n	5
G4R, M &1000	Mean	8.7
	±SD	1.2
	n	5

Group, Sex & Dose                            (mg/kg body weight)		Landing Foot Splay (cm)
G1, F & 0	Mean	7.9
	±SD	0.7
	n	5
G4, F & 1000	Mean	8.2
	±SD	1.1
	n	5
G1R, F & 0	Mean	7.9
	±SD	0.7
	n	5
G4R, F & 1000	Mean	7.3
	±SD	1.1
	n	5

TABLE 7. SUMMARY OF PHOTOACTOMETER READINGS (No. OF MOVEMENTS) RECORD

Group, Sex & Dose                                    (mg/kg body weight)		Movement Counts (no.)
G1, M & 0	Mean	670.0
	±SD	56.6
	n	5
G4, M & 1000	Mean	629.6
	±SD	56.1
	n	5
G1R, M & 0	Mean	692.2
	±SD	108.1
	n	5
G4R, M & 1000	Mean	741.0
	±SD	46.7
	n	5

Group, Sex & Dose                                    (mg/kg body weight)		Movement Counts (no.)
G1, F & 0	Mean	651.4
	±SD	78.4
	n	5
G4, F & 1000	Mean	620.0
	±SD	47.8
	n	5
G1R, F & 0	Mean	754.4
	±SD	44.3
	n	5
G4R, F & 1000	Mean	760.4
	±SD	52.9
	n	5

TABLE 8. SUMMARY OF GRIP STRENGTH (kgf) MEASUREMENT RECORD

Group, Sex & Dose (mg/kg body weight)		Fore limb	Hind limb
G1, M & 0	Mean	1.541	0.590
	±SD	0.077	0.043
	n	5	5
G4, M & 1000	Mean	1.430	0.502
	±SD	0.187	0.073
	n	5	5
G1R, M & 0	Mean	1.579	0.568
	±SD	0.038	0.020
	n	5	5
G4R, M & 1000	Mean	1.558	0.550
	±SD	0.023	0.034
	n	5	5

Group, Sex & Dose (mg/kg body weight)		Fore limb	Hind limb
G1, F & 0	Mean	1.567	0.577
	±SD	0.025	0.038
	n	5	5
G4, F & 1000	Mean	1.564	0.557
	±SD	0.011	0.031
	n	5	5
G1R, F & 0	Mean	1.565	0.556
	±SD	0.022	0.038
	n	5	5
G4R, F & 1000	Mean	1.555	0.562
	±SD	0.026	0.020
	n	5	5

TABLE 10. SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight)		Total Leucocyte Count(WBC)              (10^3cells/µL)	Total Erythrocyte Count             (RBC)               (10^6cells/µL)	Hemoglobin (HGB)  (g/dL)	Haematocrit (HCT)                   (%)	Mean Corpuscular Volume (MCV)                  (fL)	Mean Corpuscular Hemoglobin (MCH)                      (pg)	Mean Corpuscular Haemoglobin Concentration (MCHC)                (g/dL)	Platelet Count               (PLT)                (10^3cells/µL)	Mean Platelet Volume            (MPV)                 (fL)
G1, M & 0	Mean	7.99	7.17	13.50	41.82	58.28	18.82	32.32	1060.40	6.78
	±SD	0.81	0.20	0.40	1.26	0.64	0.46	0.58	111.01	0.23
	n	5	5	5	5	5	5	5	5	5
G2, M & 100	Mean	9.40	7.36	13.96	43.28	58.86	19.02	32.28	871.20	7.34
	±SD	2.60	0.31	0.57	1.72	1.43	0.61	0.38	260.67	0.34
	n	5	5	5	5	5	5	5	5	5
G3, M & 300	Mean	8.48	7.50	13.92	42.98	57.34	18.58	32.36	1073.20	7.18
	±SD	2.77	0.32	0.51	1.31	1.25	0.59	0.40	50.57	0.13
	n	5	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	7.31	7.36	13.92	42.20	57.34	18.90	32.98	953.00	7.52
	±SD	1.70	0.45	0.59	2.29	1.09	0.56	0.53	138.38	0.33
	n	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Reticulocyte   Count                       (%)	Neutrophils             (%)	Lymphocytes             (%)	Monocytes                                 (%)	Eosinophils                         (%)	Basophils                          (%)
G1, M & 0	Mean	2.74	19.30	75.76	2.36	0.68	0.18
	±SD	0.16	4.45	5.28	0.42	0.20	0.08
	n	5	5	5	5	5	5
G2, M & 100	Mean	2.55	20.10	75.26	2.12	0.86	0.26
	±SD	0.22	2.87	3.96	0.62	0.47	0.15
	n	5	5	5	5	5	5
G3, M & 300	Mean	2.56	20.46	74.46	2.78	0.70	0.22
	±SD	0.46	5.12	4.98	0.48	0.32	0.08
	n	5	5	5	5	5	5
G4, M & 1000	Mean	2.36	24.84	69.80	3.22	0.56	0.18
	±SD	0.45	3.19	3.20	0.83	0.18	0.04
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Absolute Reticulocyte Count                  (10^9cells/L)	Absolute Neutrophils              (10^3cells/µL)	Absolute Lymphocytes              (10^3cells/µL)	Absolute Monocytes              (10^3cells/µL)	Absolute Eosinophils              (10^3cells/µL)	Absolute Basophils              (10^3cells/µL)	Prothrombin Time                      (Seconds)	Activated Prothrombin Time                       (Seconds)
G1, M & 0	Mean	196.42	1.53	6.07	0.19	0.06	0.01	14.74	33.06
	±SD	6.51	0.30	0.91	0.04	0.02	0.01	0.69	32.13
	n	5	5	5	5	5	5	5	5
G2, M & 100	Mean	187.66	1.86	7.13	0.20	0.07	0.03	15.26	46.06
	±SD	17.80	0.42	2.21	0.07	0.03	0.02	1.56	6.21
	n	5	5	5	5	5	5	5	5
G3, M & 300	Mean	190.70	1.64	6.41	0.24	0.06	0.02	14.36	49.52
	±SD	27.20	0.30	2.42	0.09	0.04	0.01	0.46	2.20
	n	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	175.56	1.81	5.11	0.25	0.04	0.01	14.34	41.60
	±SD	41.83	0.45	1.20	0.11	0.02	0.00	0.79	16.11
	n	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Total Leucocyte Count              (WBC)              (10^3cells/µL)	Total Erythrocyte Count             (RBC)               (10^6cells/µL)	Hemoglobin (HGB)  (g/dL)	Haematocrit (HCT)                   (%)	Mean Corpuscular Volume (MCV)                  (fL)	Mean Corpuscular Hemoglobin (MCH)                      (pg)	Mean Corpuscular Haemoglobin Concentration (MCHC)                (g/dL)	Platelet Count               (PLT)                (10^3cells/µL)	Mean Platelet Volume            (MPV)                 (fL)
G1, F & 0	Mean	7.23	7.54	14.22	43.28	57.40	18.90	32.88	1026.80	7.76
	±SD	1.65	0.36	0.56	1.54	0.85	0.23	0.55	78.28	0.57
	n	5	5	5	5	5	5	5	5	5
G2, F & 100	Mean	5.80	7.48	14.02	42.10	56.32	18.74	33.28	1046.40	8.46
	±SD	1.01	0.17	0.52	1.04	1.19	0.51	0.36	76.03	0.34
	n	5	5	5	5	5	5	5	5	5
G3, F & 300	Mean	7.66	7.50	13.38	41.66	55.62	17.84*	32.08	991.80	7.66
	±SD	1.44	0.12	0.54	1.70	2.65	0.86	0.57	141.77	0.27
	n	5	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	8.27	7.76	13.32	41.70	53.78	17.18*	31.98*	978.20	7.12
	±SD	2.13	0.27	0.43	0.96	1.55	0.58	0.47	90.57	0.70
	n	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Reticulocyte Count                       (%)	Neutrophils             (%)	Lymphocytes             (%)	Monocytes                                 (%)	Eosinophils                         (%)	Basophils                          (%)
G1, F & 0	Mean	1.87	27.20	68.14	2.62	0.60	0.22
	±SD	0.70	12.07	12.79	1.44	0.25	0.08
	n	5	5	5	5	5	5
G2, F & 100	Mean	1.97	35.06	59.40	2.96	1.24	0.20
	±SD	0.41	4.87	4.93	0.78	0.42	0.10
	n	5	5	5	5	5	5
G3, F & 300	Mean	2.10	21.08	73.06	3.10	0.96	0.34
	±SD	0.26	4.43	3.22	0.72	0.33	0.21
	n	5	5	5	5	5	5
G4, F & 1000	Mean	2.30	21.32	72.28	3.24	1.10	0.38
	±SD	0.20	4.55	4.56	0.74	0.45	0.08
	n	5	5	5	5	5	5

Group, Sex & Dose  (mg/kg body weight)		Absolute Reticulocyte Count                  (10^9cells/L)	Absolute Neutrophils              (10^3cells/µL)	Absolute Lymphocytes              (10^3cells/µL)	Absolute Monocytes              (10^3cells/µL)	Absolute Eosinophils              (10^3cells/µL)	Absolute Basophils              (10^3cells/µL)	Prothrombin Time                      (Seconds)	Activated Prothrombin Time                       (Seconds)
G1, F & 0	Mean	140.98	2.09	4.79	0.20	0.04	0.02	15.98	49.46
	±SD	53.86	1.46	0.73	0.16	0.02	0.01	0.15	7.98
	n	5	5	5	5	5	5	5	5
G2, F & 100	Mean	147.28	2.02	3.45	0.17	0.07	0.01	14.56	51.50
	±SD	29.15	0.40	0.72	0.07	0.03	0.00	0.89	14.74
	n	5	5	5	5	5	5	5	5
G3, F & 300	Mean	157.60	1.61	5.60	0.24	0.07	0.03	14.26	60.16
	±SD	21.28	0.47	1.09	0.07	0.02	0.02	0.80	20.82
	n	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	177.92	1.77	5.98	0.27	0.09	0.03	15.26	50.46
	±SD	9.54	0.58	1.63	0.08	0.03	0.01	1.82	10.17
	n	5	5	5	5	5	5	5	5

Group, Sex & Dose  (mg/kg body weight)		Total Leucocyte Count              (WBC)              (10^3cells/µL)	Total Erythrocyte Count             (RBC)               (10^6cells/µL)	Hemoglobin (HGB)  (g/dL)	Haematocrit (HCT)                   (%)	Mean Corpuscular Volume (MCV)                  (fL)	Mean Corpuscular Hemoglobin (MCH)                      (pg)	Mean Corpuscular Haemoglobin Concentration (MCHC)                (g/dL)	Platelet Count               (PLT)                (10^3cells/µL)	Mean Platelet Volume            (MPV)                 (fL)
G1R, M & 0	Mean	7.91	7.71	14.02	43.74	56.78	18.20	32.04	863.80	6.80
	±SD	3.10	0.30	0.61	1.38	1.37	0.66	0.52	133.80	0.32
	n	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	8.04	8.05	14.26	44.94	55.86	17.74	31.76	962.60	6.94
	±SD	2.59	0.28	0.25	0.87	1.30	0.53	0.22	124.15	0.11
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	7.85	7.84	14.14	43.64	55.74	18.06	32.40	892.80	7.22
	±SD	1.83	0.58	0.65	2.09	1.52	0.71	0.45	240.59	0.34
	n	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	5.78	7.97	13.98	43.38	54.42	17.54	32.30	1025.60	7.26
	±SD	1.48	0.28	0.37	1.54	1.38	0.53	0.39	207.18	0.32
	n	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Reticulocyte Count                       (%)	Neutrophils             (%)	Lymphocytes             (%)	Monocytes                                 (%)	Eosinophils                         (%)	Basophils                          (%)
G1R, M & 0	Mean	2.27	20.62	74.00	2.56	0.96	0.20
	±SD	0.35	6.14	6.01	0.58	0.48	0.07
	n	5	5	5	5	5	5
G4R, M & 1000	Mean	2.18	19.76	76.00	1.62	0.80	0.18
	±SD	0.36	3.21	2.98	0.47	0.37	0.08
	n	5	5	5	5	5	5
G1R, F & 0	Mean	1.60	20.98	74.52	1.78	1.08	0.22
	±SD	0.41	6.18	6.25	0.22	0.29	0.04
	n	5	5	5	5	5	5
G4R, F & 1000	Mean	1.98	25.24	69.50	2.04	1.46	0.16
	±SD	0.17	7.91	7.23	0.64	0.48	0.05
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Absolute Reticulocyte Count                  (10^9cells/L)	Absolute Neutrophils              (10^3cells/µL)	Absolute Lymphocytes              (10^3cells/µL)	Absolute Monocytes              (10^3cells/µL)	Absolute Eosinophils              (10^3cells/µL)	Absolute Basophils              (10^3cells/µL)	Prothrombin Time                      (Seconds)	Activated Prothrombin Time                       (Seconds)
G1R, M & 0	Mean	174.60	1.50	5.98	0.21	0.07	0.02	16.00	47.98
	±SD	24.51	0.34	2.59	0.10	0.02	0.01	1.14	6.34
	n	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	174.66	1.58	6.11	0.14	0.06	0.02	18.50	49.32
	±SD	24.53	0.53	2.01	0.08	0.02	0.01	2.96	5.75
	n	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	124.82	1.63	5.87	0.14	0.08	0.02	16.20	49.80
	±SD	28.31	0.54	1.49	0.05	0.03	0.01	3.12	14.47
	n	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	157.46	1.43	4.04	0.13	0.09	0.01	16.60	61.16
	±SD	11.39	0.46	1.24	0.06	0.04	0.01	2.99	12.17
	n	5	5	5	5	5	5	5	5

TABLE 11. SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/kg body weight)		Glucose (GLU) mg/dL	Urea mg/dL	Creatinine (CRE)           mg/dL	Total Cholesterol (CHO) mg/dL	Trigly- cerides (TRI)              mg/dL		Total Protein (TPR)             g/dL	Albumin (ALB)           g/dL	Alanine Amino     Transferase (ALT)               U/L	Aspartate Amino Transferase (AST)           U/L	Alkaline phosphatase (ALP) U/L	Total Bilirubin (BIT) mg/dL
G1, M & 0	Mean	103.00	23.72	0.52	67.80	65.60	5.52		3.26	41.60	86.80	158.40	0.01
	±SD	12.90	2.32	0.05	6.76	18.51	0.26		0.14	3.51	14.20	35.06	0.02
	n	5	5	5	5	5	5		5	5	5	5	5
G2, M & 100	Mean	97.80	23.96	0.51	60.80	67.00	5.46		3.24	42.00	85.40	140.80	0.01
	±SD	14.81	4.36	0.04	12.46	22.62	0.25		0.16	6.20	10.95	36.68	0.01
	n	5	5	5	5	5	5		5	5	5	5	5
G3, M & 300	Mean	105.80	28.46	0.54	59.00	70.40	5.42		3.30	37.60	81.60	208.20	0.02
	±SD	18.29	4.55	0.05	8.37	34.44	0.13		0.08	5.77	13.46	43.03	0.02
	n	5	5	5	5	5	5		5	5	5	5	5
G4, M & 1000	Mean	110.00	28.40	0.51	58.40	46.80	5.56		3.18	44.60	93.00	194.60	0.03
	±SD	5.61	5.51	0.04	6.54	12.34	0.24		0.13	8.41	8.46	62.62	0.02
	n	5	5	5	5	5	5		5	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Calcium (CAL) mg/dL	Phosphorous (PHO) (mg/dL)	Globulin (g/dL)	Albumin/ Globulin Ratio	BUN (mg/dL)	Choline- esterase	Sodium (Na) mmol/L	Potassium (K)   mmol/L	Chloride (CLO) mmol/L
G1, M & 0	Mean	9.92	6.76	2.26	1.44	11.07	110.20	151.36	3.99	106.24
	±SD	0.08	0.58	0.14	0.06	1.08	31.46	0.76	0.51	0.88
	n	5	5	5	5	5	5	5	5	5
G2, M & 100	Mean	9.90	6.94	2.22	1.47	11.18	102.00	152.06	4.08	108.04
	±SD	0.77	0.46	0.17	0.13	2.04	30.34	1.15	0.40	0.67
	n	5	5	5	5	5	5	5	5	5
G3, M & 300	Mean	10.22	6.90	2.12	1.56	13.28	93.60	153.26	3.78	107.04
	±SD	0.13	0.52	0.07	0.05	2.13	13.35	2.80	0.26	2.04
	n	5	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	9.76	6.32	2.38	1.35	13.25	111.40	152.04	3.86	107.72
	±SD	0.34	0.71	0.21	0.13	2.57	19.88	1.18	0.14	0.87
	n	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Glucose (GLU) mg/dL	Urea mg/dL	Creatinine (CRE)           mg/dL	Total Cholesterol (CHO) mg/dL	Tri- glycerides (TRI)              mg/dL	Total Protein (TPR)             g/dL	Albumin (ALB)           g/dL	Alanine Amino     Transferase (ALT)               U/L	Aspartate Amino Transferase (AST)           U/L	Alkaline Phosphatase (ALP) U/L	Total Bilirubin (BIT) mg/dL
G1, F & 0	Mean	117.80	35.64	0.58	70.20	29.20	6.04	3.61	36.20	87.60	88.60	0.02
	±SD	10.64	6.91	0.08	17.68	9.31	0.46	0.26	5.26	23.46	11.41	0.02
	n	5	5	5	5	5	5	5	5	5	5	5
G2, F & 100	Mean	120.60	40.46	0.57	70.00	32.40	5.70	3.33	33.80	87.40	84.00	0.01
	±SD	15.47	6.85	0.03	5.24	6.27	0.22	0.20	3.70	9.50	20.75	0.01
	n	5	5	5	5	5	5	5	5	5	5	5
G3, F & 300	Mean	110.60	41.48	0.56	68.20	29.00	6.00	3.72	45.60	113.80	95.00	0.02
	±SD	16.20	2.41	0.03	7.50	8.66	0.44	0.34	21.62	65.57	29.72	0.03
	n	5	5	5	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	112.00	36.28	0.54	61.80	34.40	6.00	3.01	32.20	89.80	99.40	0.03
	±SD	4.74	4.48	0.03	4.38	6.58	0.10	1.09	5.40	16.28	13.99	0.01
	n	5	5	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Calcium (CAL) mg/dL	Phosphorous (PHO) (mg/dL)	Globulin (g/dL)	Albumin/Globulin Ratio	BUN (mg/dL)	Choline- esterase	Sodium (Na) mmol/L	Potassium (K)   mmol/L	Chloride (CLO) mmol/L
G1, F & 0	Mean	9.96	6.66	2.43	1.48	16.63	483.20	152.46	3.64	107.24
	±SD	0.42	0.91	0.20	0.05	3.22	127.64	1.24	0.32	1.07
	n	5	5	5	5	5	5	5	5	5
G2, F & 100	Mean	10.08	6.16	2.37	1.41	18.88	708.20	153.30	3.81	107.32
	±SD	0.45	0.59	0.19	0.15	3.20	168.26	2.00	0.26	0.90
	n	5	5	5	5	5	5	5	5	5
G3, F & 300	Mean	10.68	6.96	2.28	1.70	19.36	662.80	154.04	3.80	109.30
	±SD	0.90	0.53	0.48	0.49	1.13	264.33	1.49	0.50	1.74
	n	5	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	10.38	6.90	2.99	1.16	16.93	584.80	153.56	3.70	108.42
	±SD	1.53	0.86	1.09	0.53	2.09	134.92	1.00	0.38	1.58
	n	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Glucose (GLU) mg/dL	Urea mg/dL	Creatinine (CRE)           mg/dL	Total Cholesterol (CHO) mg/dL	Triglycerides (TRI)              mg/dL	Total Protein (TPR)             g/dL	Albumin (ALB)           g/dL	Alanine Amino     Transferase (ALT)               U/L	Aspartate Amino Transferase (AST)           U/L	Alkaline Phosphatase (ALP) U/L	Total Bilirubin (BIT) mg/dL
G1R, M & 0	Mean	107.80	28.58	0.57	57.20	39.80	5.84	3.41	45.00	97.40	218.80	0.02
	±SD	5.40	1.87	0.03	11.52	12.76	0.29	0.29	6.63	13.01	24.91	0.02
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	104.00	30.88	0.55	43.80	31.40	5.46	3.25	40.00	86.80	175.20	0.02
	±SD	6.44	2.99	0.02	5.67	7.89	0.23	0.11	6.04	10.31	38.65	0.02
	n	5	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	127.40	35.30	0.61	69.40	61.40	6.36	3.87	32.60	69.20	72.20	0.01
	±SD	5.59	5.43	0.03	13.96	32.85	0.21	0.16	2.88	18.29	9.26	0.03
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	105.00	40.52	0.61	75.00	34.40	6.18	3.83	32.00	70.80	70.80	0.05
	±SD	13.44	4.51	0.03	19.10	4.56	0.38	0.22	4.53	5.12	15.09	0.01
	n	5	5	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight)		Calcium (CAL) mg/dL	Phosphorous (PHO) (mg/dL)	Globulin (g/dL)	Albumin/ Globulin Ratio	BUN (mg/dL)	Cholinesterase	Sodium (Na) mmol/L	Potassium (K)   mmol/L	Chloride (CLO) mmol/L
G1R, M & 0	Mean	10.16	6.92	2.43	1.41	13.34	98.60	143.16	3.94	102.52
	±SD	0.69	0.80	0.21	0.19	0.87	22.41	0.22	0.26	0.58
	n	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	9.74	7.22	2.21	1.47	14.41	83.00	145.02	3.59	103.22
	±SD	0.23	0.59	0.13	0.05	1.40	17.89	0.65	0.34	0.84
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	10.24	6.26	2.49	1.56	16.48	926.80	145.42	3.35	102.90
	±SD	0.44	0.88	0.08	0.06	2.53	248.64	1.47	0.33	2.00
	n	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	10.56	6.00	2.35	1.63	18.91	872.00	144.38	3.31	102.86
	±SD	0.27	0.51	0.20	0.10	2.10	199.89	2.72	0.13	0.88
	n	5	5	5	5	5	5	5	5	5

TABLE 12. SUMMARY OF URINALYSIS RECORD

Examination	Group & Sex		G1 & M	G2 & M	G3 & M	G4 & M	G1R & M	G4R & M
	Dose (mg/kg body weight)		0	100	300	1000	0	1000
	Number of Animals		5	5	5	5	5	5
Physical	Colour	Pale Yellow	5	5	5	4	4	3
		Yellow	-	-	-	1	1	2
	Appearance	Clear	4	5	5	4	5	5
		Turbidity	1	-	-	1	-	-
	Volume (mL)	Mean	4.5	2.6	5.3	4.2	3.9	4.5
		±SD	1.7	1.1	2.6	2.1	1.3	1.4
Chemical	pH	Mean	6.7	6.8	6.9	7.1	6.8	6.8
		±SD	0.5	0.5	0.2	0.6	0.5	0.3
	Specific Gravity	Mean	1.027	1.030	1.021	1.022	1.028	1.030
		±SD	0.007	0.000	0.005	0.008	0.004	0.000
	Urobilinogen (mg/dL)	Mean	0.4	0.4	0.2	0.4	0.4	0.5
		±SD	0.4	0.4	0.0	0.4	0.4	0.4
	Bilirubin (mg/dL)	Neg	4	3	4	3	4	4
		1	1	2	1	2	1	1
	Ketones (mg/dL)	Neg	-	-	-	2	-	-
		5	5	5	5	3	5	5
	Blood (Ery/µL)	Neg	4	5	5	3	4	4
		Ca10	1	-	-	2	1	1
	Proteins (mg/dL)	Trace	4	3	5	2	1	2
		100	-	1	-	-	1
		30	1	1	-	2	3	3
		Neg	-	-	-	1	-	-
	Nitrite	Neg	1	4	3	4	3	3
		Pos	4	1	2	1	2	2
	Leucocytes (Leu/µL)	Neg	5	5	5	4	5	5
		Ca15	-	-	-	1	-	-
	Glucose (mg/dL)	Neg	5	5	5	5	5	5
Microscopic	Epithelial cells	0	-	-	1	-	-	-
		0-1	1	2	3		1	1
		1-2	3	3	1	3	4	3
		2-3	1	-	-	1	-	1
		3-4	-	-	-	1	-	-
	Casts	Absent	5	5	5	5	5	5
	Crystals	Present	5	5	5	5	5	5

TABLE 12 (Contd..,). SUMMARY OF URINALYSIS RECORD

Examination	Group & Sex		G1 & F	G2 & F	G3 & F	G4 & F	G1R & F	G4R & F
	Dose (mg/kg body weight)		0	100	300	1000	0	1000
	Number of Animals		5	5	5	5	5	5
Physical	Colour	Pale yellow	5	5	3	5	4	5
		Yellow	-	-	2	-	1	-
	Appearance	Clear	5	3	4	5	5	4
		Turbidity	-	2	1	-	-	1
	Volume (mL)	Mean	4.8	6.2	5.9	4.2	3.5	3.6
		±SD	2.1	2.5	1.7	1.6	0.8	1.3
Chemical	pH	Mean	6.8	7.1	7.1	6.7	6.8	6.8
		±SD	0.7	0.4	1.1	0.3	0.4	0.4
	Specific Gravity	Mean	1.021	1.021	1.020	1.028	1.027	1.026
		±SD	0.008	0.007	0.010	0.004	0.004	0.007
	Urobilinogen (mg/dL)	Mean	0.2	0.2	0.2	0.2	0.4	0.2
		±SD	0.0	0.0	0.0	0.0	0.4	0.0
	Bilirubin (mg/dL)	Neg	4	4	4	4	4	5
		1	1	1	1	1	1	-
	Ketones (mg/dL)	Neg	5	5	5	4	4	5
		5	-	-	-	1	1	-
	Blood (Ery/µL)	Neg	3	4	4	5	4	5
		Ca10	-	-	-	-	1	-
		Ca25	-	1	1	-	-	-
		Ca200	2	-	-	-	-	-
	Proteins (mg/dL)	Trace	2	1	2	4	5	1
		30	-	-	-	1	-	-
		Neg	3	4	3	-	-	4
	Nitrite	Neg	4	3	3	5	4	3
		Pos	1	2	2		1	2
	Leucocytes (Leu/µL)	Neg	5	5	4	5	5	5
		Ca15	-	-	1	-	-	-
	Glucose (mg/dL)	Neg	5	5	5	5	5	5
Microscopic	Epithelial cells	0-1	1	1	2	-	1	-
		1-2	4	2	3	3	3	4
		2-3	-	1	-	2	1	1
		3-4	-	1	-	-	-	-
	Casts	Absent	5	5	5	5	5	5
	Crystals	Present	5	5	5	5	5	5

TABLE 13. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight)		Adrenals	Thymus	Spleen	Epididymes	Testes	Heart	Kidneys	Brain	Liver	PSC
G1, M & 0	Mean	0.0604	0.7514	0.6342	1.0064	3.1735	1.2125	2.2590	2.0835	10.5344	2.3816
	±SD	0.0119	0.1533	0.0738	0.0640	0.1397	0.0614	0.2240	0.1289	1.1741	0.3663
	n	5	5	5	5	5	5	5	5	5	5
G2, M & 100	Mean	0.0630	0.6919	0.6735	1.0010	2.9228	1.1877	2.5969	2.1525	10.8162	2.2128
	±SD	0.0065	0.1076	0.0667	0.0443	0.1672	0.0647	0.2891	0.1217	0.9876	0.2863
	n	5	5	5	5	5	5	5	5	5	5
G3, M & 300	Mean	0.0572	0.6269	0.5922	1.0242	3.1694	1.1864	2.3507	2.0956	10.3840	2.3975
	±SD	0.0049	0.0664	0.1458	0.0415	0.2142	0.1443	0.2566	0.1243	1.4737	0.1569
	n	5	5	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	0.0605	0.5860	0.5928	1.0397	3.1413	1.2067	2.2968	2.0724	9.4292	2.2865
	±SD	0.0077	0.1830	0.1249	0.1030	0.3287	0.1716	0.1797	0.0997	0.7478	0.2833
	n	5	5	5	5	5	5	5	5	5	5

TABLE 13 (Contd..,). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose  (mg/kg body weight)		Adrenals	Thymus	Spleen	Uterus	Ovaries	Heart	Kidneys	Brain	Liver
G1, F & 0	Mean	0.0648	0.5093	0.4214	0.5513	0.1470	0.8857	1.9527	2.0259	7.4806
	±SD	0.0078	0.1181	0.0275	0.1162	0.0144	0.0824	0.1765	0.0857	0.7090
	n	5	5	5	5	5	5	5	5	5
G2, F & 100	Mean	0.0703	0.3810	0.5137	0.6402	0.1526	0.9369	1.7481	1.9522	7.5152
	±SD	0.0115	0.0378	0.0586	0.2055	0.0172	0.1019	0.1312	0.0413	0.5203
	n	5	5	5	5	5	5	5	5	5
G3, F & 300	Mean	0.0705	0.4218	0.4418	0.4521	0.1531	0.9352	1.9264	1.9350	7.3831
	±SD	0.0094	0.0893	0.0454	0.0523	0.0225	0.0600	0.1232	0.0525	0.3699
	n	5	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	0.0708	0.4073	0.4270	0.4558	0.1571	0.8627	1.7695	1.9476	7.4482
	±SD	0.0061	0.0781	0.0400	0.1060	0.0151	0.1085	0.1630	0.1456	0.6254
	n	5	5	5	5	5	5	5	5	5

TABLE 13 (Contd..,). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight)		Adrenals	Thymus	Spleen	Epididymes	Testes	Heart	Kidneys	Brain	Liver	PSC
G1R, M & 0	Mean	0.0647	0.5413	0.6230	1.2683	3.2340	1.2561	2.3803	2.1835	10.7767	2.4993
	±SD	0.0082	0.0443	0.0542	0.0911	0.2266	0.0549	0.1389	0.1071	0.9928	0.2145
	n	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	0.0624	0.5626	0.6442	1.3105	3.1893	1.2834	2.5415	2.2298	11.2594	2.9348
	±SD	0.0039	0.0494	0.0530	0.0328	0.1477	0.0958	0.1937	0.1037	1.8236	0.4075
	n	5	5	5	5	5	5	5	5	5	5

TABLE 13 (Contd..,). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose  (mg/kg body weight)		Adrenals	Thymus	Spleen	Uterus	Ovaries	Heart	Kidneys	Brain	Liver
G1R, F & 0	Mean	0.0709	0.6198	0.5040	0.5658	0.1688	1.0534	1.8789	2.0390	9.0836
	±SD	0.0059	0.0445	0.0623	0.0621	0.0205	0.0846	0.0549	0.0810	0.7625
	n	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	0.0722	0.5087	0.4635	0.5721	0.1695	1.0817	1.6534	2.0396	8.3174
	±SD	0.0036	0.0983	0.0519	0.0801	0.0085	0.1088	0.1034	0.0974	0.5321
	n	5	5	5	5	5	5	5	5	5

TABLE 15. SUMMARY OF GROSS PATHOLOGY RECORD

Organs/ Lesions	Group	G1		G1R		G2		G3		G4		G4R
	Dose (mg/kg body weight/day)	0		0		100		300		1000		1000
	Sex	M	F	M	F	M	F	M	F	M	F	M	F
	No. of Animals	5	5	5	5	5	5	5	5	5	5	5	5
No Abnormality Detected		5	5	5	5	5	5	5	5	5	5	5	5

TABLE 16. SUMMARY OF HISTOPATHOLOGY RECORD

Group No.	G1		G4
Dose (mg/kg body weight/day)	0		1000
Sex	M	F	M	F
No. of Animals	5	5	5	5
ORGANS AND OBSERVATIONS
KIDNEYS
Dilation, renal pelvis, bilateral	1	0	0	0
THYMUS
Hemorrhage	1	0	1	0
LUNGS
Alveolar macrophages	0	0	1	0
Hemorrhage	0	0	0	1
THYROID
Ultimobranchial cyst	0	1	0	0
Ectopic thymus	0	0	0	2
RECTUM
Parasite	1	0	0	0
MANDIBULAR LYMPH NODES
Sinus dilatation	0	1	0	0
UTERUS	X		X
Dilated lumen		1		0

Note: The numeral indicates number of animal/s with lesion; M: Male; F: Female; X: Organs not subjected for histopathological examination due to sex difference.

Applicant's summary and conclusion

Conclusions:

The repeated dose toxicity of the registration substance was investigated according to the Guideline OECD 407. Rats were treated at dose of up to 1000 mg/kg/day. No effect was observed. The NOAEL of 1000 mg/kg/day was obtained.

Executive summary:

The registration substance, N, N-Dimethyl-D-glucamine, was evaluated to determine the toxic potential when administered for a period of 28 consecutive days repeatedly by oral (gavage) route to Sprague Dawley rats. This study provides information on any major toxic effects, target organs, possibility of cumulative effects and also to assess the reversibility of effects after 14 days recovery period and to estimate the No Observed Adverse Effect Level (NOAEL).

A total of 60 (30 males + 30 females) Sprague Dawley rats were distributed to main and recovery groups. Each main group and recovery group consisted of 5 males and 5 females. The animals were administered N, N-Dimethyl-D-Glucamine at the dose levels of 0, 100, 300 and 1000 mg/kg/day. The vehicle and test item formulations were administered at the dose volume of 10 mL/kg body weight.

The concentration analysis of the prepared dose formulations was performed on week 1 and week 4 of the experiment as per the validated method. The results of the analysed formulations were within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is equal to or less than 10.0%.

All animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight and feed consumption. Ophthalmological examination and neurological/functional examination was carried out during week 4 for main groups and during week 6 for recovery groups. Haematology, clinical chemistry analysis, urinalysis, gross pathology and organ weighing were performed on day 29 for main groups and on day 43 for recovery groups. Histopathological examination was conducted on the tissues from the vehicle control and high dose group animals.

The animals did not reveal any clinical signs of toxicity throughout the treatment and recovery period. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, ophthalmoscopic examination, neurological/functional examination, haematology, clinical chemistry, urinalysis, organ weights (both absolute and relative) were observed. There were no gross pathological changes observed at any of the doses tested in either sex. No treatment related histopathological findings were noticed.

The NOAEL of 1000 mg/kg/day was obtained.