preclinical animal toxicity studies - International Journal of Pharma

International Journal of Pharma and Bio Sciences V1(2)2010

PRECLINICAL ANIMAL TOXICITY STUDIES REPEATED DOSE 28-DAY SUBACUTE ORAL TOXICITY STUDY OF

OXY POWDER® IN RATS

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www.ijpbs.net Pharmacology (Clinical)

Prof. Jogender K. Lalla*, Meena U.Shah* and Edward F III Group

*Corresponding Author [email protected] / [email protected]

Project Sponsor and Monitor in USA:

Dr. Edward F. Group III, DC, ND, DACBN,

CEO, GLOBAL HEALING CENTER INC. (GHC),

2040 North Loop West, Suite 108 Houston, Texas 77018,

(USA)

Tel- (713) 476–001, Fax-(713) 476–0017,

e-mail: [email protected]

Project Coordinator in India:

Prof. Jogender K. Lalla, PhD*,

Mayfair Clinical Education and Research Centre,

Sanskruti.20/701, 90 Feet Road, Thakur Complex, Kandivli

(E),Mumbai- 400 101 (INDIA)

Phone: 91(22) 28701161, Fax: 91(22) 28543643email:

[email protected] / [email protected]

Project Monitor in India:

Dr.Meena U.Shah, MD

Mayfair Clinical Education and Research Centre,

Sanskruti.20/701, 90 Feet Road, Thakur Complex, Kandivli

(E),

Mumbai- 400 101 (INDIA)

Phone: 91(22) 28701161, Fax: 91(22) 2854364

Testing Facility:

Study Director: Dr.R.M.Bhide, Ph.D

Indian Institute of Toxicology,

Kim 2057, Sadashiv Peth, VIJAYA NAGAR COLONY,

TILAK ROAD, PUNE- 411030 (INDIA)

Tel: 9822011159

e–mail: [email protected]

—————————————————————————————————————

Note: Oxy Powder® is a Registered Trade Mark of Global Healing Center Inc.

—————————————————————————————————————




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KEYWORDS

GHC’s Oxy Powder® – Dietary supplement – 28-Day Subchronic Oral Toxicity – Sprague Dawley Rats – OECD

Guidelines Test No.407

ABSTRACT:

Objectives:

1) To assess the Sub-chronic toxicity of Oxy Powder® administered orally to rats for 28 days,

2) To determine (i) Target organ toxicity (ii) “No observed effect level (NOEL)” (iii) Reversibility of signs of toxicity after

recovery period.

Background:

US FDA does not evaluate safety, efficacy and quality of dietary supplement ingredients or products. GHC decided to

study safety of Oxy Powder®, a dietary supplement used in Chronic Constipation and IBS.

Guidelines followed:

(a) OECD Guidelines No. 407, (b) WHO GCP Guidelines

(c) UCSF IACUC POLICY (d) Schedule "Y" in Drugs and Cosmetics Rules, (India).

Study Design and Controls:

1) Rats of both sexes in controlled age and body weight were selected.

2) Oxy Powder® was administered at 250, 500 and 1000 mg/kg body weight as aqueous suspensions along with a

blank

3) The results were recorded on day 0, throughout the dosing period of 28 days and during recovery period of 14 days

End Points:

1) Ophthalmoscopic examination on days 0+29,

2) Haematological analysis, biochemical analysis and urine analysis on days 0+29+43.

3) Organ weights, histopathological and gross pathological observations of sacrificed animals recorded after recovery

period.

Observations:

1) No abnormalities attributable to the treatment with Oxy Powder®

2) No target organ toxicity

3) No reversibility of any signs of toxicity at the end of recovery period of 14 days.

Conclusion:




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NOEL of Oxy Powder® administered orally to Sprague Dawley rats for 28 days was found to be 1000 mg/kg body

weight for male and female animals.

1.0 INTRODUCTION:

US FDA does not evaluate safety, efficacy and quality of dietary supplement ingredients or products.

Dietary supplements are regulated as a class of foods, not as drugs. Like foods—and unlike drugs—most dietary

supplements are not screened for safety and effectiveness by the U.S.FDA. In the US, Dietary Supplements are

covered under “Dietary Supplement Health and Education Act (DSHEA) (1) of 1994” along with ‘Guide to Nutrition

Labeling and Education Act (NLEA) Requirements, August 1994’ (2) and US FDA-CFSAN, ’Food Labeling Guide-April

2008’ (3) Under the DSHEA, if a dietary supplement has a structure or function claim the label should clearly explain the

nature of that claim and the mechanism by which it works. The manufacturer is responsible for ensuring the accuracy

and truthfulness of these claims. Potential risks involved include contamination, adulteration, and dosage inconsistency

(4). In order to be informed about the safety of dietary supplements, in addition to assessing known reported adverse

events , FDA evaluates published literature, evidence-based reports, and the known pharmacology of a compound in

order to assist in the evaluation of dietary supplement products(5). In addition to assessing known reported adverse

events, FDA promulgates regulations for ‘Dietary supplement current good manufacturing practices (cGMPs) (6a&b).

Such regulations will help ensure product quality and consistency (5).

Against this background, Global Healing Center Inc. decided to generate ‘evidence-based reports’ (as per the

Guidelines given by the Regulators) of Safety, Quality, Effectiveness and Tolerability of Oxy Powder®, a dietary

supplement marketed by them. The product is globally used by the patients in chronic constipation and constipation–

predominant IBS.

1.1 Results and Conclusions drawn from the experimental data of preceding “Preclinical Acute Toxicity study”

conducted on Oxy Powder® as per OECD Guidelines, Section 4, and Test No.420, dated 17th December, 2001.

• There was gain in the body weight of experimental rats observed in sighting at both the dose levels as well as in the

main study. In the latter study, with administration of 5000 mg/kg body weight to 4 animals, weight increase post

administration of Oxy Powder was 15.41% between 1- 7 days, 13.39% between 7- 14 days and 30.88% between

1- 14 days.

• Macroscopic examination of animals sacrificed at termination revealed no abnormalities.

• During 14 days period of study, no mortality was recorded in Sighting as well as Main study.




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• During 14 days period of study, no signs of Intoxication were observed in any rat in both the studies.

The acute oral toxicity of Oxy Powder® in these rats could be included in “Category 5” criteria of Globally Harmonized

System.

In conclusion, Oxy Powder® formulation marketed by Global Healing Center, Inc, USA exhibited high degree of safety

in Sprague Dawley rats even at dose level of 5000 g/kg body weight. This formed basis for ‘Dose Selection’ in the

present study.

1.2 Guidelines followed:

(a) OECD Guidelines No. 407, Section 4: Health Effects, 28-Day Oral Toxicity in Rodents (7)

(b) WHO GCP Guidelines described in World Health Organization’s GLP Handbook (8)

(c) UCSF IACUC POLICY– Laboratory Housing and Study Areas for Research Animals (9)

(d) Schedule "Y" in Drugs and Cosmetics (Eighth Amendment) Rules 1988, India. (10)

1.3 Profile of Experimental Product: Oxy Powder® (Marketed as capsules by Global Healing Center, Inc. USA) is an

oral dietary supplement useful in treating Chronic Constipation and Constipation – Predominant IBS.

Each capsule contains 685 mg Ozonated Magnesium Oxides, 5.5 mg Bis-carboxyethyl Germanium sesquioxide (Ge–

132) and 25 mg Natural Citric acid encapsulated in Kosher certified “00” vegetarian capsule. The Ozonated Magnesium

Oxide releases nascent oxygen in acidic pH of stomach aided by Citric acid included in the formulation to achieve pH

[11] in the gi tract. Germanium element in native form is highly toxic but Ge–132 is reported to be totally non-toxic in

mice, rats [12–15] and humans [16]. ADME of Ge-132 has been reported [17,18] Although Ge –132 is non toxic and

safe when taken orally, yet, by way of abundant precaution and to rule out toxicity on prolonged use, this study was

undertaken.

1.4 Rationale for Selection of Sprague Dawley Rat as Test System: One of the recommended rodent species by the

regulatory authorities for conducting preclinical toxicity studies among rodents is Sprague Dawley Rat, as it is a

sensitive species for expression of toxic responses.

1) Rat is recommended rodent species for conducting acute toxicity studies as per OECD guidelines.

2) Availability of the historical control data at the testing facility.

1.5 Route of Administration and Reason for Choice

Oral route of administration is the proposed therapeutic route of administration in human being.

2.0 MATERIALS AND TEST SYSTEM

2.1 Materials:




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Test Substance: Oxy powder® capsules (Marketed by Global Healing Center, Inc., USA)

2.2 Animal Test System (Source: I.I.T. Animal house):

Animals: Male and Female Sprague Dawley Rats Age: 6-8 weeks

Body Weight on Day 0: Males: Mean 96.66g [Min: 91.5 g (– 5.34%), Max: 100.5 g (+ 3.97%)]

Females: Mean 87.96g [Min: 81.7 g (– 7.12%), Max: 97.3 g (+10.62%)]

Total No. of animals: Males 30, Females 30

No. of animals per dose level: 5 per sex (dose groups sacrificed on day 29)

5 per sex (reversal groups sacrificed on day 43)

Acclimatization: Seven days prior to dosing.

Veterinary examination: Prior to and at the end of the acclimatization period.

Identification of animals: By cage number, animal number and individual marking on fur.

Diet: Pelleted feed supplied by Nav Maharashtra Chakan Oil Mills Ltd, Pune

Water: Aqua guard potable water in glass bottles ad libitum

Housing & Environment: The animals were housed 5 each, of the same sex in Polypropylene cages provided with

bedding of husk.

Housing temperature between 20 o & 24°C, Relative humidity between 30% and 70%,

Air changes 10 to 15 per hour and Dark and light cycle each of .12 hours.

2.3 Dose:

Male: 0 mg/kg and 0 mg/kg (Rev.), 250 mg/kg, 500 mg/kg, 1000 mg/kg and 1000 mg/kg (Rev.) body weight.

Female: 0 mg/kg and 0 mg/kg (Rev.), 250 mg/kg, 500 mg/kg,1000 mg/kg and 1000 mg/kg (Rev.) body weight.

2.3.1 Justification for Dose Selection:

As stated above in 1.1, results of acute toxicity studies in Sprague Dawley rats indicated that Oxy powder® was non

toxic at the dose level of 5000 mg/kg body weight. On the basis of these results, the doses selected for the study were 0

mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight. The oral route was selected for use because oral route is

considered to be a proposed therapeutic route.

2.3.2 Preparation and administration of dose:

Oxy powder® was suspended in distilled water to obtain concentrations of 200.0, 500.0, and




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1000 mg/ml. It was administered to animals at the dose levels of 250 mg/kg, 500 mg/kg and 1000 mg/kg in the dose

volume of 10 mL/kg. The test substance suspensions were freshly prepared every day for 28 days. The control animals

were administered vehicle only. Administration was by oral (gavage), once daily for 28 consecutive days.

3.0 METHODS

3.1 Randomization, Numbering and Grouping of Animals:

Sixty rats (30 male and 30 female healthy animals) were randomly divided into four groups of 5 animals per sex for

dosing up to 28 days and 5 animals per sex as reversal groups for control and high dose i.e. 0 mg/kg, 0 mg/kg (Rev.)

250 mg/kg, 500 mg/kg, 1000 mg/kg and 1000 mg/kg (Rev.) body weight. Animals were allowed acclimatization period of

7 days to laboratory conditions prior to the initiation of treatment. Five rats of same sex were housed per cage. Each

animal was fur marked with picric acid. The females were nulliparous and non-pregnant.

4.0 OBSERVATIONS:

(A) Experimental animals were kept under observation throughout the course of study for the following:

(i) Body Weight: Weight of each rat was recorded on day 0, at weekly intervals throughout the course of study and at

termination to calculate relative organ weights. From the data, group mean body weights and percent body weight gain

were calculated.

(ii) Food Consumption: The quantity of food consumed by groups consisting of five animals of each sex for different

doses ( 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg) and five animals of each sex for different doses ( 0 mg/kg

Rev. and 1000 mg/kg Rev.) was recorded at weekly interval. Food consumed per animal was calculated for control and

the treated dose groups.

(iii) Clinical signs: All animals were observed daily for clinical signs. The time of onset, intensity and duration of these

symptoms, if any, were recorded.

(iv) Mortality: All animals were observed twice daily for mortality during entire course of study.

(v) Ophthalmoscopy: The eyes of experimental animals in control as well as treated groups given different dose levels

were examined prior to the initiation of the dosing and in 4th and the 6th week (for reversal group animals) of the study.

Eye examination was carried out using a hand slit lamp after induction of mydriasis with 0.5% solution of tropicamide.




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(vi) Functional Observations: At the end of the 4th week exposure, ‘sensory reactivity’ to graded stimuli of different types

(auditory, visual and proprioceptive stimuli), ‘motor reactivity’ and ‘grip strength’ [using Grip Strength Meter Single Digital

Sensor Model 1027DSR for Rats (10lb/5Kg sensor) from Columbus Instruments, Ohio State, USA] were assessed.

(B) Terminal Studies:

Laboratory Investigations: Following laboratory investigations were carried out on day 29 and on day 43, in animals’

fasted over-night. Blood samples were collected from orbital sinus on the following morning using sodium heparin (200

IU/ml) for Blood chemistry and potassium EDTA (1.5 mg/ml) for Haematology as anticoagulant. Prothrombin time

analysis was conducted using citrate bulb (100 µl of 3.8% solution of sodium citrate for 1 ml of blood). Blood samples

were centrifuged at 3000 r.p.m. for 10 minutes.

Haematological Investigations: Haematological parameters were determined using Beckman Coulter Haematology

analyzer.

Biochemical Investigations: Biochemical parameters were determined using VeTEX Clinical Chemistry auto-analyzer

for veterinary practices, Model ACE manufactured by Alfa Wasserman.

Normal Laboratory Ranges of Rat used by Indian Institute of Toxicology are shown in Table .

Urine analysis: Urine samples were collected in week 4 and in week 6 and for estimation of normal parameters. The

estimations were performed using appropriate methodology.

Necropsy: All the animals were sacrificed on day 29 except for reversal group animals which were sacrificed on day 43

i.e. post-dosing period of 14 days, using CO2 asphyxiation technique (body weights mentioned in the table section are

fasting body weights. Necropsy of all animals was carried out and the weights of the organs including liver, kidneys,

adrenals, epididymis, thymus, spleen, brain, heart, uterus and testes/ovaries [expressed in terms of absolute values

and the relative values as per cent body weight] were recorded. The results are shown in Table .

Histopathology: Tissue samples of organs from control and treated animals at the highest dose level of 1000 mg/kg

were preserved in 10% formalin. The organs included adrenals, aorta, brain, caecum, colon, duodenum, epididymis,

gonads, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, oesophagus, prostate, rectum, sciatic nerve, spleen,

sternum with bone marrow, stomach, seminal vesicles, spinal cord, thymus, thyroid / parathyroid, trachea, urinary

bladder and uterus. They were subjected to histopathological examination.

Tissue samples of liver, kidneys, adrenals, epididymis, thymus, spleen, brain, heart, uterus and testes/ovaries of low

and intermediate dose groups’ animals were preserved for histopathology examination.

Statistical analysis: findings such as clinical signs of intoxication, body weight changes, food consumption,

haematology and blood chemistry were subjected to (co-)variance analysis followed by Student’t’ test and Cochran ’t’

test.




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The histopathological observations were used to calculate animal score according to degree and area by using

software, LABCAT Module for Histopathology from ‘Innovative Programming Associates, Inc., Princeton, New Jersey’.

5.0 RESULTS:

Randomization, Numbering and Grouping of Animals:

Allocation of animals to different groups is recorded inTabel No 1

Clinical signs:

Male animals: All animals in Group I (0 mg/kg, animal nos.1 to 5), Group II (0 mg/kg, Reversal*, animal nos.11 to 15),

Group III (250 mg/kg, animal nos.21 to 25), Group IV (500 mg/kg, animal nos.31 to 35),




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Group V (1000 mg/kg, animal nos.41 to 45) and Group VI (1000 mg/kg, Reversal*, animal nos.51 to 55) were free of

toxic clinical signs throughout the dosing period of 28 days.

* In case of Reversal Groups II and VI, additional observations during the recovery period of 14 days

also did not exhibit any toxic clinical signs.

Female animals: All animals in Group I (0 mg/kg, animal nos. 6 to 10), Group II (0 mg/kg, Reversal*, animal nos. 16 to

20), Group III (250 mg/kg, animal nos. 26 to 30), Group IV (500 mg/kg, animal nos. 36 to 40), Group V (1000 mg/kg,

animal nos. 46 to 50) and Group VI (1000 mg/kg, Reversal*, animal nos. 56 to 60) were free of toxic clinical signs

throughout the dosing period of 28 days.

* In case of Reversal Groups II and VI, additional observations during the recovery period of 14 days

also did not exhibit any toxic clinical signs.

Mortality: All animals from control and all the treated dose groups survived throughout the dosing period of 28 days and

the post-dosing recovery period of 14 days.

Body weight: Results of body weight determination of animals Table 2 from control and different dose groups exhibited

comparable body weight gain throughout the dosing period of 28 days.

During the post-dosing recovery period, animals from 1000 mg/kg Reversal Group VI exhibited normal body weight gain

when compared with those of respective controls.




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Food consumption: During dosing and the post-dosing recovery period, the quantity of food consumed by animals

from different dose groups was found to be comparable with that by control animals.

Ophthalmoscopy: Ophthalmoscopic examination of animals in control and test product– treated groups did not reveal

any abnormality.

Functional Observations: These tests conducted on the experimental animals at termination and recorded in Table 2

to 5 did not reveal any abnormalities.




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Haematological investigations: The results of haematological investigations Table 6

conducted on day 29 and on day 43 (for reversal group animals), revealed following significant changes in the values of

different parameters investigated when compared with those of respective controls; However, the increase or decrease

in the values obtained was within normal biological and laboratory limits or the effect was not dose dependent.




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Parameters Laboratory Range

MCV 44.5 to 69.0 (µm3)

MCH 12.0 to 24.5 (pg)

MCHC 21.6 to 42.0 (%)

Total WBC 3.00 to 19.00 (x103/uL)




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Male animals:

Decreased values of MCHC were obtained for animals in dose groups administered 500 mg/ kg (P<0.05) and 1000

mg/kg (P<0.01) body weight of Oxy powder® sacrificed on day 29 (P<0.05)

Increase in total WBC count values were obtained for animals in the dose group of 500 mg/kg sacrificed on day 29

(P<0.05).

Female animals:

Increased MCV values were obtained for animals in dose groups administered 500 mg/kg sacrificed on day 29 (P<0.05)

Increased MCV and MCH values were obtained for animals in dose groups administered 1000 mg/kg reversal group

sacrificed on day 43 (P<0.05).

Biochemical Investigations: Results of Biochemical investigations conducted on days 29 and 43 (for reversal group

animals) and recorded in Table 7 revealed the following significant changes in the values of different parameters

studied when compared with those of respective controls; however, the values obtained were within normal biological

and laboratory limits. In other words, the effect was not dose-dependent.




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Parameters Laboratory Range

Alkaline Phosphatase 50 to 80 (IU/L)

BUN 20 to 50 (mg%)

AST 30 to 70 (IU/L)

LDH 300 to 400 (IU/L)




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Male animals:

Blood Urea Nitrogen showed elevated levels in animals in 500 mg/kg dose group sacrificed on day 29 (P<0.05),

LDH levels were elevated in animals in1000 mg/kg dose group sacrificed on day 29 (P<0.05).

Aspartate Aminotransferase levels decreased in animals in 1000 mg/kg reversal group sacrificed on day 43 (P<0.01).

Female animals:

Alkaline Phosphatase exhibited elevated levels in animals in 1000 mg/kg reversal group sacrificed on day 43 (P<0.05).

Urine analysis: Urine analysis data Table 8 of control group and treated group of animals determined in week 4 and

reversal group of animals in week 6 did not reveal any abnormalities.




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Organ Weight: Group Mean Relative Organ Weights (%of body weight) are recorded in Table No.9. Comparison of

organ weights of treated animals with respective control animals on day 29 was found to be comparable Similarly,

organ weight data of animals’ in 1000 mg/kg reversal group was comparable with respective control group on day 43.




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Histopathology: In histopathological examination (Table 10) . focal lymphocytic infiltration and/or necrosis in liver,

lymphocytic infiltration in the kidneys, focal lymphocytic infiltration in the heart, gliosis in the brain, interstitial




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pneumonitis in the lungs, eosinophillic infiltration in uterus were observed in few male and female animals in control and

treated animals in 1000 mg/kg dose group of animals with similar quality and quantity and are considered incidental,

gender and physiology related and are covered in the background data of the pathology.




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28





29





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Necropsy: Gross pathological examination of animals in control as well as the treated groups did not reveal any

abnormalities (Table 11)

6.0 DISCUSSION:

As stated above, US FDA does not evaluate safety, efficacy and quality of dietary supplement ingredients or

products(19). As with most foods, there is no requirement for manufacturers of most dietary supplements to provide

evidence of product safety to FDA prior to marketing (15). Accordingly, FDA regulates the safety of dietary supplements

primarily through a post-marketing surveillance of whether the product is adulterated fewer than one of the provisions of

the FD&C Act. In order to be informed about the safety of dietary supplements, in addition to assessing known reported

adverse events, FDA evaluates published literature, evidence-based reports, and the known pharmacology of a

compound in order to assist in the evaluation of dietary supplement products (16).

Adverse event reports (AERs) are an important tool for developing a “signal” which can help FDA to identify potential

safety problems with dietary supplements. And, because dietary supplements are not subject to standardized quality




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control measures, contamination, adulteration, and dosage inconsistency are common (14). Under DSHEA, another

important arm of FDA’s regulatory and surveillance activities used to help ensure the safety of dietary supplement

products is the Agency’s authority to promulgate regulations for dietary supplement current good manufacturing

practices (CGMPs). Such regulations will help ensure product quality and consistency. FDA published a proposed rule

for dietary supplement CGMPs on March 13, 2003, which would establish standards to ensure that dietary supplements

and dietary ingredients are not adulterated with contaminants or impurities, and are labeled to accurately reflect the

active ingredients and other ingredients in the product (16).

1) All the male and female animals from control and all the treated dose groups up to 1000 mg/kg survived throughout

the dosing period of 28 days and the recovery period of 14 days.

2) No signs of intoxication were observed in male and female animals from different dose groups during the dosing

period of 28 days and during the recovery period of 14 days.

3) Male and female animals from all the treated dose groups exhibited comparable body weight gain with that of controls

throughout the dosing period of 28 days and the recovery period of 14 days.

4) Food consumption of control and treated animals was found to be comparable throughout the dosing period of 28

days and the recovery period of 14 days.

5) Ophthalmoscopic examination, conducted prior to and at the end of dosing period on animals from control and all the

treated dose groups did not reveal any abnormality.

6) Haematological analysis conducted at the end of the dosing period on day 29 and at the end of recovery period on

day 43, revealed no abnormalities attributable to the treatment.

7) Biochemical analysis conducted at the end of the dosing period on day 29 and at the end of recovery period on day

43, revealed no abnormalities attributable to the treatment.

8) Functional battery observation tests conducted at termination revealed no abnormalities.

9) Urine analysis, conducted at the end of the dosing period in week 4 and at the end of recovery period in week 6,

revealed no abnormality attributable to the treatment.

10) Organ weight data of male and female sacrificed at the end of the dosing period and at the end of the recovery

period was found to be comparable with that of respective controls.

11) Gross pathological examination did not reveal any abnormality.

12) Histopathological examination did not reveal any abnormality.

7.0 Conclusion:




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Based on these findings, the no observed effect level (NOEL) of Oxy Powder marketed by Global Healing Center Inc.

USA, to Sprague Dawley rats via oral route over a period of 28 days was found to be 1000 mg/kg body weight for both

male and female animals.

REFERENCES

1) Dietary Supplement Health and Education Act of 1994. Public Law 103-417,103rd Congress

(Available at www.fda.gov/opacom/laws/DSHEA.html).

2) GUIDE TO NUTRITION LABELING AND EDUCATION ACT (NLEA) REQUIREMENTS-August 1994 (Available

at www. fda.gov/ora/inspect_ref/igs/nleatxt.html).

3) US FDA-CFSAN-A Food Labeling Guide-April 2008(Available at www.cfsan.fda.gov/guidance.html).

4) New York State Task Force on Life & the Law, “Dietary Supplements, Balancing Consumer Choice & Safety” pp.

39, Oct.2005; New York State Department of Health, Albany, NY 12237, [Available at http://

www.health.state.ny.us/nysdoh/task force]

5) Robert E. Brackett,"Dietary Supplement Safety Act: How is FDA Doing after 10 Years?" Testimony

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6a) Dietary Supplement Current Good Manufacturing Practices (cGMPs) and Interim Final Rule (IFR) Fact sheet,

22nd June 2007 (Available at www.cfsan.fda.gov/~dms/dscgmps 6.html)

6b) Backgrounder- Final Rule for Current Good Manufacturing Practices (cGMPs) for Dietary Supplements, 22nd

June 2007 (Available at www.cfsan.fda.gov/~dms/dscgmps 7.html)

(7) OECD Guidelines for the Testing of Chemicals (No. 407, Section 4: Health Effects) "Repeated Dose 28-Day Oral

Toxicity in Rodents" (Adopted on 12 May 198 1 and Updated on 27 July 1995.)

(8) UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR)

HANDBOOK OF GOOD LABORATORY PRACTICE (GLP), Quality practices for regulated non-clinical research

and development

(9) UCSF IACUC POLICY– Laboratory Housing and Study Areas for Research Animals (Adopted on May 2001,

Revised December 2003).

(10) Schedule "Y" in Drugs and Cosmetics (Eighth Amendment) Rules 1988, Ministry of Health and Family Welfare,

Government of India.




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(11) Independent Laboratory Tests on Oxygen Based Colon Cleansers; Scientific Analysis of Commercial Products for

Oxidative Ability” quoted In ‘Oxy powder TESTED AGAINST COMPETITION COMES OUT #1 IN LAB TESTS’ at

http://www.greenwillowtree.com/Page.bok

(12) Asai K., “Miracle Cure: Organic Germanium”, pp 59-66, Japan Publications Inc., 1980.(Through

www.regenerativenutrition.com/content).

(13) Raisin J, Hess B, Blatter M, et al., Toxicity of an organic germanium compound: deleterious consequences of a

"natural remedy." Schweiz Med Wochenschr, 1992, 122:11-13 (PDR Health(Available at www.pdrhealth.com)

(14) Samochowiec L and Wojcicki J. Basic pharmacological properties of Sanumgerman. In: 1st Int. Conf. on

Germanium, Hanover; October 1984. (Lekim & Samochowiec eds., Semmelweis-Verlag, 1985 (available in

Sandra Goodman,” The health and life enhancer (GERMANIUM)”, CHAPTER 11)

(15) Dluzniewski A, Gastol-Lewinska L, Buczynska B, Monicqewski A. Teratogenic effects of Sanumgerman in rats. In:

1st Int. Conf. on Germanium. Hanover, Oct. 1984. Lekim & Samochowiec, eds. Semmelweis-Verlag. 1985.

(16) Miyao K, Onishi T, Asai K. Tomizawa S & Suzuki F. Toxicology and Phase I Studies on a Novel

Organogermanium Compound, Ge-132., Current Chemotherapy & Infectious Diseases Proceedings, 11th ICC &

19th ICAAC, Amer.Soc.Microbiology, 80-82, 1980.

17) Lekim D. & Kehlbeck H. 1985. “The biological activity of Germanium discussed in 1st International Conference on

Germanium, Hanover, Oct. 1984” in Lekim & Samochowiec, eds. Semmelweis-Verlag (Available in Sandra

Goodman, “The health and life enhancer (GERMANIUM)”, CHAPTER 11).

(18) Mizushima M, Satoh H, and Miyao K. Some pharmacological and clinical aspects of a novel organic germanium

compound Ge-132 in ‘1st Int. Conf. on Germanium. Hanover, Oct. 1984’, (Lekim & Samochowiec eds.,

Semmelweis-Verlag, 1985, GE-132; PHAR. NER.).

(19) New York State Task Force on Life & the Law, “Dietary Supplements, Balancing Consumer Choice & Safety”

p.39, Oct.2005; New York State Department of Health, Albany, NY 12237,[Available at http://

www.health.state.ny.us/nysdoh/task force]

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preclinical animal toxicity studies - International Journal of Pharma