8/3/2019 OCED Pre Clinical http://slidepdf.com/reader/full/oced-pre-clinical 1/33 International Journal of Pharma and Bio SciencesV1(2)2010PRECLINICAL ANIMAL TOXICITY STUDIES REPEATED DOSE 28-DAY SUBACUTE ORAL TOXICITY STUDY OF OXY POWDER ® IN RATS1 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.DIndian 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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International Journal of Pharma and Bio Sciences V1(2)2010
PRECLINICAL ANIMAL TOXICITY STUDIESREPEATED DOSE 28-DAY SUBACUTE ORAL TOXICITY STUDY OF
OXY POWDER ® IN RATS
5www.ijpbs.net Pharmacology (Clinical)
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 20o
& 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
International Journal of Pharma and Bio Sciences V1(2)2010
PRECLINICAL ANIMAL TOXICITY STUDIESREPEATED DOSE 28-DAY SUBACUTE ORAL TOXICITY STUDY OF
OXY POWDER ® IN RATS
7www.ijpbs.net Pharmacology (Clinical)
(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 l 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,
International Journal of Pharma and Bio Sciences V1(2)2010
PRECLINICAL ANIMAL TOXICITY STUDIESREPEATED DOSE 28-DAY SUBACUTE ORAL TOXICITY STUDY OF
OXY POWDER ® IN RATS
9www.ijpbs.net Pharmacology (Clinical)
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
International Journal of Pharma and Bio Sciences V1(2)2010
PRECLINICAL ANIMAL TOXICITY STUDIESREPEATED DOSE 28-DAY SUBACUTE ORAL TOXICITY STUDY OF
OXY POWDER ® IN RATS
18www.ijpbs.net Pharmacology (Clinical)
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.
International Journal of Pharma and Bio Sciences V1(2)2010
PRECLINICAL ANIMAL TOXICITY STUDIESREPEATED DOSE 28-DAY SUBACUTE ORAL TOXICITY STUDY OF
OXY POWDER ® IN RATS
31www.ijpbs.net Pharmacology (Clinical)
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 28days 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.