diabliss: the world's fist diabetic friendly sugar product performance report

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DIABLISS CONSUMER PRODUCTS PVT LTD

DiaBliss Herbal Blended Cane Sugar

Product Performance Report

8/3/2013

For the first time in the world we are rendering pure cane sugar, carbohydrates and fructose diabetic

friendly and making available products that can provide safe and limitless food choices. Long term

product tests showing safety and efficacy of the product in diabetics are summarized. Diabetics can

now consume a limitless choice of foods safely without any compromise in taste factors.

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TABLE OF CONTENTS

Section Sub

Section

Topic Page

Number

1 1.1 Executive Summary 3

1.2 Company Background 4

2 Diabetes Background 6

2.1 Diabetes Trends 6

2.2 Diabetes - Lifestyle Issues 9

3 DiaBliss Herbally Blended Products: An Integrated Food System for

Sugar, Carbohydrate and Fructose Management

9

3.1 DiaBliss Product Versatility & Competitive Differentiation 11

4 DiaBliss Product Performance 12

4.1 Product Characterisation at SGS 12

4.2 Animal Trials at Sugen Life Sciences 13

4.2a Summary of Acute Toxicity Trials 13

4.2b Summary of Sub-Acute Toxicity Trials 14

4.3

4.4

Glycemic Index Studies of DiaBliss Herbal Treated Sugar

GTT Test with Diabliss based sweets on diabetics

15

16

4.5 Short Term Glucose Tolerance Tests 17

4.5a GTT Blood Sugar Levels 18

4.5b Extended GTT Tests – Urine Glucose (Glycosuria) Levels 19

4.6 Long Term Test Results 20

4.7 Fasting and Post Prandial Response – Type 1 and Type 2 Diabetics 23

4.8 Fasting, Post Prandial and HbA1c Correlations: Type 1 & Type 2

Diabetics

23

4.9 DiaBliss Sugar Long Term Use in Juvenile Diabetic 24

5 References 25

A1 Appendix 1A: Metals Analysis of Diabliss Sugar analyzed at SGS

Laboratories

Appendix 1B: Summary of Herbicide Analysis of Herbal Solution

analyzed at SGS Laboratories

26

29

A2 Appendix 2: ACUTE ORAL TOXICITY STUDY OF DIABLISS

SUGAR IN WISTAR RATS

34

A3 Appendix 3: SUB-ACUTE ORAL TOXICITY STUDY OF

DIA BLISS SUGAR IN WISTAR RATS

60

A4

Appendix 4: Glycemic Index Measurement of Diabliss Sugar 121

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Section 1:

1.1 Executive Summary:

DIABLISS HERBAL CANE SUGAR – SUMMARY OF PRODUCT PERFORMANCE

For the first time in the world we are able to render pure cane diabetic friendly sugar by blending

sugar with a proprietary blend of herbal extracts using techniques from traditional Indian medicine

system. The herbal extract is derived from several herbs and spices (black pepper, turmeric,

fenugreek, ginger, pomegranate, etc) using water and no other chemicals or solvents. The herbal

extract is colorless, odorless and tasteless and thereby restores the original taste factor of cane sugar

& derivative products.

We have successfully completed acute and sub-acute oral toxicity tests in mice. The product has

been extensively analyzed for heavy metals, pesticides and insecticides and proven to be fit for

human consumption. The product has been tested in thousands of people from periods ranging from

3 to 36 months and data to date indicates that the product is safe and in fact lowers long term blood

sugar levels as measured by HbA1c. The product has been tested in subjects ranging in age from 7 to

82 and is effective in Type 1 & Type 2 diabetics.

The attached report summarizes product performance test results.

We have also developed and tested a range of products for carbohydrate & fructose

management. These products enable diabetics to consume an unlimited choice of foods without

compromising on taste factors and thereby restore serious lifestyle constraints that diabetics face

every day with limited food choices. We are the only product that provides sugar, carbohydrate and

fructose management system thereby providing a complete and integrated approach to diabetic

friendly foods. Over 20 products have been formulated and tested. They include salt, rice, multi

grain porridges, instant beverages (lemon tea, masala tea, coffee, lime juice mix), chocolates,

premixed sweet mixes (kheer mix, kesari bath mix, etc). These products provide comprehensive

carbohydrate, fructose and glucose management.

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1.2 Company Background:

DiaBliss Consumer Products Pvt Ltd is a private limited company formed to commercialize

DiaBlissTM

brand consumer products to meet the extensive and unmet needs of diabetics all over the

world. The company has developed herbal treated sugar and a variety of derivative products that

allow diabetics to consume a limitless variety of foods without compromising taste factors normally

associated with artificial sugar substitutes. DiaBliss Consumer Products is committed to improving

the lifestyle of diabetics all over the world by providing safe, holistic and integrated sugar and sugar

based foods.

Management Team & Board of Directors:

V R Ramanathan is CEO & Board Member of DiaBliss Consumer Products. He also serves as the CEO of Electron India and has been involved in manufacturing since1974. He has been involved in manufacture of photoelectric cells, electronic head for fuel pumps at retail outlets. For the last 12 years he is involved in manufacture of RFID related products for physical access, logical access, vehicle access & traking, asset tracking, e-cash solution on smart cards. Ramanathan has been working on Diabliss herbal treated sugar for over four years in all aspects of animal & human safety testing, long term subject trials, product testing, and derivative products development. He holds a BE degree in Electronics Engineering from Guindy College of Engineering.

Siva Vallabhaneni is COO & Board Member of DiaBliss Consumer Products Pvt Ltd. He served as a senior executive in several MNCs including DuPont, Tronox, Cristal Global. Most recently he served as Chief, Global Intelligence at Cristal Global serving as an advisor to Chairman, President and Senior Executives in the areas of business transformation and strategic growth. He has 25 years’ experience in strategic planning, supply chain management, operational excellence, and general business management, M&A, Manufacturing, R&D in Chemicals, FMCG industries. He is holder of two worldwide patents. He holds a B Tech in Chemical Engineering from Andhra University and M. S. in Chemical Engineering from Clarkson University, USA

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Dr. Arun Chockalingam, MS, PhD, FACC, FAHA is a Board Member of DiaBliss

Consumer Products Pvt. Ltd. He is a world renowned expert in health policy,

epidemiology, nutrition and global prevention of non-communicable diseases.

Dr. Chockalingam serves as Secretary General of the World Hypertension

League. He also serves as a Professor of Global Health at Mount Sinai

Medical School, New York, NY, USA. Dr. Chockalingam was the Founding

Director of the Office of Global Health at the National Heart, Lung, and Bl ood

Institute at the U. S. National Institutes of Health in Bethesda, Maryland from

2010-2013. He was also Founding Director of Continuing Public Health

Education and Global Health at the Faculty of Health Sciences at the Simon Fraser University,

Vancouver, Canada. He had 130 publications in the areas of Cardiovascular and Diabetic Medicine

and an author of 5 books. Dr. Chockalingam serves as a consultant to the World Health Organization,

UN, governments of India and China. He received his undergraduate degree in Electronics and

Communication Engineering from the University of Madras, India (1970); an M.S. degree in

Biomedical Engineering from the Indian Institute of Technology, Madras, India (1978); a Ph.D. in

Cardiac Physiology and Pharmacology from Memorial University of Newfoundland (1982); and

completed his F.A.H.A. Fellowship in Cardiovascular Epidemiology and Prevention with the American

Heart Association (1986) and F.A.C.C. Fellowship in Cardiology with the American College of

Cardiology (1999).

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Section 2. Diabetes Background1

Diabetes, also known as Diabetes Mellitus, is a chronic health condition where the body is unable to

produce enough insulin and properly break down sugar (glucose) in the blood. Glucose comes from

food and is used by the cells for energy. Glucose is also made in the liver. Insulin is a hormone

produced by the pancreas, a large gland behind the stomach. Insulin is needed to move sugar into the

cells where it can be used for energy needed for bodily processes.

After digestion of food, glucose passes into the bloodstream. For glucose to get into cells, insulin

must be present. Throughout the pancreas are clusters of cells called the islets of Langerhans. Islets

are made up of several types of cells, including beta cells that make insulin. When normal individuals

eat, beta cells in the pancreas automatically produce the right amount of insulin to move glucose from

blood into the cells of the body. In individuals with diabetes, however, the pancreas either produces

little or no insulin, or the cells do not respond appropriately to the insulin that is produced. Glucose

builds up in the blood, overflows into the urine, and passes out of the body in the urine. Thus, the

body loses its main source of fuel even though the blood contains large amounts of glucose. Glucose

may also interact with cells, especially those in very narrow blood vessels. This process may lead to

neuropathies and decreased immune function.

With Type 1 diabetes, the body does not make any insulin. With Type 2 diabetes, the more common

type, the body does not make or use insulin properly. Without enough insulin, glucose stays in the

blood and causes a condition called hyperglycemia, or high blood sugar levels.

Diabetes is associated with long-term complications that affect almost every part of the body. The

disease often leads to blindness, heart and blood vessel disease, stroke, kidney failure, amputations,

and nerve damage. Uncontrolled diabetes can complicate pregnancy, and birth defects are more

common in babies born to women with diabetes. Pregnant women can temporarily develop

gestational diabetes, a type of diabetes that begins late in pregnancy.

2.1 Diabetes Trends2,3

:

Although increases in both the prevalence and incidence of type 2 diabetes have occurred globally,

they have been especially dramatic in societies in economic transition in much of the newly

industrialized world and in developing countries. Worldwide, the number of cases of diabetes is

currently estimated to be around 150 million as per the World Health Organization. This number is

predicted to double by 2025, with the greatest number of cases being expected in China and India.

These numbers may represent an underestimate and there are likely to be many undiagnosed cases.

Previously a disease of the middle aged and elderly, type 2 diabetes has recently escalated in all age

groups and is now being identified in younger age groups, including adolescents and children,

especially in high-risk populations.

India is now being called the diabetes capital of the world. India accounts for the largest number of

the diabetic population in the world: 62.4 million live with diabetes (5.4% of Indian population), 77.2

million people (6.4% of Indian population) live with pre-diabetes with high propensity to acquire the

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disease. This is followed by China (43.2 million) and the United States (26.8 million), as per figures

released by the International Diabetes Federation (IDF).

Another major region where diabetes is expected to reach epidemic levels is the Middle East and

Northern Africa (MENA) Region. IDF estimates 32.6 million or 9.1% of the population now have

diabetes and this number is expected to double in less than 20 years. In most parts of the world, for

every diabetic there is a pre diabetic with propensity to acquire the disease. For example India’s

diabetic population is estimated at 7.1% of the population and pre diabetic levels are estimated at 7%

of the population.

IDF estimates that 347 million people worldwide have diabetes. By 2030, nearly 9 per cent of the

country’s population is likely to be affected from the disease, warns the fourth edition of the World

Diabetes Atlas launched by the IDF at the 20th World Diabetes Congress in Montreal, Canada. New

data projected that the number of people with diabetes will rise to 552 million by 20304.

In fact the prevalence of diabetes in India has almost doubled in a period of 11 years as per ICMR-

INDIAB study3. This alarming increase in diabetes could be partially related to better diagnostic

infrastructure, but largely related to life style factors combined with genetic propensity for the

disease. Diabetes in India is also no longer the disease of the middle aged. Like developed countries,

the disease has become prevalent among people in their 20s, 30s and 40s. In fact we are also

beginning to see an alarming increase in diabetes among teenagers, following similar trends being

observed in United States and other western countries.

.

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In India, genetic disposition, coupled with lifestyle factors such as stress and eating habits are largely

contributing towards the rapid spread of the disease

Data on sugar consumption has shown significant increase in per capita sugar consumption across the

world. The following data indicates a 350% increase in per capita sugar consumption in India in the

last 50 years.

Per Capita Sugar Consumption in India is 18-20 kg/year and about 5 kg/yr of Gur & Khandasari

bringing total consumption to approximately 25 kg/yr/per person (67g/person/day)

US per capita sugar consumption is 71 kg/yr/person (194g/person/day). As we are moving towards a

more hectic life style, there has been a corresponding multi fold increase in per capita sugar

consumption as there is sugar in almost every prepared food to impart the desired taste factor. If we

only study the urban population in tier 1 cities in India, we can expect per capital sugar consumption

approaching the same levels in the US where the per capital consumption is almost 300% higher than

the average per capital consumption in India. We are following similar eating patterns of fast foods

and prepared foods, all of which contain sugar.

Currently, up to 11 per cent of India’s urban population and 3 per cent of rural population, above the

age of 15 years, have diabetes. Diabetes affects all people in the society, not just those who live with

it. The World Health Organization estimates that mortality from diabetes and heart disease cost India

about $210 billion every year and is expected to increase to $335 billion in the next ten years. These

estimates are based on lost productivity, resulting primarily from premature death.

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2.2 Diabetes – Lifestyle Issues:

Most diabetics undergo a significant change in lifestyle after being diagnosed with the disease:

i. They cannot consume sugar or sweets

ii. They cannot consume foods rich in carbohydrates

As a result, their life style is severely impeded.

Diabetics typically use sugar substitutes such as Aspertame, Sucralose, Saccharin, Stevia, Levulose,

etc. that go by various trade names. These products are either derived from chemical routes or

extracts from fruits. Some diabetics consume sugar in limited quantities for their coffee or tea every

day, as they are used to the natural cane sugar taste and are not able to be satisfied by the sweeteners.

Products made with these substitutes have a serious after taste, but diabetics normally do not have an

option but to consume these products. In some cases diabetics occasionally consume sugar based

sweets to quench their craving for sugar. All these sweets or product preparations only address the

sugar portion of the diet, but when a diabetic consumes foods rich in carbohydrates or fruits, they are

not able to address carbohydrate and fructose management issues as these materials are next in line

with respect to breakdown into blood sugars due to their rapid breakdown and conversion into

glucose which becomes available in the blood stream.

Section 3: DiaBliss Herbal Blended Products: An Integrated Food System for Sugar,

Carbohydrate and Fructose Management

In order to address the diabetes epidemic, the inventors of DiaBliss turned back to the classical

Indian medical sciences system to understand the disease. After 10 years of R&D and 4 years of

product testing, for the first time in the world, we are able to render sugar diabetic friendly by

blending with herbal extracts. The herbal extracts are derived from herbs and spices (Black Pepper,

Turmeric, Fenugreek, Ginger, Gooseberry, Cinnamon and Pomegranate) using water and no other

chemicals or solvents and retains the original taste of cane sugar.

Herbs have been used as food and for medicinal purposes for centuries. Herbal medicine is based on

the premise that plants contain natural substances that can promote health, wellness and alleviate

illness. In different herbs, a wide variety of active phytochemicals, including the flavonoids,

terpenoids, lignans, sulfides, polyphenolics, carotenoids, coumarins, saponins, plant sterols,

curcumins, and phthalides have been identified. Many of these herbs contain potent antioxidant

compounds that provide significant protection against chronic diseases. These compounds may

protect LDL cholesterol from oxidation, inhibit cyclooxygenase and lipoxygenase enzymes, improve

metabolism, enhance insulin sensitivity, inhibit lipid peroxidation, or have antiviral or antitumor

activity. Today we are witnessing a great deal of public interest in the use of herbal remedies and

ingredients in foods. There has been considerable research in the efficacy of the various components

used in the Diabliss herbal extract. The following summarizes some of the key findings from

research into efficacy of these herbs and spices.

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1. Black Pepper has anti-inflammatory and antioxidant properties, and so helps to prevent the

risks of cancers. It also may have anti-microbial properties, and it contains vanadium which is

being researched for its potential benefits in improving insulin sensitivity and blood sugar

levels in people with Type -2 diabetes.

2. Turmeric – Curcumin, an ingredient derived from turmeric, combined with extracts from

black pepper and onion skin has been found to be effective against obesity, diabetes and

abnormal cholesterol levels. The combination which goes by acronym CPQ - curcumin,

piperine (derived from black pepper) and quercetin (found in onion and skinks of some fruits)

- could control diet-induced changes in body weight, blood glucose, triglyceride, cholesterol,

and low-density lipoprotein (LDL).

3. Fenugreek – Trigonella foenum graecum – The seeds of this plant contain an alkaloid

trigonelline and another compound known as choline. These seeds have been reported to be

diuretic, anti-tussive and hypoglycaemic in nature. Research studies show that fenugreek

seeds decrease blood glucose and triglyceride levels and have significant effect on LDL or

HDL cholesterol when consumed at higher doses.

4. Ginger - The active ingredient in ginger, the pungent phenolic gingerol constituents were

identified as the major active compounds in the ginger extract enhancing glucose uptake.

According to researchers, extracts from ginger are able to increase the uptake of glucose into

muscle cells independently of insulin.

5. Indian gooseberry (Amla) has been used widely in ayurvedic medicine for thousands of

years. It has been shown to be effective in lowering cholesterol, “hardening of the arteries”

(atherosclerosis), diabetes, pain and swelling of the pancreas (pancreatitis), cancer, upset

stomach, eye problems, joint pain, diarrhea, dysentery, obesity, and “organ restoration.” Some

research studies also indicate that amla may prevent age-related hyperlipidaemia through

attenuating oxidative stress in the ageing process.

6. Cinnamon – Polyphenols from cinnamon impacts glucose metabolism through helping in the

following five pathways: 1. Increase in glucose metabolism about 20-fold, which

significantly improves blood sugar regulation. 2. Polyphenols in Cinnamon has been found to

have "insulin-like effects" due to a bioactive compound, qualifying it as a candidate for an

insulin substitute. 3. Cinnamon slows the emptying of your stomach to reduce sharp rises in

blood sugar following meals, and improves the effectiveness, or sensitivity, of insulin. 4.

Cinnamon actually enhances antioxidant defenses. Several studies have shown that daily

consumption of cinnamon lowers serum glucose, triglyceride, LDL cholesterol, and total

cholesterol in people with type 2 diabetes and suggest that the inclusion of cinnamon in the

diet of people with type 2 diabetes will reduce risk factors associated with diabetes and

cardiovascular diseases.

7. Pomagranate - Consumption of pomegranate (Punica granatum, Punicaceae) fruit and/or

juice has been reported to reduce the risk of CVD and prostate cancer. Each pomegranate

contains 600–800 seeds and when purified, oil from these seeds (POMo) is a rich naturally

occurring source of the bioactive compound 9-cis, 11-trans conjugate linolenic acid (CLA).

Studies have shown that Pomogranate Seed Oils showed improvement of insulin sensitivity

which is associated with a decrease in the risk of developing type 2 diabetes.

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While consumption of these herbs and spices are very beneficial, the issue of how they can all be

consumed daily to deliver the desired efficacy, given the strong taste factor, places a practical

limitation. For example studies on use of fenugreek seeds have shown daily consumption of 50g of

fenugreek seeds have shown beneficial results. Along similar lines 6g of cinnamon consumption per

day has shown beneficial results in terms of lowering blood sugar levels and cholesterol.

DiaBliss has spent the last 10 years in R&D and developed a proprietary process to extract these

beneficial active ingredients into a herbal solution using water and no other chemicals or solvents.

Most importantly, DiaBliss is able to extract the goodness of key active ingredients into a colorless,

odorless and tasteless solution which can be combined with any foods to provide diabetic friendly and

a host of wellness properties to the food without altering the native taste of the food.

In addition to herbal blended sugar, we have come up with other variants which allow us to deliver

diabetic friendly foods. Herbal solution has been blended with rice and flour to impart diabetic

friendly features to allow carbohydrate management. We have also blended fruit extracts with herbal

solution so that juices, jams, ketchup and other derivative products which contain sugars can be

rendered diabetic friendly. We therefore have created a complete system of foods with sugar,

carbohydrate and fructose management, thereby allowing complete flexibility of food choices among

diabetics. Another interesting variant is herbal blended salt. We have found that this salt, when used

in cooking also provides carbohydrate management by rendering carbohydrates and sugars found in

vegetables diabetic friendly. Products based on these integrated systems are in the commercialization

pipeline and will be introduced into the market.

3.1 DiaBliss Product Versatility & Competitive Differentiation

DiaBliss is the first diabetic friendly product in the world made with natural cane sugar. Diabetics can

now enjoy pure cane sugar without any guilt or fear of their blood sugar shooting up. DiaBliss sugar

allows diabetics to consume any amount of sugar or sugar-based sweets. The herbal-treated sugar

does not taste differently from untreated or normal sugar and does not leave an after-taste, retaining

the original coloring.

The next question is why to try this herbal remedy when so many other substitutes for sugar are

readily available in the market?

The product is pure cane sugar blended with herbal extracts. It therefore does not have strong after

tastes of traditional sweeteners. Unlike sweeteners which are made with synthetic materials, DiaBliss

is 100% natural. You can enjoy all the sweets you have been craving for without compromising on

the taste or the risk of your blood sugar shooting up. DiaBliss can be used for your entire family for

all your sugar needs and is not age sensitive. Those who have propensity for diabetes can greatly

benefit by consuming DiaBliss Sugar as it also delivers a preventive function for people who are at

high risk of onset of diabetes.

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There are two primary strengths of traditional forms of Indian medicine: One, there are no side-

effects of any kind and secondly, it has been seen that DiaBliss holds a long-term therapeutic value as

different from short-lived, repetitive curative intervention. Additionally, in most cases, there is no

‘dependence syndrome’. Here, a patient cannot afford to miss even a single dose of medication if he

is not to develop the symptoms of his disease.

DiaBliss Products can be used wherever cane sugar is used. We have developed a complete line of

products with DiaBliss Sugar. They include Sugar, Salt, Rice, Sweets, Chocolate, Coffee & Tea

Mixes, Multi Grain Porridges, Jams, Ketchup, Milk Shakes, Ice Cream Mixes, etc.

Table 1 is a comparative analysis of various classes of sweeteners relative to DiaBliss. Key

differentiation attributes are:

Natural Taste of Pure Cane Sugar with no after taste characteristics of sweeteners

High degree of product versatility in terms of usage in baking, cooking, low and high

temperature, wide pH ranges, etc, similar to Splenda®

100% Natural - Made from pure cane sugar and natural herbal extracts

No chemical processes in herbal extraction, only uses water in extraction

Only product that provides sugar, carbohydrate and fructose management system thereby

providing a complete and integrated approach to diabetic friendly foods

Product showing positive efficacy in terms on long term blood sugar control (ie lower

levels of HbA1c, Fasting and Post prandial levels) in type 1 and type 2 diabetics based on

consumption periods from three months to three years. (details of long term testing

summarized in following sections)

Content analysis, acute and sub-acute trials and histopathology studies on animals indicate

product is safe for human consumption.

Section 4: DiaBliss Product Performance

DiaBliss Sugar has undergone extensive and structured tests for the past four years. Hundreds of

diabetics are continuing to consume the products and we have followed their blood parameters over

this period and have found exceptional product performance. Subjects ranging in age from 7 to 82

and type 1 and type 2 diabetics have safely consumed the product. The following sections discuss

their results in detail.

4.1. Product Characterisation at SGS

We have tested Cane Sugar, DiaBliss Herbal Blended Sugar, Herbal Solution at SGS Labs. SGS is a

US based global testing and analysis laboratory. SGS analyzed for metals including Arsenic,

Calcium, Copper, Iron, Lead, Magnesium, Potassium, Selenium, Sodium, Tin, Zinc, Vitamin A,

Vitamin C and Energy. Metals analysis in cane sugar and DiaBliss Herbal blended sugar respectively

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and shows all metal levels indicate that the product is safe for human consumption. In addition to

metals, (Arsenic, Copper, Lead, Tin and Zinc) the analysis looked for a total of 45 pesticides and

found no trace of any pesticides.

The following analytical test reports are attached for reference:

Appendix 1A: Summary of Metals Analysis of Commercial Cane Suga

Appendix 1B: Summary of Metals Analysis of DiaBliss Herbal Cane Sugar

Appendix 1C: Summary of Herbicide Analysis of Herbal Solution

4.2 Animal Trials at Sugen Life Sciences

In order to ascertain that the DiaBliss product is safe for human consumption, we have undertaken

sub-acute and acute toxicity studies at Sugen Life Sciences, Pvt. Ltd based in Tirupathy, AP. Sugen

Life Sciences (SLS) is a Contract Research Organization with key focus on Pre-Clinical safety,

efficacy toxicology testing and Clinical services to biopharma industries. All studies are conducted in

accordance with the requirements of the national and international regulatory guidelines like OECD,

Schedule Y, FDA, EPA, ICH-GCP. SLS provides comprehensive drug testing services involving

laboratory-based studies, vertebrate animal models and clinical studies, under regulatory compliance.

They follow Good Laboratory Practices (GLP), current Good Manufacturing Practices (cGMP) and

Good Clinical Practices (GCP).

4.2a Summary of Acute Toxicity Trials

This study was performed to assess the acute oral toxicity of DiaBliss Sugar supplied by DiaBliss

Consumer Products Pvt. Ltd in Wistar Rats.

Wistar rats fasted overnight, were dosed with DiaBliss Sugar dissolved in sterile distilled water as

single oral gavage, using intubation cannula. The food was withheld until 4 hours of post dosing. The

first group of three female rats was given a single dose of 2000 mg of DiaBliss Sugar per kg body

weight. No mortality was observed at this dose level; hence the Group II was administered at the

same dose level. No mortality was observed at this dose level, either. Hence, further testing was

essential. The animals did not show any clinical signs of toxicity when treated with 2000 mg of

DiaBliss Sugar per kg body weight. At the end of 14 days of observation period, all the rats were

subjected to gross pathological examination and the symptoms were physiological variation in nature,

hence there were no test item related changes. Therefore, the test item is considered to be safe.

The acute oral median lethal dose LD50 (cut off value) of DiaBliss Sugar in Wistar rats was found to

be > 2000-5000 mg/kg body weight

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DiaBliss Sugar is being classified as follows :

Globally Harmonised System (GHS) : Low Toxicity

Appendix 2 summmarises Acute Oral Toxicity trial report of DiaBliss Herbal Cane Sugar in Wistart

rats.

4.2b Summary of Sub-Acute Toxicity Trials

In the sub acute toxicity trials, 28 Day Repeated Dose Oral Toxicity Study of DiaBliss Sugar in

Wistar Rats was conducted. This study was conducted to assess the toxic effects of DiaBliss Sugar in

Wistar Rats, when administered orally for a period of 28 consecutive days.

Three groups of Rats comprising of 6 males and 6 females per group were treated with DiaBliss

Sugar at 180.0, 540.0 and 1800.0 mg/kg bd. wt. for a period of 28 consecutive days. A concurrent

control group, comprising of the same number of animals was also maintained in the study.

Rats were observed individually for visible signs of reaction to the treatment twice a day throughout

the study period. Individual body weight and feed consumption were monitored weekly.

Haematological and biochemical analyses of blood samples were performed on all animals at the end

of the treatment period. All the surviving rats were sacrificed and subjected to a gross pathological

examination at the end of the treatment period. Absolute organ weights were recorded and relative

organ weights were calculated for the organs viz., liver, kidneys, adrenals, testis/ovary,

epididymides/uterus, thymus, spleen, brain and heart in all rats.

The animals exposed to test item did not show any specific treatment related clinical changes in all

the treated groups. No significant changes were observed in the mean body weight and food

consumption in any of the treated group animals.

Haematological analysis and clinical chemistry analysis did not reveal any treatment related changes

in any of the parameters at various dose levels studied in either sex.

The absolute and relative organ weights did not reveal any significant difference in the treated

groups’ animals when compared with the control group animals in either sex. Gross pathological

examination of the animals did not reveal any treatment related effects in any of the dose group.

Histopathological examination of the high dose group animals did not reveal any treatment related

effects compared to control group animals. There was no test item related toxicity recorded in any of

the treatment groups under the experimental conditions.

Appendix 3 summmarises Sub-Acute Oral Toxicity trial report of DiaBliss Herbal Cane Sugar in

Wistart rats.

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4.3 Glycemic Index Studies of DiaBliss Herbal Cane Sugar

The effect of Natural Sugar on the alterations in the blood glucose levels as measured by Oral

Glucose Tolerance Test (OGTT) was compared against a glucose standard in a non-blind, repeated

measure, crossover design trial. Twelve healthy subjects with a mean age of 30.25 (SD 4.55) years

were recruited. Subjects were served with either 75 grams of Natural Sugar (Test food) or a standard

substance glucose weighing 75 grams, on two different days. Blood glucose was determined from

venous blood samples in fasted subjects (0 min) and at every 15 min interval in first one hour and

every 30 minute intervals in second hour after the consumption of Natural Sugar or glucose. The GI

value was calculated geometrically by expressing the incremental area under the blood glucose curve

(iAUC) as a percentage of each subject’s average iAUC for the standard food. The GI value of the

Natural Sugar was found to be 53.93 (range 26.04-77.60), and can be classified under low glycemic

food.

Results

Table1: Calculation of iAUC after supplementation with glucose and DiaBliss Herbal Cane Sugar

(Data as Mean±S.D)

--------------------------------------------------------------------------------------------------------------------------

Blood glucose concentration (mg %) Mean iAUC GI

------------------------------------------------------------------------------------------------

Time periods N 0 15 30 45 60 90 120

(min)

Glucose 12 68.00 107.42 140.08 136.67 120.50 121.17 102.00 86.33

Bal ±11.40 ±13.15 ±16.34 ±21.56 ±25.72 ±21.94 ±25.25

DiaBliss 12 67.83 96.25 110.75 102.83 96.67 91.58 85.08 53.93

Sugar ±11.42 ±13.80 ±11.93 ±14.66 ±15.84 ±10.63 ±13.61 47.08

--------------------------------------------------------------------------------------------------

N = Sample size, iAUC = Incremental Area under the Curve, GI=Glycemic Index, min=minutes

Inference: The GI value of the DiaBliss Herbal Cane Sugar tested in the present study was found to be 53.93.

Therefore it is classified as a food / nutrient with low GI.

As per the Food and Agriculture Organization GI cut-off values are as follows:

Low <55; Medium 56–69 inclusive; High >70 (Brand-Miller et al. 2003).

GI values of other foods: Glucose: 99; Fructose: 19 ; Sucrose: 69-80 (Table sugar); Maltose:

105; Lactose: 46

16 | P a g e

Detailed test report on Glycemic Index studies on Diabliss Sugar conducted at Sugen Life Sciences

may be found in Appendix

4.5 GTT Test with Untreated Sugar & Diabliss Sugar based sweets on diabetics

A similar curve was observed when a GTT test was conducted on four diabetcis using an Indian

sweet meat (Ravva laddu).

In the first testing diabetics were given four ravva laddus containing 60g of ravva mix along with 60g

of untreated sugar. The blood sugar levels were measured with a strip glucometer every ½ hour for a

period of two hours. The following day, the same ravva laddu was made with 60g of Diabliss Sugar.

Also, the carbohydrate in the ravva was addressed by treating it with herbal solution at the rate of

20ml/kg.

The average blood sugar levels were normalized and data in Figure below clearly demonstrates the

increased rate of absorption of the Diabliss based sweets.

17 | P a g e

4.5 Short Term Glucose Tolerance Tests

We initiated a short term glucose tolerance tests with 11 subjects, 9 of whom were known diabetics.

Also, two of the subjects were regular consumers of alcohol. Each subject was given 50 grams of

DiaBliss Herbal Cane Sugar solution dissolved in water. Blood samples were collected prior to

intake (i.e., fasting level) and at 1, 2 and 3 hours. Blood samples were collected by an independent

laboratory and blood sugar levels were measured by colorimetric technique.

Table 1 summarizes the details of the test candidates. In order to preserve the confidentiality, we

have not shown the names of the subjects. Please note, subjects 3 & 6 are regular consumers of

alcohol whose liver function may have already been impeded and alcohol load in the system may

already be high.

Table 1: Details of Subjects for extended GTT Test:

ID Age Sex How long

diagnosed

with

diabetes?

(yrs)

Did you

consume

Medication?

If yes, Specify

Type 1 Type 2 Salt Sugar

1 58 M x 8 Yes Amaril, 1000mg, 1-0-1 Yes Yes

2 54 M x 15 None No No

3 49 M No No

4 48 M x No No

5 65 M x 3 Yes Yes Yes

6 49 M x 17 Yes Metformin 500mg 1-0-1 Yes Yes

7 45 M x 3 Yes Yes Yes

8 80 M x 7 Yes No No

What Diabiss

Products are you

cosuming

Type of Diabestes

Not Diabetic

18 | P a g e

All subjects who consumed 50g of the DiaBliss sugar did not complain of any discomfort after

consuming this quantity of sugar. In addition, subject 9, a 45 year old male consumed both normal

cane sugar and DiaBliss sugar on two separate occasions.

4.5a GTT Blood Sugar Levels

The blood sugar profile of the subjects is summarized in Figure 1 and Table 2 below:

Table 2: Blood Sugar Profiles of Subjects consuming 50g of DiaBliss Herbal Blended Sugar:

We can see from the data above that even after consuming 50g (over 200% of daily sugar

consumption in a single dose), all subjects, with the exception of the two alcoholics, have shown that

0

50

100

150

200

250

300

0 20 40 60 80 100 120 140 160 180 200

Blo

od

Su

gar

Re

adin

gs, m

g/d

l

Time, Minutes

Fig 1: Extended GTT Tests with 50g intake of Diabliss Sugar Among Group of Diabetic & Non-diabetic Subjects

1 2 3 4 5 6 7 (Reg Sugar) 7 (Diabliss) 8

Subject

Number

1 2 3 4 5 6 7 (Reg

Sugar)

7

(Diabliss)

8

Diabetic Diabetic Non-Diabetic Diabetic Diabetic Diabetic Diabetic

Time, min

0 116 142 104 156 135 121 142 81 140

60 221 233 154 261 224 179 255 203 266

120 164 158 97 217 142 191 226 134 189

180 123 118 90 164 112 103 108 70 171

Diabetic

Blood Sugar Readings with Calorimeter, mg/dl

19 | P a g e

the blood sugar levels have dropped below or very close to fasting sugar levels confirming the

excellent absorption characteristics of DiaBliss Herbal treated sugar.

4.5b Extended GTT Tests – Urine Glucose (Glycosuria) Levels

The data summarized in table 3 shows urine glucose (glycosuria) levels of the subjects during the

tests. When the blood sugar levels typically exceed 180-200 mg/l level, the kidneys start to excrete

glucose from the urine as glycosuria5.

Table 3: Urine Analysis from Extended GTT Test: Glycosuria Profiles in Test Subjects:

The above data on the ranges of glycosuria characterized by the measurements indicate interesting

trends:

1. Urine glucose levels of all subjects, with the exception of the two chronic alcoholics in terms

of not seeing major spikes, as well as returning back to levels at fasting is a further indication

of excellent sugar absorption from the herbal treated sugar.

2. Subject number 7 shows an interesting glycisuria level. This is the subject who participated in

two extended GTT tests: Data labeled 7 (Regular Sugar) and 9(Diabliss) characterizes

glycosuria profiles with regular cane sugar and DiaBliss herbal treated sugar respectively. It

is interesting to note a lower glycosuria profile with herbal treated sugar suggesting good

blood sugar control which therefore does not require triggering of the body safety mechanism

to reject excess blood glucose through urine as glucosuria.

4.6 Long Term Test Results

DiaBliss herbal cane sugar has been consumed by hundreds of diabetics over the course of last four

years. We have also followed their blood sugar readings over the years and the following sections

summarize a sampling of the results among type 1 and type 2 diabetics. In the current data summary,

the consumption time line varied from 3 months to 34 months. Subjects varied in age from 7 to 72,

type 1 and type 2 diabetics.

Subject Note:

Number Fasting 60 min 120 min 180 min Reading

1 Nil + Trace Nil Trace

3 Nil Trace Nil Nil ++

4 Trace ++ + Trace +++

5 Nil + Nil Nil

6 Nil Trace + Nil

7 (Regular Sugar) Nil ++ + Nil

7 (DiaBliss) Nil Trace Trace Nil

8 Nil Trace Nil Nil

9 Nil + Trace

500-1,000 mg/dl

Urine Analysis

Up to 250 mg/dl

Urine Glucose (Glycosuria)

1,000-1,500 mg/dl

20 | P a g e

We have measured fasting blood sugar, post prandial blood sugar and HbA1c (long term, i.e., 3

month blood sugar average). We have found the following from this long term study:

Product lowered HbA1c levels within 60-90 days from consumption of DiaBliss products

Fasting & PP levels showed decreasing trends immediately

Subjects report more energetic feeling; Major lifestyle improvements were observed,

especially with juvenile diabetics

HbA1c or glycated hemoglobin or glycosylated hemoglobin is a form of hemoglobin that is measured

primarily to identify the average plasma glucose concentration over prolonged periods of time,

typically two to three months. Normal levels of glucose produce a normal amount of glycated

hemoglobin. As the average amount of plasma glucose increases, the fraction of glycated hemoglobin

increases in a predictable way. This serves as a marker for average blood glucose levels over the

previous months prior to the measurement.

Recommended HbA1c readings fall within the reference range of 6.5 to 7%. This implies that for

every 100 red blood cells, 6-7 cells have glucose attached to them. Mean blood sugar levels can be

better understood from the following table7.

Table 4 summarises fasting, post prandial (2 hour readings) and HbA1C levels of various subjects

who are continuing to consume DiaBliss Herbal treated sugar for periods ranging from 3 months to

36 months. These subjects ranged in age between 7 years and 82 years. We also selected data to

illustrate product performance among type 1 and type 2 diabetic

% mmol/L mg/dL Inference

3-4 2-4 36-72 Extremely low to low blood sugar

4-5 5.5 99-100 Perfect!

4.5-5 5-6 90-108 Normal range for non-diabetics (pre-prandial)

5.5 - 6 7 – 8 126 - 144 Normal post-prandial in non-diabetics

6.5 – 7 9-10 162 - 180 Maximum post-prandial in non-diabetics

6.5 – 7.5 9 – 11 162 - 198 High, even for diabetics

7.5 – 9.5 11 – 15 198 - 270 Indicates to poorly controlled BG

12.5 - 25 20 - 25 360 - 450 Extremely high

> 19 33 or > 33 > 594 High possibility of serious electrolyte imbalance!!

HbA1c-Average Blood Glucose Reading

21 | P a g e

The above data is presented in the following plots to give us a better graphical characterization of

DiaBliss product performance among a vastly different population. Figure 2 compares HbA1c levels

between type 1 and type 2 diabetics. Data plotted below show profiles prior to start of consumption

of DiaBliss herbal treated sugar and after commencement of regular consumption of sugar.

The following are details of the subjects:

1. Male, Type 2 diabetic, Age 51, diabetic since 1 year. He has been consuming Diabliss since

10 months. HbA1c levels have dropped from 7.4 to 6.2 or 16% reduction within three months

from start of consumption of DiaBliss.

2. Male, Type 2 diabetic, Age 50, diabetic since 14 years. DiaBliss consumption since 30

months. HbA1C levels varied between 7.0 and 8.2 pror to consumption of DiaBliss Sugar.

His current levels have stabilized around 6.7 currently. In the graph, there is a step change

reduction in HbA1c levels since November 2011. The subject reports increasing DiaBliss

consumption since this period and a step reduction in HbA1c has been recorded. Over the

course of 30 months, average reduction in HbA1c is from about 7.5 to 6.7 or 11%

3. Male, age 8 years, Type 1 Juvenile diabetic, consuming DiaBliss Sugar since 18 months.

HbA1c levels have dropped from 11.5 to 6.5 or about 45%.

4. Female, age 59 years, Type 1 dabetic, consuming DiaBliss sugar since 9 months. HbA1c

levels have dropped from 8.9 to 6.1 or about 31%.

Figure 2: HbA1C Profiles: Type 1 vs. Type 2 Diabetics

Table 4: Representative Sampling of Fasting, Post Prandial and HbA1c levels of long term consumers of DiaBlissNo Age Sex Profession Type of

Diabetic

Diabetic

Since

How Long

Consuming

Herbal Sugar

Before

Herbal

Sugar

After

Herbal

Sugar

Latest

Reading

HbA1c HbA1c HbA1c Fasting

Average

PP

Average

Fasting

Average

PP

Average

Medication Details

1 54 M 1 Many

Years

12 months 5.4 5.4 210 231 98 137 Insulin 25 units Morning &

Evening before herbal sugar, 15

units morning & evening

2 51 M Consulting

Engineer

2 1 Year 10 months 6.8 6.3 6.2 136 185 122 141 Continuing medication Glycomet

500mg 1-0-1

3 45 M Traveling

Sales

2 3 Years 34 months 8.8 6.7 6.9 Amryl m - 2 mg

4 61 F House Wife 2 8 Years 36 months 7.0 7.0 107 137

5 50 M Engineer 14 Years 30 months 7.6 7.0 6.7 107 177 101 157 Iscept Fort 1- 0 -1 and Pioglit

6 58 M Catering

Business

2 25 months 7.2 6.3 6.3 121 182 114 140 Glycomet 500mg 1-0-1 lowered

to 1/2-0-1/2

7 7.5 M School 1 22 months 18 months 11.5 8.2 7.9 204 281 Child started going to school

after starting herbal treated

sugar; Weight Gain of 2.5 kg

(+10%), Insulin dose increased

8 59 F Housewife 1 7 months 8.9 7.0 6.1 140 200 90 139

9 66 M 2 3 Years 6.6 6.6 101 121

10 58 M 2 2 Years 18 months 7.2 7.2 124 177 No Medication

11 82 M Retired 20 Years 3 Months 8.4 228 372 93 139 Started noticing significant

increase in energy levels

Before Herbal

Sugar

After Herbal Sugar

22 | P a g e

So, in the four subjects we have seen HbA1c levels dropped by 11-16% for type 2 diabetics and about

45% for type 1 diabetics over DiaBliss consumption period ranging from 9 months to 30 months

4.7 Fasting and Post Prandial Response – Type 1 and Type 2 Diabetics

Fasting and Post Prandial (PP) response has the potential to immediately show the impact of

consuming DiaBliss sugar. However, both Fasting and PP are subject to considerable variability. For

example if the subject, the previous night may have indulged in eating foods high in sugar or

carbohydrates, the fasting readings could be skewed.

Figure 3 compares typical fasting and PP plots for type 1 and type 2 diabetics. We can clearly see

immediate responses for both the subjects. In the case of the 82 year old type 2 diabetic, his blood

sugar levels were not responding to medications. Within a short period of consuming DiaBliss Sugar,

he reported feeling more energetic. The other subject, a 53 year old type 1 diabetic who has been

consuming DiaBliss sugar for over 12 months, has lowered Insulin intake from 25 units to 18 units, or

a 28% reduction in Insulin intake.

This response is important as the product seems to have a very visible short term benefit that can be

readily measured. This can be augmented by HbA1c measurements which will start to show longer

term benefit, within 60-90 days from the start of consumption of the DiaBliss product.

23 | P a g e

Figure 3: Fasting & Postprandial response of type 1 & type 2 diabetics

4.8 Fasting, Post Prandial and HbA1c Correlations: Type 1 & Type 2 Diabetics

Figure 4 below shows the plot of Fasting, PP and HbA1c for the type 1 diabetic. We can clearly see

correlation between Fasting, PP and HbA1c levels for this type 1 diabetic. Similarly, Figure 5 shows

Fasting, PP and HbA1c level plots are shown for a type 2 diabetic, a 58 year old who has been

consuming Diabliss Sugar since 15 months. He also shows a similar correlation. In case of this

subject, his diabetes medication level has been lowered by 50%, in close consultation with his

physician.

Figure 4: Fasting, PP, HbA1c Profiles for Type 1 diabetic

Figure 5: Fasting, PP, HbA1c Profiles for Type 2 diabetic

0

50

100

150

200

250

300

350

9.9.06 22.1.07 21.9.07 28.6.09 13.11.2011

Blo

od

Su

gar,

mg

/dl

Type 1 Diabetic, Male Age 52 - Fasting & PP Profiles

Fasting

PP

Started consuming DiaBlissHerbal Cane Sugar15 months consumption

Insulin 25 Units Morning & EveningGemerfort 1-0-1

Insulin 18 Units Morning & Evening Gemerfort 1-0-1HbA1c (4/6/12) 5.4

0

50

100

150

200

250

300

350

400

16/6/2012 5/7/2012 25/7/2012 22/8/2012

Blo

od

Su

gar,

mg/

dl

Type 2 Diabetic, Male Age 82 - Fasting & PP profiles

Fasting

PP

Started Consuming DiaBliss Herbal Cane Sugarin early/mid-June 2012; 4 months; Baseline HbA1c = 8.4

Note: January 2013 HbA1c = 6.3

0

50

100

150

200

250

4/3/2012 14/8/2012 15/9/2012 17/9/2012

Blo

od

Su

gar

Leve

ls, m

g/d

l

Type 1 Diabetic, Female, age 58 - Fasting & PP Profiles

Fasting

PP

Started consuming DiaBliss Herbal Cane Sugarfor Tea & Coffee

6

6.5

7

7.5

8

8.5

9

4/3/2012 6/6/2012 14/8/2012 17/9/2012

Hb

A1

c

Type 1 Diabetic, Female, age 58 - HbA1c Profile

Started consuming DiaBliss Herbal Cane Sugar for Tea & Coffee

24 | P a g e

As we can see from the above charts there is a direct correlation (barring some day to day variability

due to lifestyle or diet factors) between Fasting/Post Prandial blood sugar levels and HbA1c readings

for both type 1 and type 2 diabetics.

4.9 DiaBliss Sugar Long Term Use in Juvenile Diabetic

We wanted to share the impact of DiaBliss product on a Juvenile diabetic. The subject is a 7.5 year

Juvenile diabetic whose life style has significantly improved since consuming DiaBliss. Figure 6

summarises his HbA1c profile. The juvenile diabetic is now able to go to school on a full time basis

(which was not the case prior to use of DiaBliss), gain weight and able to enjoy all kinds of food

preparations at home. During this period the child has gained 2.5kg of body weight (10% increase).

Figure 6: Data from Juvenile Diabetic

100

110

120

130

140

150

160

170

180

190

11.9.2011 19.12.2011 20.4.2012 20.04.2012 2.10.2012

Blo

od

Su

gar,

mg/

dl

Type 2 Diabetic, Male, Age 51: Fasting & PP Profiles

Fasting

PP

Started DiaBliss Herbal Cane Sugar26 months consumption

6

6.2

6.4

6.6

6.8

7

7.2

7.4

7.6

11.9.2011 19.12.2011 20.04.2012 2.10.2012

Hb

A1

c

Type 2 Diabetic, Male, Age 51: HbA1c Profile

Started DiaBliss Herbal Cane Sugar26 months consumption

8 year old juvenile

diabetic

Two years on

DiaBliss Sugar

Major lifestyle

improvement

Able to go to

school full-time

Able to enjoy all

kinds of foods &

sweets

Gaining body

weight without

significant increase

in insulin dosage

45% reduction in

HbA1c levels

25 | P a g e

5: References

1. National Standard, The Authority on Integrative Medicine, taken

fromhttp://www.naturalstandard.com/demo/demo-mc-diabetes.asp

2. WHO Report Series 913, “DIET, NUTRITION AND THE PREVENTION OF CHRONIC

DISEASES”, Section on Diabetes – Pages 72-78, 2003

3. Diabetologia 2011

4. International Diabetic Foundation Annual Report 2011. Web Site Address

http://www.idf.org/sites/default/files/attachments/IDF-AR2011-EN.pdf

5. Glycosuria, Wikepedia, http://en.wikipedia.org/wiki/Glycosuria

6. Comparison of Accuracy of Five Glucometers in South Africa, JEMDSA 2009;14(2):102-105

7. Decoding HbA1c Test for Blood Sugar - Normal reading for the HbA1c Calculator

Medindia http://www.medindia.net/patients/patientinfo/hba1c-blood-sugar-test-normal-

reading.htm#ixzz2BhvlSsjy

http://www.medindia.net/patients/patientinfo/hba1c-blood-sugar-test-normal-reading.htm#ixzz2BhvlSsjy

http://www.medindia.net/patients/patientinfo/hba1c-blood-sugar-test-normal-reading.htm#ixzz2BhvlSsjy

26 | P a g e

Appendix 1: Comprehensive Analysis at SGS Laboratories:

APPENDIX 1A: METALS ANALYSIS OF DIABLISS CANE SUGAR

Appendix 1B: Summary of Herbicide Analysis of Herbal

Solution analyzed at SGS Laboratories

29

Appendix 2: ACUTE ORAL TOXICITY STUDY OF DIABLISS SUGAR

IN WISTAR RATS

34

SLS Study Number : SLS/043-11

Sugen Life Sciences Pvt Ltd Page 1 of 26

CONFIDENTIAL TITLE PAGE

REPORT

ACUTE ORAL TOXICITY STUDY OF DIA BLISS SUGAR IN WISTAR RATS

DATA REQUIREMENT GUIDELINE: OECD N° 423

Study Director: Syed Nasir Ahamed, M.Sc

Report Submission: February 07, 2012

SPONSOR

En-Joy Consumer Products Pvt Ltd 1/283, Shripuram Street

Shripuram, Chennai- 600 097

Tamil Nadu

TESTING FACILITY

Sugen Life Sciences Pvt. Ltd #4/86, S.V. Nagar

Perumalla palli (post) Tirupati – 517 505

Andhra Pradesh India



STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information contained in this study on the basis of its falling within the scope of FIFRA Section 10 (d) (1) (A), (B), or (C). Sponsor En-Joy Consumer Products Pvt Ltd

1/283, Shripuram Street


Tamil Nadu

Signature __________________________

Sponsor/Submitter Date ___________________________



STATEMENT OF COMPLIANCE

Study Title : Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats This study was conducted according to Good Laboratory Practice Principles as published by the OECD in 1998, N° 1 [ENV/MC/CHEM (98)17]. This study was conducted in accordance with the written study plan, authorized by the sponsor and SLS management, and following the standard operating procedures of SLS. There were no known circumstances that may have affected the quality or integrity of the data. Test Item characterization (identity, strength, purity and composition), stability, and method of synthesis and location of documents for the synthesis is the responsibility of the Sponsor All raw data, including any storage medium for electronically recorded data, documentation, the study plan, study plan amendments, the final report, and proportionate amount of the test item will be retained in the Archives at SLS. I accept responsibility for the conduct of the study and hereby declare that the study was performed under my direction according to the procedures described herein. This report represents a true and accurate record of the results obtained. Syed Nasir Ahamed, M.Sc Study Director Date:



STATEMENT OF QUALITY ASSURANCE UNIT

Study Title : Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats This study has been audited and the final report is examined with respect to the study plan, standard operating procedures and raw data. The report is a true reflection of the raw data. The audits were carried out according to the standard operating procedures of the Quality Assurance Unit of Sugen Life Sciences Pvt Ltd (SLS). Findings resulting from the audits were reported to the Study Director and the Management on the dates specified below. These reports are kept in the Archives at SLS. During the course of the study, the following areas were audited:

Audit

No

Details

Dates of Audit

Dates Reported to the

Study Director

Management

1 Draft Study Plan 29/12/2011 29/12/2011 29/12/2011

2 Dosing Activity 18/01/2012 18/01/2012 18/01/2012

3 Gross Pathology and Necropsy

01/02/2012 01/02/2012 01/02/2012

4 Draft Report 04/02/2012 04/02/2012 04/02/2012

K.S.V.P. Ratnam Quality Assurance Unit Date:



TABLE OF CONTENTS

TITLE PAGE ............................................................................................................................. 1 STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS ................................................. 2 STATEMENT OF COMPLIANCE ............................................................................................ 3 STATEMENT OF QUALITY ASSURANCE UNIT .................................................................. 4 TABLE OF CONTENTS............................................................................................................ 5 PERSONNEL INVOLVED IN THE STUDY ............................................................................ 7 LIST OF ABBREVIATIONS ..................................................................................................... 8 SUMMARY ............................................................................................................................... 9

1.0 INTRODUCTION ...................................................................................................... 10 1.1 Study Objective ....................................................................................................... 10 1.2 Study Guidelines ..................................................................................................... 10 1.3 Justification for Selection of Test System ................................................................ 10 1.4 Testing Facility and Study Period ............................................................................ 10 1.5 Archives .................................................................................................................. 10

2.0 EXPERIMENTAL PROCEDURE ............................................................................. 11 2.1 Test Item................................................................................................................. 11 2.2 Instruments and Equipments .................................................................................... 11 2.3 Solvent and Chemical .............................................................................................. 11 2.4 Animals ................................................................................................................... 11 2.5 Acclimatization ....................................................................................................... 12 2.6 Husbandry Practices ................................................................................................ 12 2.7 Animal Identification ............................................................................................... 12 2.8 Feed and Water ....................................................................................................... 12 2.9 Environmental Conditions ....................................................................................... 12 2.10 Experimental Activities............................................................................................ 14 2.11 Preparation of Dosing Solution................................................................................ 14 2.12 Dose Administration ................................................................................................ 14 2.13 Experimental Design ............................................................................................... 14 2.14 Observations ........................................................................................................... 14

3.0 RESULTS .................................................................................................................. 15 3.1 Mortalities ............................................................................................................... 15 3.2 Clinical Observations ............................................................................................... 15 3.3 Body Weights ......................................................................................................... 15 3.4 Terminal Studies ..................................................................................................... 15

4.0 CONCLUSION .......................................................................................................... 15 5.0 REFERENCES ........................................................................................................... 16

TABLE 1 - Mortality ................................................................................................................ 17 TABLE 2 - Mean Body Weights ............................................................................................... 18 APPENDIX 1 ........................................................................................................................... 19 Clinical Observations of Individual Animal ................................................................................ 19 APPENDIX 2 ........................................................................................................................... 20



Individual Animal Body Weight Data ........................................................................................ 20 APPENDIX 3 ........................................................................................................................... 21 Necropsy Findings of Individual Animal .................................................................................... 21 APPENDIX 4 ........................................................................................................................... 22 Classification ............................................................................................................................ 22 APPENDIX 5 ........................................................................................................................... 23 Record of Deviation from the Study Protocol............................................................................ 23 APPENDIX 6 ........................................................................................................................... 24 Feed Analysis Report…………………………………………………………………. ............... 26 APPENDIX 7……………………………………………………………………………………...25 Water Analysis Report………………………………………………………………………….....25 APPENDIX ………………………………………………………………………………….…...26 Certificate - Department of Science and Industrial Research……………………………………..26



PERSONNEL INVOLVED IN THE STUDY

Signature Study Director Syed Nasir Ahamed, M.Sc _________________

Study Personnel O. Lokanatha, M.Sc _________________

REPORT APPROVAL

This study report is approved by:

C. Sreenivasulu Reddy, B.Com Test Facility Management



LIST OF ABBREVIATIONS

CPCSEA Committee for Purpose of Control and Supervision of Experiments on Animals

IAEC Institutional Animal Ethics Committee

Mg Milligram

Kg Kilogram

B.wt. Body weight

GHS Globally Harmonised System

SLS Sugen Life Sciences Pvt Ltd

NA Not Available

NAD No Abnormalities Detected



SUMMARY

This study was performed to assess the acute oral toxicity of Dia Bliss Sugar supplied by Nityananda Sweets and Snacks in Wistar Rats. The method followed was as per the guideline of

OECD N° 423, "Acute Oral Toxicity–Acute Toxic Class Method”. The Organisation for Economic Co-operation and Development (OECD) guidelines for the Testing of Chemicals, adopted by the council on 17th December, 2001.

Wistar rats fasted overnight, were dosed with Dia Bliss Sugar dissolved in sterile distilled water as single oral gavage, using intubation cannula. The food was withheld until 4 hours of post dosing. The first group of three female rats was given a single dose of 2000 mg of Dia Bliss Sugar per kg body weight. No mortality was observed at this dose level; hence the Group II was administered at the same dose level. No mortality was observed at this dose level also. Hence, further testing was not essential (Appendix 4). The animal’s din’t show any clinical signs of toxicity when treated with 2000 mg of Dia Bliss Sugar per kg body weight. (Appendix1). At the end of 14 days observation period, all the rats were subjected to gross pathological examination (Appendix 1) and the symptoms were physiological variation in nature, hence there were no test item related changes. So, the test item is considered to be safe.

The acute oral median lethal dose LD50 (cut off value) of Dia Bliss Sugar in Wistar rats was found to be > 2000-5000 mg/kg body weight Dia Bliss Sugar is being classified as follows: Globally Harmonised System (GHS) : “Category 5”



1.0 INTRODUCTION

1.1 Study Objective

This objective of this study was to assess the acute oral toxicity (LD50) as a result of single dose administration of Dia Bliss Sugar in Wistar rats. The study was conducted in compliance with the OECD Principles of GLP (1998).

1.2 Study Guidelines

The present study was conducted following the guideline:

The Organisation for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, N° 423 (Adopted by the Council on 17th December, 2001) "Acute Oral Toxicity - Acute Toxic Class Method".

1.3 Justification for Selection of Test System

The rat (Rattus norvegicus) was selected as test system because it is a readily available rodent species. It has been historically shown to be a suitable model for acute oral toxicity assessment and is recommended by the OECD and other regulatory authorities. The results of the study are expected to be of value in predicting the acute toxicity of the test item in humans beings.

1.4 Testing Facility and Study Period

The study was performed at Sugen Life Sciences, Tirupati – 517 505, Andhra Pradesh, India, and all data generated and recorded during this study will be stored in the Archives at SLS

Study Initiation : December 29, 2011 Acclimatization Start : January 11, 2012 Experiment Start : January 18, 2012 Experiment Termination : February 02, 2012 Study Completion : February 07, 2012

1.5 Archives

All original raw data including any storage medium for electronically recorded data, documentation, the signed study plan, the draft report, a copy of the final report and a one gram sample of the test substance will be retained in the Archives at Sugen Life Sciences Pvt Ltd for a period of five years. At the end of this period, the Sponsor's instructions will be



sought for archiving period.

2.0 EXPERIMENTAL PROCEDURE

2.1 Test Item

Details of the test substance provided by the Sponsor

Test Item Name : Dia Bliss Sugar C.A. Name : NA CAS N° : NA Analysed Concentration : NA Batch/Lot N° : NA Supplied by : Nityananda Sweets and Snacks Date of Manufacture : NA Date of Expiry : NA Appearance : Crystal (White color) Storage Condition : Preserve in well closed container and away from moisture

2.2 Instruments and Equipments

Balance : Electronic Weighing Balance, TP4101DE (Capable of measuring 0.1 g to 4.1 kg) Electronic Balance, GE7101 (2.0g to 7.1Kg) Ohaus Micro Balance, PO214C (Capable of measuring 0.1 mg to 200.0 g) Intubation Cannula : Metal Cannula, Size 18 G x 5 cm, Syringe : “Top” Hypodermic syringe, Boro-silicate hard glass, Size 5 ml

2.3 Solvent and Chemical

Distilled water : Glass apparatus Disinfectant Solution : Labolene

2.4 Animals

Test Species : Rat (Rattus norvegicus) Strain : Wistar Animal Source : Sri Venkateswara Enterprises, Bangalore.



Total Number of Animals Used : 06 Females (nulliparous and non-pregnant) Body Weight (g) before Dosing : Minimum: 171.6, Maximum: 175.5. Age of the Animals : 8 to 10 weeks at the time of dosing.

2.5 Acclimatization

Rats were allowed to acclimatize to the experimental room conditions for a period of five days prior to randomization for group I and group-II prior to commencement of dosing. During the acclimatization period, the rats were observed daily twice for clinical signs of disease. Prior to randomization, a detailed physical examination was performed on all animals.

2.6 Husbandry Practices

Caging : Polypropylene rat cages covered with stainless steel grid top were used. Autoclaved clean paddy husk was used as the bedding material.

Water Bottle : Each cage was supplied with a polypropylene water bottle with a stainless steel nozzle

Housing : Three rats per cage Room Sanitation : Each day, the floor of the experiment room was swept and all work

tops and the floor were mopped with a disinfectant solution.

2.7 Animal Identification

Individual animal was identified with number marked on the tail using permanent marker pen and coloured cage label showing study number, study code, test item code, group number, number of animals, sex, species, strain, dose, cage number and animal number.

2.8 Feed and Water

The animals were provided ad libitum laboratory rodent pellet feed supplied by Provimi Animal Nutrition India Pvt. Ltd., Bangalore and charcoal filtered, UV sterilized water (Aqua guard water filter system). Fresh feed was supplied at least once a week and water bottles were refilled daily or whenever required.

2.9 Environmental Conditions

Rats were maintained in an environment-controlled room. The experimental room temperature and humidity were recorded daily. The temperatures recorded were in the range of 23.12 – 24.15 °C respectively. The relative humidity recorded was in the range of 54.43 – 61.69%. In the experimental room, 12 hours of artificial lighting and 12 hours darkness were



maintained, light hours being 6.00 to 18.00h.



2.10 Experimental Activities

The following observations/activities were performed during the experimental period:

Daily - animal observations - providing feed and water - floor sweeping and mopping - rack cleaned - recording room temperature and relative humidity Weekly - recording body weight

- recording photoperiod

2.11 Preparation of Dosing Solution

The test item was found to be soluble in sterile distilled water, so the actual dosing solution was prepared using sterile distilled water as vehicle. Required quantity of Dia Bliss Sugar was mixed in distilled water and the final volume was made up to 10 ml/kg. Gavage solutions were prepared freshly prior to dosing on all the occasions.

2.12 Dose Administration

A dose volume of 10 ml/kg body weight was maintained for each rat. All rats were dosed by gavage (day 0) using a metal cannula attached to a Boro-silicate hard glass syringe, which was graduated up to 5 ml. Rats were fasted overnight prior to dosing till four hours post-dosing.

2.13 Experimental Design

The Group I of three female rats was given a single dose of 2000 mg of Dia Bliss Sugar per kg body weight. No mortality was observed at this dose level, hence the group II was administered at the same dose level, no mortality was observed at this dose level hence, further testing was not essential (Table 1 and Appendix 4).

2.14 Observations

The rats were observed for signs of toxicity and mortality at specific time points for 30 minutes, 1, 2, 3, and 4 hours post dosing on the day of dosing (Appendix 1). Subsequently, the rats were observed twice a day for morbidity and mortality for a period of 14 days following oral dosing (Table 1). The clinical signs were recorded twice a day. Individual body



weights were recorded prior to dosing on days 0, 7 and 14 (Appendix 2).

3.0 RESULTS

3.1 Mortalities

No mortality was observed in the rats from group I and from Group II treated at 2000mg of Dia Bliss Sugar per kg B.wt. (Table 1).

3.2 Clinical Observations

No behavioral changes were observed as clinical sign in the rats from group I and group II treated with Dia Bliss Sugar per kg B.wt between day 0 to day 14. All rats survived and appeared normal from post dosing till to the termination of the experiment (Appendix 1).

3.3 Body Weights

Positive gain in body weights were recorded in all the rats treated with Dia Bliss Sugar at the dose level of 2000 mg /kg body weight (Appendix 2).

3.4 Terminal Studies

External

External examination of rats sacrificed terminally did not reveal any lesion of pathological significance (Appendix 3).

Internal

Visceral examination of rats sacrificed terminally did not show any lesions when treated with Dia Bliss Sugar at the dose level of 2000 mg/kg b.wt.

4.0 CONCLUSION

The acute oral median lethal dose LD50 (cut off value) of Dia Bliss Sugar in Wistar rats was found to be > 2000 - 5000 mg/kg body weight

Dia Bliss Sugar is being classified as follows:

Globally Harmonised System (GHS) : “Category 5”



5.0 REFERENCES

5.1 OECD, 1998 : OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, Number 1, “OECD Principles on Good Laboratory Practice” ENV/MC/CHEM(98)17 (as revised in 1997).

5.2 OECD, 2001 : OECD N° 423, "Acute Oral Toxicity – Acute Toxic Class Method”. The Organisation for Economic Co-operation and Development (OECD) Guidelines for the Testing of Chemicals, Adopted by the Council on 17th December 2001.



Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

TABLE 1 - Mortality

F- Female h- Hours

0- No Mortality

Group Dose

(mg/kg)

Animal No

& Sex 1 h 2 h 3 h 4 h 24 h Day 1-7 Day 8-14

I

II

2000.0 1F-3F 0 0 0 0 0 0 0

2000.0 4F-6F 0 0 0 0 0 0 0




TABLE 2 - Mean Body Weights

Group Dose (mg/kg)

Animal No & Sex

Mean body weight (g)

Day 0 Day 7 Day 14

I

II

2000.0 1F-3F 172.90±1.12 178.73±0.83 185.96±1.26

2000.0 4F-6F 173.47±1.79 180.67±1.72 187.10±2.23

Percent Mean Body Weight Changes

Group Dose (mg/kg)

Animal No & Sex

Percentage Mean body weight Change on

Day 7 Day 14

I 300.0 1F-3F 3.4±0.2 7.6±0.2

II 50.0 4F-6F 4.2±0.1 7.9±0.7

F- Female



Acute Oral Toxicity Study of Dia Bliss Sugar in Rats

APPENDIX 1

Clinical Observations of Individual Animal

Group – I

Animal N° & Sex

Dose Level

(mg/kg)

Clinical Signs Observed Post Dosing

At Hour (Day 0) On Day

0 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1F

2000.0

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2F 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3F 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Group – II

Animal N° & Sex

Dose Level

(mg/kg)

Clinical Signs Observed Post Dosing

At Hour (Day 0) On Day

0 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1F

2000.0

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2F 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3F 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 = Normal




APPENDIX 2

Individual Animal Body Weight Data

Group Animal No & Sex

Dose (mg/kg)

Body weight (g)

% Body Weight Change on Day

% Body Weight Change on Day

Day 0 Day 7 Day 14 0 – 7 7 – 14

I

1F

2000.0

171.6 177.8 184.6 3.6 7.6

2F 173.5 179 186.2 3.2 7.3

3F 173.6 179.4 187.1 3.3 7.8

II

4F

2000.0

172.1 179.3 184.6 4.2 7.3

5F 172.8 180.1 187.8 4.2 8.7

6F 175.5 182.6 188.9 4.0 7.6




APPENDIX 3

Necropsy Findings of Individual Animal

Group N°

Animal No & Sex

Dose (mg/kg)

Mode of Death External Internal

I

II

1F

2000.0

TS NAD NAD

2F TS NAD NAD

3F TS NAD NAD

4F

2000.0

TS NAD NAD

5F TS NAD NAD

6F TS NAD NAD

NAD- No Abnormalities Detected; TS: Terminal Sacrifice




APPENDIX 4

Classification

Test Procedure with a Starting Dose of 2000 mg/kg Body Weight (Source: OECD Guideline N° 423)


APPENDIX 5

Record of Deviation from the Study Protocol It is declared that there were no study plan deviations during conduct of the study.




APPENDIX 6

Feed Analysis Report




APPENDIX 7

Water Analysis Report




APPENDIX 9

Certificate – Department of Science and Industrial Research

Appendix 3: SUB-ACUTE ORAL TOXICITY STUDY OF DIABLISS

SUGAR IN WISTAR RATS

60

SLS Study Number: SLS/042-11


CONFIDENTIAL TITLE PAGE

REPORT

28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

DATA REQUIREMENT GUIDELINE: OECD N° 407

Study Director: Syed Nasir Ahamed, M.Sc

Report Submission: 01/12/2011

SPONSOR

En-Joy Consumer Products Pvt Ltd



Tamil Nadu

TESTING FACILITY

Sugen Life Sciences Pvt. Ltd #4/86, S.V. Nagar

Perumalla palli (post) Tirupati – 517 505

Andhra Pradesh India



STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information in this study on the basis of its falling within the scope of FIFRA Section 10 (d) (1) (A), (B), or (C). Sponsor En-Joy Consumer Products Pvt Ltd



Tamil Nadu

Signature ____________________ Sponsor/Submitter Date _____________________



STATEMENT OF COMPLIANCE

Study Title : 28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in

Wistar Rats

This study was conducted in accordance with the written study plan, authorized by the sponsor and SLS management, and following the standard operating procedures of SLS. There were no known circumstances that may have affected the quality or integrity of the data. All raw data, including any storage medium for electronically recorded data, documentation, the study plan, study plan amendments, the final report, slides and proportionate amount of the test item will be retained SLS Archives. I accept responsibility for the conduct of the study and hereby declare that the study was performed under my direction according to the procedures described herein. This report represents a true and accurate record of the results obtained. Syed Nasir Ahamed, M.Sc Study Director



STATEMENT OF QUALITY ASSURANCE UNIT

Study Title : 28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in

Wistar Rats This study has been audited and the final report was examined with respect to the study plan, standard operating procedures and raw data. The report is a true reflection of the raw data. The audits were carried out according to the standard operating procedures of the Quality Assurance Unit of Sugen Life Sciences (SLS). Findings resulting from the audits were reported to the Study Director and the Management on the dates specified below. These reports are kept in the Archives at SLS. During the course of the study, the following areas were audited:

Audit

No Details Dates of Audit

Dates Reported to the

Study Director

Management

1 Randomization, Body Weight, Dosing 18/10/2011 18/10/2011 18/10/2011

2 Dosing, Body Weight and Food Consumption

24/10/2011 24/10/2011 24/10/2011

3 Necropsy, Clinical Chemistry 15/11/2011 15/11/2011 15/11/2011

4 Draft Report 30/11/2011 30/11/2011 30/11/2011

K.S.V.P. Ratnam Quality Assurance Unit Date:



TABLE OF CONTENTS

TITLE PAGE........................................................................................................................................ 1 STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS ..................................................... 2 STATEMENT OF COMPLIANCE .................................................................................................... 3 STATEMENT OF QUALITY ASSURANCE UNIT........................................................................ 4 Table of Contents ................................................................................................................................. 5 PERSONNEL INVOLVED IN THE STUDY ................................................................................... 7 LIST OF ABBREVIATIONS ............................................................................................................. 8 SUMMARY........................................................................................................................................ 10

1.0 INTRODUCTION ................................................................................................................ 11 1.1 Study Objective ............................................................................................................... 11 1.2 Guideline ......................................................................................................................... 11 1.3 Justification for Selection of the Test System ............................................................... 11 1.4 Testing Facility and Study Period .................................................................................. 11

2.0 EXPERIMENTAL PROCEDURE ...................................................................................... 11 2.1 Test Item .......................................................................................................................... 11 2.2 Animals ............................................................................................................................ 12 2.3 Acclimatization ............................................................................................................... 12 2.4 Environmental Conditions .............................................................................................. 12 2.5 Housing ............................................................................................................................ 12 2.6 Feed and Water................................................................................................................ 12 2.7 Grouping .......................................................................................................................... 13 2.8 Animal Identification ...................................................................................................... 13 2.9 Experimental Outline ...................................................................................................... 13 2.10 Route of Administration ................................................................................................. 13 2.11 Duration of Treatment .................................................................................................... 13 2.12 Selection of Vehicle ........................................................................................................ 13

3.0 OBSERVATIONS .............................................................................................................. 14 3.1 Clinical Signs .................................................................................................................. 14 3.2 Body Weight.................................................................................................................... 14 3.3 Food Consumption .......................................................................................................... 14 3.4 Clinical Pathology Observations .................................................................................... 14 3.5 Gross Pathology .............................................................................................................. 15 3.6 Evaluation of Results ...................................................................................................... 15

4.0 ARCHIVES ......................................................................................................................... 16 5.0 RESULTS AND DISCUSSION ........................................................................................ 16

5.1 Mortalities ........................................................................................................................ 16 5.2 Clinical Observations ...................................................................................................... 16 5.3 Body Weight.................................................................................................................... 16 5.4 Food Consumption .......................................................................................................... 16 5.5 Haematology ................................................................................................................... 16 5.6 Clinical Chemistry .......................................................................................................... 16 5.7 Organ Weight .................................................................................................................. 17 5.8 Relative Organ Weight ................................................................................................... 17 5.9 Gross Pathological Findings ........................................................................................... 17 5.10 Histopathology ................................................................................................................ 17



6.0 CONCLUSION .................................................................................................................. 17 7.0 REFERENCES .................................................................................................................... 18 TABLE 1 - Clinical Signs – Summary by Group ........................................................................ 19 TABLE 2 - Body Weight (g) - Group Mean Values .................................................................... 20 TABLE 3 - Food Consumption (g) - Group Mean Values .......................................................... 22 TABLE 4 - Hematology - Group Mean Values ........................................................................... 24 TABLE 5 - Clinical Chemistry - Group Mean Values ................................................................ 26 TABLE 6 - Organ Weights (g) - Group Mean Values ................................................................. 28 TABLE 7 - Relative Organ Weights (%) - Group Mean Values ................................................ 30 TABLE 8 - Gross Pathology Findings – Summary by Group ..................................................... 32

APPENDIX – A ................................................................................................................................. 33 Clinical Signs of Individual Animals ........................................................................................ 33

APPENDIX – B ................................................................................................................................. 35 Individual Animal Body Weight (g) ......................................................................................... 35

APPENDIX – C ................................................................................................................................. 37 Food Consumption (g/cage)....................................................................................................... 37

APPENDIX – D ................................................................................................................................. 39 Individual Animal Haematological Values ............................................................................... 39

APPENDIX – E .................................................................................................................................. 43 Individual Animal Clinical Chemistry Values ......................................................................... 43

APPENDIX – F .................................................................................................................................. 47 Individual Animal Organ Weights (g) ...................................................................................... 47

APPENDIX – G ................................................................................................................................. 51 Individual Animal Relative Organ Weights (%) ...................................................................... 51

APPENDIX – H ................................................................................................................................. 55 Gross and Microscopic Findings of Individual Animals ......................................................... 55

APPENDIX – I ................................................................................................................................... 59 Feed Analysis Report ................................................................................................................. 59

APPENDIX – J ................................................................................................................................... 60 Water Analysis Report ............................................................................................................... 60

APPENDIX – L .................................................................................................................................. 61 Certificate – Department of Science and Industrial Research ................................................. 61



PERSONNEL INVOLVED IN THE STUDY

Signature

Study Director Syed Nasir Ahamed, M.Sc _________________

Study Personnel O. Lokanatha, M.Sc _________________

Pathology K. Poorani, M.V.Sc _________________

Clinical Chemistry S. Mamatha, M.Sc _________________

Statistical Evaluation I. Nagaraju, M.Sc _________________

REPORT APPROVAL

This study report is approved by:

C. Sreenivasulu Reddy, B.Com. Test Facility Management



LIST OF ABBREVIATIONS

AD - Average Dose ALB - Albumin ALP - Alkaline Phosphatase B.wt - Body weight Cfu - colony forming units CPCSEA - Committee for Purpose of Control and Supervision of Experiments on

Animals CR - Creatinine dl - Deciliter EDTA - Ethylene Diamine Tetra Acetic Acid F - Female F.C - Food Consumption fl - Femtolitre g - Grams Hb - Hemoglobin HCT - Haematocrit HD - High Dose IAEC - Institutional Animal Ethics Committee IU/l - International Unit per Litre kg - Kilogram M - Male MCH - Mean Corpuscular Hemoglobin MCHC - Mean Corpuscular Hemoglobin Concentration MCV - Mean Corpuscular Volume mg - Milligrams mm3 - cubic millimeter N° - Number NAD - No Abnormalities Detected pg - Pictogram PLT - Platelet Count ppb - parts per billion RBC - Red Blood Cells SD - Standard Deviation SGOT - Serum Glutamate Oxaloacetate Transaminase SGPT - Serum Glutamate Pyruvate Transaminase SLS - Sugen Life Sciences TABC - Total Aerobic Bacterial Count TD - Therapeutic Dose



LIST OF ABBREVIATIONS (Continued) TI - Test Item TLC - Thin Layer Chromatography TP - Total Protein VC - Vehicle Control WBC - White Blood Cells % - Percent µ - Micron



SUMMARY This study was conducted to assess the toxic effects of Dia Bliss Sugar in Wistar Rats, when administered orally for a period of 28 consecutive days. The methods followed were as per the guideline:

The Organization for Economic Co-operation and Development (OECD) for Testing of Chemicals No 407, entitled "Repeated Dose 28-day Oral Toxicity Study in Rodents", adopted on July 27, 1995.

Three groups of Rats comprising 6 male and 6 females per group were treated with Dia Bliss Sugar at 180.0, 540.0 and 1800.0 mg/kg bd. wt. for a period of 28 consecutive days. A concurrent control group, comprising the same number of animals was also maintained in the study.

Rats were observed individually for visible signs of reaction to the treatment twice a day throughout the study period. Individual body weight and feed consumption were monitored weekly. Haematological and biochemical analyses of blood samples were performed on all animals at the end of the treatment period. All the surviving rats were sacrificed and subjected to a gross pathological examination at the end of treatment period. Absolute organ weights were recorded and relative organ weights were calculated for the organs viz., liver, kidneys, adrenals, testis/ovary, epididymides/uterus, thymus, spleen, brain and heart in all Rats.

The animals exposed to test item did not show any specific treatment related clinical changes in all the treated groups. No significant changes were observed in the mean body weight and food consumption in any of the treated group animals.

Haematological analysis and clinical chemistry analysis did not reveal any treatment related changes in any of the parameter at various dose levels studied in either sex.

The absolute and relative organ weights did not reveal any significant difference in the treated groups’ animals when compared with the control group animals in either sex. Gross pathological examination of the animals did not reveal any treatment related effects in any of the dose group. Histopathological examination of the high dose group animals did not reveal any treatment related effects compared to control group animals. There was no test item related toxicity recorded in any of the treatment group under the experimental conditions.



1.0 INTRODUCTION

1.1 Study Objective

The objective of this study was to determine the adverse effects occurring as a result of the repeated oral administration of Dia Bliss Sugar in Wistar Rats for a period of 28 consecutive days.

1.2 Guideline

The study was conducted following the guideline:

The Organisation for Economic Co-operation and Development (OECD) for Testing of Chemicals No 407, entitled "Repeated Dose 28-day Oral Toxicity Study in Rodents", adopted on July 27, 1995.

1.3 Justification for Selection of the Test System

Rats were selected as test system because it is a readily available laboratory rodent species. It has been historically shown to be a suitable model for repeated dose toxicity assessment and is recommended by the OECD and other regulatory authorities. The results of the study are believed to be of value in predicting the toxicity of the test item in human beings.

1.4 Testing Facility and Study Period

The study was performed at Sugen Life Sciences, Tirupathi – 517 505, Andhra Pradesh, India, and all data generated and recorded during this study will be stored SLS Archives.

Study Initiation : 08 October, 2011 Acclimatization Start : 13 October, 2011 Experiment Start : 18 October, 2011 Date of Sacrifice : 15 November, 2011 Study Completion : 01 December, 2011

2.0 EXPERIMENTAL PROCEDURE

2.1 Test Item

Details of the test item as provided by the sponsor are: Test Item Name : Dia Bliss Sugar Supplied by : Nityananda Sweets and Snacks, Shripuram Manufactured by : Nityananda Sweets and Snacks, Shripuram Date of Manufacture : NA Appearance : Small white crystals.



Solubility : Slightly soluble in water Storage Condition : At room Temperature

2.2 Animals

24 male and 24 female healthy, young Rats (Rattus norvegicus) of Wistar strain, obtained from CPCSEA approved vendor, were used for the study. Female Rats were nulliparous and non-pregnant. All animals were examined for good health at the time of receipt. Age of the animals at the start of treatment was approximately 9 weeks.

2.3 Acclimatization

Rats were allowed to acclimatize to the experimental room conditions for a period of five days prior to randomization. During the acclimatization period, the rats were observed daily twice for clinical signs of disease. Prior to randomization, a detailed physical examination was performed on all animals.

2.4 Environmental Conditions

Rats were maintained in an environment-controlled room. The experimental room temperature and humidity were recorded daily. The temperatures recorded were in the range of 22.0 – 25.0 °C respectively. The relative humidity recorded was in the range of 42.6 – 56.73%. In the experimental room, 12 hours of artificial lighting and 12 hours darkness were maintained, light hours being 6.00 to 18.00h. The experimental room was cleaned and mopped with a disinfectant daily.

2.5 Housing

The animals were housed in group of two of same sex per cage in solid floor polypropylene Rats cages. Each cage was fitted with a stainless steel top grill and a polypropylene water bottle with stainless steel drinking nozzle. The bottom of the cage was layered with clean, sterilized paddy husk. The cages were kept on 5 tier racks and their positions were rotated weekly.

2.6 Feed and Water

The animals were provided laboratory rodent pellet feed supplied by Provimi Animal Nutrition India Pvt. Ltd., Bangalore and charcoal filtered, UV sterilized water (Aqua guard water filter system) ad libitum. Fresh feed was supplied at least once a week and water bottles were refilled daily or whenever required. Feed, drinking water and paddy husk were analyzed for microbial contaminants at six-month intervals and the results of the recent analyses are furnished in the appendix (Appendices I and J).



2.7 Grouping

The animals were allocated to different groups using the principle of randomization. The animals were equally distributed to four groups viz., control (G1), therapeutic dose (G2), average dose (G3) and high dose (G4). Each group was comprised of 6 male and 6 female rats. At the time of treatment commencement, the body weight variation among the animals was within ± 20% of the mean body weight for each sex. Individual body weights of the animals ranged between 164.8 to 204.9 g for males and152.0 to 168.0 g for females at the time of treatment commencement.

2.8 Animal Identification

Individual animal was identified with number marked on the tail using permanent marker pen and coloured cage label showing study N°, study code, test item code, group N°, N° of animals, sex, species, strain, dose, cage N° and animal N°.

2.9 Experimental Outline

Group N° Group

Dose (mg/kg

b.wt./day)

Number of Rats (Males + Females)

Treated Clinical Pathology Histo-pathology

G1

G2

G3

G4

Control

Therapeutic dose

Average dose

High dose

0.0

180.0

540.0

1800.0

6 +6

6 +6

6 +6

6 +6

6 +6

6 +6

6 + 6

6 + 6

6 + 6

-

-

6 + 6

2.10 Route of Administration

The route of administration was oral through oral gavage. The oral administration represents a possible route of human exposure and was agreed by the study sponsor.

2.11 Duration of Treatment

Animals were administered with Dia Bliss Sugar mixed with sterile distilled water for a period of 28 days.

2.12 Selection of Vehicle

Vehicle was selected based on the solubility of the test item. Dia Bliss Sugar being a water soluble test item, water was used as vehicle.



3.0 OBSERVATIONS

3.1 Clinical Signs

Animals were observed for mortality and morbidity twice a day. All visible signs and symptoms such as skin and fur changes, eye and mucous membrane changes, respiratory, behavioral pattern and general changes were recorded twice a day.

3.2 Body Weight

Individual body weight was recorded for all animals on the day of commencement of treatment and at weekly intervals thereafter.

3.3 Food Consumption

The weekly food consumption of the animals was calculated through out the study.

3.4 Clinical Pathology Observations

Clinical pathological tests were conducted on blood samples collected from all the surviving animals at the end of the treatment period. Animals were deprived of food overnight and blood samples were collected by puncturing the orbital sinus plexus with the help of a fine capillary tube.

Around 0.8 ml of blood was collected in vials containing EDTA for haematology analysis.

Two to three ml blood was collected from each animal in clean centrifuge tubes. The blood was allowed to clot at room temperature and the serum was separated by centrifugation at low speed. The serum thus separated was used for all clinical chemistry analyses.

Clinical pathology parameters studied and instrument used are given below: ---------------------------------------------------------------------------------------------------------------- Parameter Sample Type Instrument Used ----------------------------------------------------------------------------------------------------------------

Haematology Leucocyte count (WBC) Whole blood Mindray BC-2800Vet Erythrocyte count (RBC) Whole blood Mindray BC-2800 Vet Haemoglobin (Hb) Whole blood Mindray BC-2800 Vet Haematocrit (HCT) Whole blood Mindray BC-2800 Vet Mean Corpuscular Volume (MCV) Whole blood Mindray BC-2800 Vet Mean Corpuscular Haemoglobin (MCH) Whole blood Mindray BC-2800 Vet Mean Corpuscular Haemoglobin Concentration Whole blood Mindray BC-2800 Vet (MCHC) Platelet (Plt) Whole blood Mindray BC-2800 Vet ----------------------------------------------------------------------------------------------------------------



---------------------------------------------------------------------------------------------------------------- Parameter Sample Type Instrument Used ---------------------------------------------------------------------------------------------------------------- Clinical Chemistry

Alkaline Phosphatase (ALP) Serum Merck – Microlab 300 Albumin Serum Merck – Microlab 300 Triglycerides Serum Merck – Microlab 300 Creatinine Serum Merck – Microlab 300 Glucose Serum Merck – Microlab 300 Serum Glutamate Oxaloacetate Transaminase Serum Merck – Microlab 300 (SGOT) Serum Glutamate Pyruvate Transaminase Serum Merck – Microlab 300 (SGPT) Total protein Serum Merck – Microlab300 Urea Serum Merck – Microlab 300

---------------------------------------------------------------------------------------------------------------- 3.5 Gross Pathology

All the animals were euthanized using anesthetic ether and subjected to a complete necropsy under the direct supervision of the veterinary pathologist at the end of the treatment period. The animals were examined carefully for external abnormalities before the necropsy. The thoracic, abdominal and cranial cavities were then cut open and thorough examinations of the organs were carried out to detect changes or abnormalities, if any. Absolute weights of adrenals, brain, uterus, ovaries, testes, epididymides, heart, kidneys, liver, spleen and thymus were recorded immediately after dissection of all animals. Paired organs were weighed together. Relative weights of these organs were calculated later.

3.6 Evaluation of Results

Raw data were processed by the Department of Statistics and Systems Management, Sugen Life Sciences to give group means and standard deviations for comparison between the control and treated groups, using SPSS statistical software. All the parameters characterized as continuous data such as, body weight, feed consumption, organ weight, relative organ weight, hematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. Where the data did not meet the homogeneity of variance, Kruskal-Wallis test was performed to calculate the significance.



4.0 ARCHIVES

All raw data, the signed study plan, study plan amendment, a copy of the final report, slides and a proportionate amount of the test item will be retained in the Archives at Sugen Life Sciences for a period of five years. At the end of this period, the sponsor's instructions will be sought to either extend the archiving period or return the archived material to the sponsor or for the material to be disposed off.

5.0 RESULTS AND DISCUSSION

5.1 Mortalities

No mortality was observed in rats exposed to test item at 180.0, 540.0 and there was mortality observed at 1800.0 mg/kg bd. wt. orally for a period of 28 days , but the mortality was not due to test item toxicity.

5.2 Clinical Observations

No treatment related clinical signs were recorded in any of the animal administered with different doses of Dia Bliss Sugar as compared to control group animals in either sex. (Table 1, Appendix A)

5.3 Body Weight

No statistical significant variation was observed in the average weekly body weight of animals treated with Dia Bliss Sugar as compared to control animals while in female animals also, no such variation was observed. (Table 2; Appendix B)

5.4 Food Consumption

No significant difference was observed in the average food consumption in treated group animals of either sex when compared to the control group animals. (Table 3; Appendix C)

5.5 Haematology

No significant difference was observed in all the heamatological parameters of the animals administered with different doses of Dia Bliss Sugar as compared to the control group animals in either sex. (Table 4; Appendix D)

5.6 Clinical Chemistry

No significant difference was observed in all the clinical chemistry parameters of the animals treated with different doses of Dia Bliss Sugar as compared to the control group animals in either sex. All the clinical chemistry parameters were found to be within the normal range. (Table 5; Appendix E)



5.7 Organ Weight

No significant difference was observed in the absolute organ weights of the animals treated with different doses of Dia Bliss Sugar as compared to the control group animals in either sex. (Table 6; Appendix F)

5.8 Relative Organ Weight

No significant difference was observed in the relative organ weights of the animals treated with different doses of Dia Bliss Sugar as compared to the control group animals in females while a low variation was observed in Heart and Thymus in males. (Table 7; Appendix G)

5.9 Gross Pathological Findings

5.9.1 External

The external examination of the carcasses of all animals did not reveal any changes.

5.9.2 Internal

After the external examination, the carcass was cut open and examined for gross pathology of visceral organs. The lobes of the liver were examined for changes and collected. The spleen, pancreas were examined for changes. The small intestine and large intestine were observed and samples of jejunum, ileum, cecum, colon and rectum were collected. The kidneys were removed and examined for changes. The larynx, trachea, esophagus, thymus and thyroids were collected along with lungs. The brain was collected and examined. No significant lesions or abnormalities like changes in size, color, congestion, hemorrhage, inflammation and necrosis were detected in all the organs. Gross examination at necropsy did not reveal any Dia Bliss Sugar treatment related abnormalities.

5.10 Histopathology

The animals treated with Dia Bliss Sugar at dose level of 1800.0 mg/kg bd. wt did not reveal any histopathological changes in either sex as compared to control group animals. Results of the histopathological examination are shown in Table 8 and Appendix H.

6.0 CONCLUSION

Wistar Rats treated with Dia Bliss Sugar orally for 28 days at various dose levels did not reveal any treatment related adverse/toxic effects under the experimental conditions.



7.0 REFERENCES

7.1 Physician’s Desk Reference, 1999, 53rd ed., 1667-76

7.2 Martindale, 32nd ed., K. Parfitt ed., 44.

7.3 Paget and Barnes (1964): Evaluation of drug activities Pharma Eds. Laurance and Bacharach, Vol.1, Academic Press, New York.

7.4 OECD Guidelines for Testing of Chemicals, No 407 entitled, "Repeated Dose 28-day Oral Toxicity Study in Rodents", adopted on July 27, 1995.




TABLE 1 - Clinical Signs – Summary by Group

Normal Day of Observation

0 7 14 21 28

Male

G1 6 6 6 6 6

G2 6 6 6 6 6

G3 6 6 6 6 6

G4 6 6 5 5 5

Female

G1 6 6 6 6 6

G2 6 6 6 6 6

G3 6 6 6 6 6

G4 6 6 6 6 6 Note: Number of animals with clinical sign at least once during the week interval.




TABLE 2 - Body Weight (g) - Group Mean Values

Sex: Male

Day N°

G1 G2 G3 G4

Mean SD N Mean SD N Mean SD N Mean SD N

0 174.18 17.41 6 166.28 13.41 6 176.08 10.19 6 177.93 17.43 6

7 208.32 18.10 6 202.47 17.88 6 199.07 19.53 6 198.13 27.71 6

14 226.82 24.15 6 224.55 19.53 6 218.25 16.54 6 227.54 20.10 5

21 238.23 23.96 6 233.30 20.08 6 226.98 17.22 6 237.52 21.41 5

28 247.08 25.32 6 243.45 20.34 6 235.47 17.77 6 245.96 21.92 5

Average Body Weight - Male

Average Body Weight- Male

140.00

160.00

180.00

200.00

220.00

240.00

260.00

280.00

300.00

Day 0 Day 7 Day 14 Day 21 Day 28Days

Ave

rage

Foo

d C

onsu

mpt

ion

(g)

G1G2G3G4



Table 2 - Body Weight (g) - Group Mean Values (Continued)

Sex: Female

Day N°

G1 G2 G3 G4


0 159.57 5.20 6 156.52 3.52 6 158.02 6.51 6 158.47 5.04 6

7 183.55 6.67 6 179.88 6.33 6 188.75 12.57 6 182.60 5.83 6

14 191.02 7.23 6 186.22 7.69 6 196.93 17.20 6 189.37 5.77 6

21 195.73 7.95 6 190.43 8.40 6 202.77 21.52 6 194.43 6.14 6

28 204.27 7.87 6 197.73 8.23 6 210.35 21.55 6 201.73 8.97 6

Average Body Weight – Female

Average Food Consumption- Females

120.00

140.00

160.00

180.00

200.00

220.00

240.00

260.00

280.00

Day 0 Day 7 Day 14 Day 21 Day 28

Days

Ave

rage

Foo

d Co

nsum

ptio

n (g

)

G1G2G3G4




TABLE 3 - Food Consumption (g) - Group Mean Values

Sex: Male

Day N°

G1 G2 G3 G4


7 206.17 8.84 6 215.37 12.51 6 222.07 12.09 6 223.87 11.39 6

14 258.80 35.51 6 247.20 36.03 6 247.63 20.08 6 220.30 11.59 5

21 237.93 23.48 6 231.87 17.22 6 253.20 5.66 6 214.60 12.10 5

28 246.23 25.52 6 239.20 19.79 6 261.07 6.30 6 217.90 15.34 5

Average Food Consumption – Male

Average Food Consumption- Males

150.00

190.00

230.00

270.00

310.00

7 14 21 28Days

Aver

age

Food

Con

sum

ptio

n (g

)

G1G2G3G4



TABLE 3 - Food Consumption (g) - Group Mean Values (Continued)

Sex: Female

Day N°

G1 G2 G3 G4


7 165.07 3.70 6 165.53 7.71 6 162.47 1.88 6 149.33 4.09 6

14 180.97 1.46 6 179.10 9.81 6 176.80 1.44 6 172.83 6.62 6

21 195.37 3.39 6 188.20 5.80 6 187.83 3.39 6 181.17 6.67 6

28 204.00 2.21 6 200.97 5.06 6 197.83 6.31 6 193.93 7.26 6

Average Feed Consumption – Female

Average Food Consumption-Females

100.00

120.00

140.00

160.00

180.00

200.00

220.00

240.00

260.00

7 14 21 28Days

Ave

rage

Foo

d C

onsu

mpt

ion

(g)

G1G2G3G4




TABLE 4 - Hematology - Group Mean Values

Sex: Male

Parameter G1 G2

Mean SD N Mean SD N

RBC (1x106/mm3) 7.29 0.46 6 7.12 0.92 6

WBC (1x103/mm3) 12.77 1.23 6 14.00 1.37 6

Hb (g/dl) 16.55 1.72 6 16.93 1.15 6

HCT (%) 38.40 5.38 6 40.37 1.28 6

MCV (um3) 50.02 1.17 6 50.65 2.02 6

MCH (pg) 25.65 1.34 6 25.17 1.44 6

MCHC (g/dl) 54.17 1.42 6 54.33 1.35 6

Plt (1x103/mm3) 393.15 35.44 6 401.63 73.40 6

Parameter G3 G4

Mean SD N Mean SD N

RBC (1x106/mm3) 6.90 0.53 6 7.79 0.52 5

WBC (1x103/mm3) 13.40 1.24 6 13.96 1.39 5

Hb (g/dl) 17.78 0.90 6 16.49 0.71 5

HCT (%) 40.57 1.52 6 40.38 1.25 5

MCV (um3) 51.42 1.04 6 49.56 1.25 5

MCH (pg) 24.73 0.89 6 25.50 0.95 5

MCHC (g/dl) 55.53 1.59 6 54.20 1.91 5

Plt (1x103/mm3) 373.58 72.48 6 387.72 54.34 5



TABLE 4 - Hematology - Group Mean Values (Continued)

Sex: Female

Parameter G1 G2

Mean SD N Mean SD N

RBC (1x106/mm3) 6.65 0.64 6 6.60 0.74 6

WBC (1x103/mm3) 12.84 0.94 6 14.31 1.53 6

Hb (g/dl) 16.00 1.49 6 16.08 1.47 6

HCT (%) 40.35 1.26 6 39.57 1.13 6

MCV (um3) 49.77 1.19 6 50.18 1.55 6

MCH (pg) 25.52 1.08 6 24.93 0.97 6

MCHC (g/dl) 55.20 1.06 6 55.47 1.08 6

Plt (1x103/mm3) 401.13 34.44 6 370.38 67.16 6

Parameter G3 G4

Mean SD N Mean SD N

RBC (1x106/mm3) 6.89 0.84 6 6.87 0.77 6

WBC (1x103/mm3) 15.23 2.03 6 14.35 1.11 6

Hb (g/dl) 15.93 1.25 6 16.48 1.27 6

HCT (%) 39.77 1.73 6 40.28 1.19 6

MCV (um3) 50.05 1.57 6 50.52 1.26 6

MCH (pg) 25.58 1.03 6 25.30 0.76 6

MCHC (g/dl) 54.15 0.72 6 55.35 1.68 6

Plt (1x103/mm3) 385.20 26.49 6 390.27 44.32 6




TABLE 5 - Clinical Chemistry - Group Mean Values

Sex: Male

Parameter G1 G2

Mean SD N Mean SD N

Glucose (mg/dl) 98.00 11.87 6 79.00 11.51 6

SGOT (IU/l) 48.50 1.87 6 49.00 2.00 6

SGPT (IU/l) 31.17 3.82 6 30.00 4.05 6

ALP (IU/l) 312.58 12.89 6 292.00 10.47 6

Total Protein (g/dl) 6.00 0.41 6 6.13 0.37 6

Albumin (g/dl) 4.45 0.39 6 4.65 0.43 6

Triglycerides (mg/dl) 65.00 9.61 6 62.83 5.31 6

Creatinine (mg/dl) 0.57 0.24 6 0.57 0.24 6

Urea (mg/dl) 42.17 2.56 6 42.67 3.33 6 .

Parameter G3 G4

Mean SD N Mean SD N

Glucose (mg/dl) 98.80 8.01 6 64.20 20.73 5

SGOT (IU/l) 48.33 3.01 6 48.00 3.54 5

SGPT (IU/l) 33.83 5.38 6 36.40 3.36 5

ALP (IU/l) 302.92 17.40 6 322.52 15.93 5


Albumin (g/dl) 4.43 0.23 6 4.42 0.18 5


Creatinine (mg/dl) 0.53 0.25 6 0.54 0.24 5

Urea (mg/dl) 42.33 2.07 6 43.60 3.51 5



TABLE 5 - Clinical Chemistry - Group Mean Values (Continued)

Sex: Female

Parameter G1 G2

Mean SD N Mean SD N

Glucose (mg/dl) 91.50 8.38 6 93.17 13.70 6

SGOT (IU/l) 42.67 2.58 6 47.83 2.79 6

SGPT (IU/l) 40.50 3.56 6 38.33 3.44 6

ALP (IU/l) 306.80 26.77 6 304.48 12.33 6


Albumin (g/dl) 4.40 0.24 6 4.20 0.23 6


Creatinine (mg/dl) 0.43 0.18 6 0.43 0.20 6

Urea (mg/dl) 37.67 4.08 6 39.17 2.64 6

Parameter G3 G4

Mean SD N Mean SD N

Glucose (mg/dl) 97.17 11.43 6 99.67 21.65 6

SGOT (IU/l) 47.50 3.27 6 45.67 4.27 6

SGPT (IU/l) 41.17 3.19 6 40.83 3.25 6

ALP (IU/l) 313.02 17.32 6 313.87 11.21 6


Albumin (g/dl) 4.33 0.23 6 4.50 0.21 6


Creatinine (mg/dl) 0.45 0.16 6 0.67 0.28 6

Urea (mg/dl) 37.83 3.82 6 39.17 2.79 6




TABLE 6 - Organ Weights (g) - Group Mean Values

Sex: Male

Parameter G1 G2

Mean SD N Mean SD N

Brain 1.760 0.100 6 1.727 0.067 6

Heart 0.822 0.071 6 0.750 0.093 6

Liver 8.362 0.939 6 7.903 0.622 6

Kidneys 1.758 0.175 6 1.728 0.109 6

Adrenals 0.037 0.019 6 0.037 0.033 6

Spleen 1.310 0.197 6 1.310 0.162 6

Thymus 0.218 0.086 6 0.247 0.049 6

Testis 3.122 0.277 6 3.047 0.218 6

Epididymis 1.110 0.068 6 1.131 0.058 6

Parameter G3 G4

Mean SD N Mean SD N

Brain 1.645 0.148 6 1.912 0.190 5

Heart 0.707 0.018 6 0.810 0.155 5

Liver 7.327 0.769 6 9.062 1.148 5

Kidneys 1.675 0.096 6 1.940 0.274 5

Adrenals 0.037 0.014 6 0.046 0.017 5

Spleen 1.348 0.121 6 1.438 0.209 5

Thymus 0.242 0.058 6 0.202 0.089 5

Testis 2.853 0.260 6 2.958 0.188 5

Epididymis 1.112 0.084 6 1.138 0.048 5



TABLE 6 - Organ Weights (g) - Group Mean Values (Continued)

Sex: Female

Parameter G1 G2

Mean SD N Mean SD N

Brain 1.790 0.097 6 1.660 0.043 6

Heart 0.593 0.088 6 0.537 0.057 6

Liver 6.305 1.034 6 6.246 0.181 6

Kidneys 1.337 0.101 6 1.317 0.073 6

Adrenals 0.052 0.016 6 0.035 0.010 6

Spleen 0.822 0.208 6 0.737 0.078 6

Thymus 0.209 0.058 6 0.238 0.045 6

Ovaries 0.098 0.020 6 0.123 0.026 6

Uterus 0.523 0.211 6 0.324 0.086 6

Parameter G3 G4

Mean SD N Mean SD N

Brain 1.637 0.054 6 1.733 0.114 6

Heart 0.582 0.048 6 0.707 0.052 6

Liver 6.118 0.113 6 6.295 0.455 6

Kidneys 1.285 0.029 6 1.303 0.140 6

Adrenals 0.052 0.022 6 0.053 0.005 6

Spleen 0.820 0.069 6 0.892 0.113 6

Thymus 0.269 0.033 6 0.242 0.059 6

Ovaries 0.125 0.012 6 0.087 0.021 6

Uterus 0.275 0.022 6 0.450 0.209 6




TABLE 7 - Relative Organ Weights (%) - Group Mean Values

Sex: Male

Parameter G1 G2

Mean SD N Mean SD N

Brain 0.723 0.070 6 0.718 0.045 6

Heart 0.336 0.015 6 0.312 0.043 6

Liver 3.425 0.385 6 3.273 0.066 6

Kidneys 0.718 0.041 6 0.717 0.032 6

Adrenals 0.015 0.008 6 0.016 0.015 6

Spleen 0.541 0.109 6 0.542 0.036 6

Thymus 0.089 0.031 6 0.101 0.012 6

Testis 1.281 0.138 6 1.263 0.055 6

Epididymis 0.455 0.038 6 0.469 0.020 6

Parameter G3 G4

Mean SD N Mean SD N

Brain 0.704 0.026 6 0.787 0.078 5

Heart 0.304 0.021 6 0.332 0.053 5

Liver 3.137 0.207 6 3.732 0.479 5

Kidneys 0.719 0.046 6 0.798 0.104 5

Adrenals 0.015 0.005 6 0.019 0.006 5

Spleen 0.577 0.012 6 0.589 0.059 5

Thymus 0.103 0.018 6 0.084 0.039 5

Testis 1.222 0.067 6 1.218 0.097 5

Epididymis 0.477 0.036 6 0.470 0.047 5



TABLE 7 - Relative Organ Weights (%) - Group Mean Values (Continued)

Sex: Female

Parameter G1 G2

Mean SD N Mean SD N

Brain 1.122 0.057 6 1.061 0.017 6

Heart 0.373 0.066 6 0.342 0.030 6

Liver 3.964 0.747 6 3.993 0.164 6

Kidneys 0.838 0.069 6 0.842 0.061 6

Adrenals 0.032 0.011 6 0.022 0.006 6

Spleen 0.554 0.135 6 0.470 0.047 6

Thymus 0.127 0.033 6 0.152 0.026 6

Ovaries 0.554 0.135 6 0.079 0.016 6

Uterus 0.330 0.135 6 0.208 0.058 6

Parameter G3 G4

Mean SD N Mean SD N

Brain 1.037 0.038 6 1.095 0.081 6

Heart 0.368 0.031 6 0.446 0.028 6

Liver 3.876 0.148 6 3.976 0.309 6

Kidneys 0.814 0.041 6 0.822 0.071 6

Adrenals 0.033 0.015 6 0.034 0.004 6

Spleen 0.520 0.057 6 0.564 0.082 6

Thymus 0.170 0.017 6 0.153 0.040 6

Ovaries 0.079 0.009 6 0.055 0.012 6

Uterus 0.174 0.014 6 0.286 0.140 6




TABLE 8 - Gross Pathology Findings – Summary by Group

Organs &

Lesions

Group G1 G2 G3 G4

Sex M F M F M F M F N° of Animals 6 6 6 6 6 6 5 6

Brain NAD NAD NAD NAD NAD NAD NAD NAD

Liver NAD NAD NAD NAD NAD NAD NAD NAD

Kidney NAD NAD NAD NAD NAD NAD NAD NAD

Heart NAD NAD NAD NAD NAD NAD NAD NAD

Thymus NAD NAD NAD NAD NAD NAD NAD NAD

Adrenals NAD NAD NAD NAD NAD NAD NAD NAD

Testis NAD X NAD X NAD X NAD X

Epididymis NAD X NAD X NAD X NAD X

Ovaries X NAD X NAD X NAD X NAD

Oviduct & Uterus X NAD X NAD X NAD X NAD

Pancreas NAD NAD NAD NAD NAD NAD NAD NAD

Key: M= Male, F= Female, NAD = No Abnormality Detected Note: “X” organs not examined as per sex difference.




APPENDIX – A

Clinical Signs of Individual Animals

Group: G1 (Control) Animal

N° Day of Observation

0 7 14 21 28 Male

1 1 1 1 1 1 2 1 1 1 1 1 3 1 1 1 1 1 4 1 1 1 1 1 5 1 1 1 1 1 6 1 1 1 1 1

Female 7 1 1 1 1 1 8 1 1 1 1 1 9 1 1 1 1 1 10 1 1 1 1 1 11 1 1 1 1 1 12 1 1 1 1 1

Group: G2 (Therapeutic Dose)

Animal N°

Day of Observation 0 7 14 21 28

Male 13 1 1 1 1 1 14 1 1 1 1 1 15 1 1 1 1 1 16 1 1 1 1 1 17 1 1 1 1 1 18 1 1 1 1 1

Female 19 1 1 1 1 1 20 1 1 1 1 1 21 1 1 1 1 1 22 1 1 1 1 1 23 1 1 1 1 1 24 1 1 1 1 1

Key: 1 – Normal



APPENDIX – A (Continued)

Clinical Signs of Individual Animals Group : G3 (Average Dose)

Animal N°


Male 25 1 1 1 1 1 26 1 1 1 1 1 27 1 1 1 1 1 28 1 1 1 1 1 29 1 1 1 1 1 30 1 1 1 1 1

Female 31 1 1 1 1 1 32 1 1 1 1 1 33 1 1 1 1 1 34 1 1 1 1 1 35 1 1 1 1 1 36 1 1 1 1 1

Group : G4 (High Dose)

Animal N°


Male 37 1 1 1 1 1 38 1 1 1 1 1 39 1 1 2 2 2 40 1 1 1 1 1 41 1 1 1 1 1 42 1 1 1 1 1

Female 43 1 1 1 1 1 44 1 1 1 1 1 45 1 1 1 1 1 46 1 1 1 1 1 47 1 1 1 1 1 48 1 1 1 1 1

Key: 1 – Normal, 2 - Dead




APPENDIX – B

Individual Animal Body Weight (g)

Group : G1 (Control)

Animal N°

Day of Observation Terminal 0 7 14 21 28 Male

1 161.8 194.7 214.6 228.4 236.2 234.1 2 160.5 192.2 206.6 214.5 221.6 220.1 3 169.9 217.3 238.6 251.2 260.3 258.3 4 172.6 207.3 211.7 223.5 231.2 229.6 5 172.2 198.3 218.7 231.4 241.6 239.5 6 208.1 240.1 270.7 280.4 291.6 288.7

Female 7 152.0 179.3 187.1 193.5 201.4 200.1 8 156.1 177.0 183.2 188.0 196.2 194.2 9 159.4 186.7 193.3 196.0 206.4 205.1 10 162.5 183.9 194.6 197.5 204.9 202.9 11 160.3 195.1 202.6 210.1 218.3 216.4 12 167.1 179.3 185.3 189.3 198.4 196.2

Group : G2 (Therapeutic Dose) Animal

N° Day of Observation Terminal 0 7 14 21 28

Male 13 150.1 178.3 200.4 208.6 219.2 217.1 14 152.8 182.3 204.4 212.3 221.1 219.5 15 164.0 205.4 220.0 228.4 239.6 237.6 16 171.1 220.3 233.3 243.6 252.1 250.6 17 174.4 212.8 241.7 250.1 261.3 259.7 18 185.3 215.7 247.5 256.8 267.4 265.2

Female 19 150.7 174.8 180.7 184.3 189.2 188.4 20 155.1 177.7 182.1 186.1 196.3 197.9 21 155.5 171.7 177.2 181.0 188.2 187.4 22 158.8 188.9 197.6 203.5 208.9 206.4 23 158.5 182.6 192.2 196.4 204.5 202.3 24 160.5 183.6 187.5 191.3 199.3 197.8



APPENDIX – B (Continued)

Individual Animal Body Weight (g) Group : G3 (Average Dose)

Animal N°


25 165.0 170.4 195.4 203.4 213.1 211.4 26 179.8 183.0 223.5 234.6 241.6 239.4 27 162.8 200.7 222.8 231.2 239.8 237.2 28 177.8 222.3 243.5 252.7 263.5 261.9 29 182.1 215.6 218.7 226.8 234.7 232.6 30 189.0 202.4 205.6 213.2 220.1 218.8

Female 31 153.7 184.5 185.9 189.3 195.6 193.4 32 156.0 172.7 175.5 178.0 186.4 184.3 33 154.2 179.0 188.2 191.0 199.1 198.1 34 159.7 189.9 202.1 208.5 216.2 214.9 35 154.0 203.2 206.4 211.3 218.3 217.6 36 170.5 203.2 223.5 238.5 246.5 244.7


Animal N°


37 153.7 180.6 196.6 206.1 214.6 212.1 38 176.7 202.4 244.1 256.7 264.7 262.4 39 166.3 153.7 FD FD FD FD 40 180.2 210.6 219.9 227.5 234.6 233.1 41 185.8 207.3 232.1 240.6 248.7 246.2 42 204.9 234.2 245.0 256.7 267.2 265.4

Female 43 152.8 179.3 185.3 189.6 196.4 195.1 44 152.8 179.5 186.2 190.0 194.7 199.7 45 156.4 178.4 185.6 191.5 198.7 206.7 46 158.5 179.5 190.3 196.0 204.6 210.6 47 162.1 185.7 188.3 193.5 201.3 209.5 48 168.2 193.2 200.5 206.0 214.7 220.7

Key: FD : Found Dead




APPENDIX – C

Food Consumption (g/cage)

Group : G1 (Control) Animal


7 14 21 28 Male

1 196 217.8 213.4 218.6 2 3 210.5 279 260.2 268.9 4 5

212 279.6 240.2 251.2 6 Female

7 161.3 179.8 191.5 201.7 8

9 168.7 182.6 197.8 206.1 10

11 165.2 180.5 196.8 204.2 12

Group : G2 (Therapeutic Dose) Animal


7 14 21 28 Male

13 208.6 284 251.1 261.9 14

15 207.7 212 217.9 225.6 16 17 229.8 245.6 226.6 230.1 18

Female 19 171.4 185.4 192.3 200.1 20 21 156.8 167.8 184.1 196.4 22 23

168.4 184.1 195..9 206.4 24



APPENDIX – C (Continued)

Food Consumption (g/cage) Group : G3 (Average Dose)

Animal N°

Day of Observation 7 14 21 28

Male 25 219.3 234.1 259 267.5 26 27 235.3 238.1 247.7 254.9 28 29 211.6 270.7 252.9 260.8 30

Female 31 163.4 175.6 184.7 192.3 32 33 160.3 178.4 187.1 196.5 34 35 163.7 176.4 191.4 204.7 36


Animal N°

Day of Observation 7 14 21 28

Male 37 233 217.9 208.9 209.6 38 39 227.5 210.1 206.4 208.5 40 41

211.1 232.9 228.5 235.6 42 Female

43 145.6 165.6 173.8 185.7 44 45

153.7 178.6 182.9 196.7 46 47 148.7 174.3 186.8 199.4 48




APPENDIX – D

Individual Animal Haematological Values

Group : G1 (Control)

Animal No

Haematological Parameters

RBC (1x106/mm3)

WBC (1x103/mm3)

Hb (g/dl)

HCT (%)

MCV (um3)

MCH (pg)

MCHC (g/dl)

Plt (1x103/mm

3)

Male

1 7.73 14.60 17.50 38.60 51.30 23.80 53.20 365.40

2 6.59 12.50 14.60 42.30 48.60 26.40 52.60 389.40

3 6.84 13.70 18.60 41.70 50.20 25.40 54.60 351.20

4 7.53 12.80 16.30 38.60 48.70 25.10 55.30 389.70

5 7.51 11.70 14.50 41.30 50.10 27.80 53.10 451.20

6 7.56 11.30 17.80 27.90 51.20 25.40 56.20 412.00

Female

7 6.45 12.36 16.50 42.30 48.90 25.10 56.50 365.30

8 6.82 12.78 14.60 41.00 50.30 24.60 54.60 421.30

9 6.91 13.45 18.40 39.40 51.40 26.40 55.30 421.60

10 7.63 11.63 16.70 38.70 50.70 27.10 55.00 435.60

11 5.69 12.48 14.60 40.10 48.70 24.30 53.60 412.00

12 6.42 14.32 15.20 40.60 48.60 25.60 56.20 351.00



APPENDIX – D (Continued)


Group : G2 (Therapeutic Dose)

Animal No


RBC (1x106/mm3)

WBC (1x103/mm3)

Hb (g/dl)

HCT (%)

MCV (um3)

MCH (pg)

MCHC (g/dl)

Plt (1x103/mm3)

Male

13 7.48 12.40 15.60 41.30 52.40 26.40 54.60 356.70

14 7.63 16.30 17.60 42.10 53.10 23.50 55.50 402.30

15 8.54 14.50 15.90 38.40 51.40 24.10 52.30 465.30

16 6.35 14.30 17.80 39.70 50.20 27.30 53.20 512.30

17 6.48 13.40 16.30 40.50 48.90 25.10 54.60 345.60

18 6.21 13.10 18.40 40.20 47.90 24.60 55.80 327.60

Female

19 6.47 12.45 18.60 38.50 47.90 26.10 54.60 346.50

20 6.93 15.30 14.70 40.20 50.20 24.60 56.30 256.70

21 7.65 14.80 16.30 41.20 51.30 24.50 55.40 347.90

22 5.39 16.40 14.60 38.40 50.70 25.40 57.10 421.60

23 6.42 12.70 15.80 40.20 52.10 23.40 55.20 423.10

24 6.73 14.20 16.50 38.90 48.90 25.60 54.20 426.50





Group : G3 (Average Dose)

Animal No


RBC (1x106/mm3)

WBC (1x103/mm3)

Hb (g/dl)

HCT (%)

MCV (um3)

MCH (pg)

MCHC (g/dl)

Plt (1x103/mm3)

Male

25 6.41 14.50 18.40 39.40 52.10 24.10 55.10 456.30

26 6.21 12.70 17.60 42.60 53.10 23.60 57.60 356.20

27 6.89 12.50 18.40 40.10 51.20 24.70 53.60 285.60

28 7.32 12.70 18.60 38.70 50.20 26.20 54.40 294.70

29 7.63 12.60 17.50 42.10 51.20 24.70 55.20 423.10

30 6.92 15.40 16.20 40.50 50.70 25.10 57.30 425.60

Female

31 6.49 14.50 17.40 37.90 50.30 24.60 54.60 347.60

32 6.43 16.70 15.60 42.10 51.40 24.80 52.80 396.10

33 5.69 18.40 14.50 40.30 51.80 26.50 54.70 378.90

34 7.21 14.30 14.60 41.10 50.30 25.10 53.90 363.80

35 7.63 14.90 16.30 39.40 48.90 25.30 54.60 412.50

36 7.91 12.60 17.20 37.80 47.60 27.20 54.30 412.30






Animal No


RBC (1x106/mm3)

WBC (1x103/mm3)

Hb (g/dl)

HCT (%)

MCV (um3)

MCH (pg)

MCHC (g/dl)

Plt (1x103/mm3)

Male

37 8.64 14.30 15.40 41.70 50.20 24.10 56.30 347.90

38 7.89 14.70 16.40 40.30 50.90 26.30 54.00 421.30

39 D D D D D D D D

40 7.42 15.70 17.20 38.50 47.90 25.60 54.20 312.40

41 7.63 12.40 17.07 40.10 48.60 26.40 55.30 435.60

42 7.35 12.70 16.40 41.30 50.20 25.10 51.20 421.40

Female

43 6.27 14.70 17.60 41.20 48.70 24.60 55.40 326.40

44 6.37 14.90 15.30 42.10 50.30 24.80 55.30 357.80

45 5.94 15.80 14.60 40.30 51.20 26.10 55.60 396.40

46 7.62 12.70 17.40 38.90 52.40 24.60 56.10 402.30

47 7.73 13.40 16.50 39.40 50.70 25.40 52.30 402.40

48 7.26 14.60 17.50 39.80 49.80 26.30 57.40 456.30

D- Dead




APPENDIX – E

Individual Animal Clinical Chemistry Values

Group: G1 (Control)

Animal No

Clinical Chemistry Parameters

Glucose (mg/dl)

SGOT (IU/l)

SGPT (IU/l)

Total Protein (g/dl)

Creatinine (mg/dl)

Albumin (g/dl)

ALP (IU/l)

Urea (mg/dl) Triglycerides

Male

1 119.00 46.00 34.00 5.50 0.80 4.20 296.50 38.00 62

2 86.00 49.00 33.00 5.90 0.60 4.30 297.50 42.00 59

3 93.00 51.00 26.00 6.20 0.40 3.90 312.40 46.00 71

4 104.00 50.00 28.00 6.70 0.90 4.60 321.40 42.00 63

5 95.00 47.00 36.00 5.80 0.40 4.70 326.40 43.00 54

6 91.00 48.00 30.00 5.90 0.30 5.00 321.30 42.00 81

Female

7 88.00 43.00 36.00 5.80 0.30 4.60 268.90 39.00 56

8 88.00 42.00 39.00 6.20 0.30 4.20 312.60 31.00 69

9 84.00 46.00 42.00 5.40 0.40 4.30 325.80 35.00 71

10 97.00 41.00 46.00 6.20 0.60 4.20 343.60 41.00 53

11 86.00 39.00 42.00 6.30 0.30 4.80 287.30 38.00 72

12 106.00 45.00 38.00 6.00 0.70 4.30 302.60 42.00 56



APPENDIX – E (Continued)

Individual Animal Clinical Chemistry Values Group: G2 (Therapeutic Dose)

Animal No


Glucose (mg/dl)

SGOT (IU/l)

SGPT (IU/l)


Creatinine (mg/dl)

Albumin (g/dl)

ALP (IU/l)


Male

13 74.00 49.00 27.00 5.70 0.50 5.20 285.40 38.00 63

14 71.00 48.00 36.00 5.90 0.70 4.90 294.60 42.00 57

15 77.00 52.00 28.00 6.70 0.40 4.70 302.10 46.00 61

16 91.00 46.00 29.00 6.20 0.80 4.60 305.70 42.00 72

17 95.00 50.00 34.00 6.40 0.20 3.90 284.60 41.00 59

18 66.00 49.00 26.00 5.90 0.80 4.60 279.60 47.00 65

Female

19 102.00 48.00 33.00 5.90 0.60 4.10 289.60 41.00 59

20 85.00 43.00 37.00 5.40 0.50 4.00 312.70 40.00 63

21 100.00 48.00 38.00 6.20 0.70 4.60 324.60 35.00 71

22 98.00 51.00 43.00 5.80 0.30 4.20 301.40 37.00 56

23 105.00 50.00 41.00 6.40 0.20 4.30 300.50 42.00 63

24 69.00 47.00 38.00 6.10 0.30 4.00 298.10 40.00 62




Individual Animal Clinical Chemistry Values Group: G3 (Average Dose)

Animal No


Glucose (mg/dl)

SGOT (IU/l)

SGPT (IU/l)


Creatinine (mg/dl)

Albumin (g/dl)

ALP (IU/l)


Male

25 89.00 49.00 34.00 5.90 0.60 4.30 281.50 42.00 63

26 94.00 43.00 40.00 6.20 0.70 4.70 283.60 46.00 71

27 93.00 48.00 38.00 6.30 0.20 4.50 325.40 41.00 56

28 93.00 52.00 36.00 6.70 0.40 4.70 302.40 43.00 81

29 103.00 50.00 29.00 6.50 0.90 4.20 312.20 42.00 53

30 111.00 48.00 26.00 6.40 0.40 4.20 312.40 40.00 56

Female

31 85.00 46.00 37.00 6.20 0.40 4.50 345.60 32.00 53

32 103.00 42.00 46.00 6.10 0.30 4.30 312.40 37.00 69

33 96.00 49.00 43.00 6.30 0.60 4.70 302.40 38.00 56

34 91.00 51.00 42.00 5.90 0.30 4.30 298.70 42.00 57

35 91.00 50.00 39.00 5.40 0.40 4.10 302.60 36.00 63

36 117.00 47.00 40.00 5.60 0.70 4.10 316.40 42.00 61




Individual Animal Clinical Chemistry Values Group: G4 (High Dose)

Animal No


Glucose (mg/dl)

SGOT (IU/l)

SGPT (IU/l)


Creatinine (mg/dl)

Albumin (g/dl)

ALP (IU/l)


Male

37 78.00 46.00 34.00 5.60 0.60 4.60 302.40 46.00 72

38 89.00 43.00 42.00 6.30 0.70 4.20 326.40 42.00 83

39 D D D D D D D D D

40 43.00 50.00 34.00 6.70 0.40 4.30 314.70 43.00 63

41 43.00 49.00 35.00 6.40 0.80 4.40 345.60 48.00 59

42 68.00 52.00 37.00 6.30 0.20 4.60 323.50 39.00 71

Female

43 139.00 42.00 37.00 5.40 0.40 4.70 312.40 37.00 56

44 99.00 50.00 46.00 6.00 0.30 4.60 306.40 42.00 59

45 81.00 51.00 42.00 6.20 0.80 4.20 296.40 40.00 61

46 105.00 43.00 38.00 5.90 0.90 4.50 326.10 39.00 84

47 80.00 47.00 42.00 5.70 1.00 4.70 318.20 35.00 53

48 94.00 41.00 40.00 5.60 0.60 4.30 323.70 42.00 61

D- Dead




APPENDIX – F

Individual Animal Organ Weights (g)

Group: G1 (Control) Animal

No Organ Weights (g)

Male

Brain Heart Liver Kidneys Adrenals Spleen Thymus Testis Epididymis

1 1.750 0.820 9.460 1.830 0.020 1.240 0.090 3.340 1.150

2 1.800 0.750 7.550 1.470 0.020 1.200 0.220 3.180 1..118

3 1.930 0.820 8.370 1.870 0.030 1.250 0.160 3.130 1.170

4 1.640 0.750 6.970 1.650 0.060 1.710 0.240 3.020 1.020

5 1.690 0.850 8.850 1.770 0.060 1.240 0.260 2.640 1.030

6 1.750 0.940 8.970 1.960 0.030 1.220 0.340 3.420 1.170

Female

Brain Heart Liver Kidneys Adrenals Spleen Thymus Ovaries Uterus

7 1.750 0.740 8.150 1.420 0.070 0.950 0.220 0.12 0.460

8 1.870 0.610 6.000 1.330 0.030 0.950 0.160 0.12 0.750

9 1.720 0.580 6.010 1.340 0.070 1.030 0.174 0.11 0.440

10 1.800 0.620 6.460 1.300 0.040 1.070 0.230 0.08 0.350

11 1.670 0.500 4.990 1.170 0.050 0.510 0.160 0.08 0.820

12 1.930 0.510 6.220 1.460 0.050 0.780 0.310 0.08 0.320



APPENDIX – F (Continued)

Individual Animal Organ Weights (g) Group: G2 (Therapeutic Dose) Animal


Male


13 1.650 0.630 7.340 1.650 0.100 1.120 0.180 2.860 1.054

14 1.670 0.780 7.020 1.560 0.020 1.232 0.210 2.890 1.062

15 1.780 0.860 7.860 1.720 0.040 1.240 0.230 2.954 1.148

16 1.690 0.720 8.210 1.860 0.010 1.260 0.260 3.010 1.186

17 1.820 0.840 8.350 1.780 0.020 1.560 0.290 3.120 1.169

18 1.750 0.670 8.640 1.800 0.030 1.450 0.310 3.450 1.167

Female


19 1.620 0.480 6.340 1.430 0.020 0.640 0.178 0.130 0.345

20 1.630 0.520 6.480 1.360 0.030 0.710 0.189 0.140 0.470

21 1.640 0.510 6.020 1.240 0.040 0.810 0.260 0.090 0.360

22 1.710 0.560 6.047 1.260 0.030 0.690 0.240 0.100 0.270

23 1.640 0.510 6.241 1.270 0.050 0.850 0.270 0.120 0.240

24 1.720 0.640 6.345 1.340 0.040 0.720 0.290 0.160 0.260




Individual Animal Organ Weights (g) Group : G3 (Average Dose) Animal


Male


25 1.530 0.710 6.850 1.680 0.020 1.210 0.190 2.410 1.027

26 1.620 0.720 7.540 1.710 0.040 1.340 0.220 2.890 1.096

27 1.670 0.690 6.480 1.560 0.030 1.380 0.230 2.880 1.078

28 1.930 0.720 8.640 1.840 0.060 1.560 0.340 3.210 1.154

29 1.560 0.720 7.560 1.640 0.040 1.340 0.280 2.790 1.260

30 1.560 0.680 6.890 1.620 0.030 1.260 0.194 2.940 1.055

Female


31 1.540 0.640 6.210 1.270 0.060 0.890 0.211 0.120 0.260

32 1.690 0.620 6.240 1.290 0.080 0.730 0.270 0.110 0.270

33 1.620 0.530 6.024 1.340 0.070 0.860 0.260 0.140 0.250

34 1.670 0.560 6.024 1.280 0.040 0.760 0.280 0.140 0.310

35 1.630 0.530 6.000 1.260 0.040 0.890 0.280 0.120 0.290

36 1.670 0.610 6.210 1.270 0.020 0.790 0.310 0.120 0.270




Individual Animal Organ Weights (g) Group : G4 (High Dose) Animal


Male


37 1.680 0.670 8.050 1.720 0.030 1.100 0.170 2.710 1.110

38 2.110 1.060 10.480 2.350 0.030 1.450 0.240 2.930 1.120


40 2.011 0.841 9.650 2.000 0.050 1.560 0.340 2.960 1.140

41 2.020 0.780 9.400 1.970 0.050 1.430 0.120 3.240 1.220

42 1.740 0.700 7.730 1.660 0.070 1.650 0.142 2.950 1.100

Female


43 1.720 0.730 6.510 1.230 0..060 0.930 0.150 0.070 0.44

44 1.810 0.730 6.200 1.290 0.050 0.910 0.330 0.100 0.840

45 1.520 0.630 6.190 1.350 0.050 1.050 0.250 0.070 0.240

46 1.830 0.690 6.620 1.170 0.060 0.870 0.270 0.090 0.450

47 1.800 0.700 5.490 1.220 0.050 0.700 0.230 0.070 0.300

48 1.720 0.790 6.760 1.560 0.050 0.890 0.220 0.120 0.430

D- Dead




APPENDIX – G

Individual Animal Relative Organ Weights (%)

Group : G1 (Control) Animal

No Relative Organ Weights (%)

Male


1 0.748 0.350 4.041 0.782 0.009 0.530 0.038 1.427 0.491

2 0.818 0.341 3.430 0.668 0.009 0.545 0.100 1.445 0.508

3 0.747 0.317 3.240 0.724 0.012 0.484 0.062 1.212 0.453

4 0.714 0.327 3.036 0.719 0.026 0.745 0.105 1.315 0.444

5 0.706 0.355 3.695 0.739 0.025 0.518 0.109 1.102 0.430

6 0.606 0.326 3.107 0.679 0.010 0.423 0.118 1.185 0.405

Female


7 1.151 0.487 5.362 0.934 0.046 0.625 0.124 0.625 0.303

8 1.198 0.391 3.844 0.852 0.019 0.609 0.102 0.609 0.480

9 1.079 0.364 3.770 0.841 0.044 0.646 0.109 0.646 0.276

10 1.108 0.382 3.975 0.800 0.025 0.658 0.142 0.658 0.215

11 1.042 0.312 3.113 0.730 0.031 0.318 0.100 0.318 0.512

12 1.155 0.305 3.722 0.874 0.030 0.467 0.186 0.467 0.192



APPENDIX – G (Continued)

Individual Animal Relative Organ Weights (%)

Group: G2 (Therapeutic Dose) Animal


Male


13 0.760 0.290 3.381 0.760 0.046 0.516 0.083 1.317 0.485

14 0.761 0.355 3.198 0.711 0.009 0.561 0.096 1.317 0.484

15 0.749 0.362 3.308 0.724 0.017 0.522 0.097 1.243 0.483

16 0.674 0.287 3.276 0.742 0.004 0.503 0.104 1.201 0.473

17 0.701 0.323 3.215 0.685 0.008 0.601 0.112 1.201 0.450

18 0.660 0.253 3.258 0.679 0.011 0.547 0.117 1.301 0.440

Female


19 1.075 0.319 4.207 0.949 0.013 0.425 0.118 0.086 0.229

20 1.051 0.335 4.178 0.877 0.019 0.458 0.122 0.090 0.303

21 1.055 0.328 3.871 0.797 0.026 0.521 0.167 0.058 0.232

22 1.077 0.353 3.808 0.793 0.019 0.435 0.151 0.063 0.170

23 1.035 0.322 3.938 0.801 0.032 0.536 0.170 0.076 0.151

24 1.072 0.399 3.953 0.835 0.025 0.449 0.181 0.100 0.162




Individual Animal Relative Organ Weights (%) Group: G3 (Average Dose) Animal


Male


25 0.724 0.336 3.240 0.795 0.009 0.572 0.090 1.140 0.486

26 0.677 0.301 3.150 0.714 0.017 0.560 0.092 1.207 0.458

27 0.704 0.291 2.732 0.658 0.013 0.582 0.097 1.214 0.454

28 0.737 0.275 3.299 0.703 0.023 0.596 0.130 1.226 0.441

29 0.671 0.310 3.250 0.705 0.017 0.576 0.120 1.199 0.542

30 0.713 0.311 3.149 0.740 0.014 0.576 0.089 1.344 0.482

Female


31 1.002 0.416 4.040 0.826 0.039 0.579 0.137 0.078 0.169

32 1.083 0.397 4.000 0.827 0.051 0.468 0.173 0.071 0.173

33 1.051 0.344 3.907 0.869 0.045 0.558 0.169 0.091 0.162

34 1.046 0.351 3.772 0.802 0.025 0.476 0.175 0.088 0.194

35 1.058 0.344 3.896 0.818 0.026 0.578 0.182 0.078 0.188

36 0.979 0.358 3.642 0.745 0.012 0.463 0.182 0.070 0.158




Individual Animal Relative Organ Weights (%) Group: G4 (High Dose) Animal


Male


37 0.792 0.316 3.795 0.811 0.014 0.519 0.080 1.278 0.523

38 0.804 0.404 3.994 0.896 0.011 0.553 0.091 1.117 0.427


40 0.863 0.361 4.140 0.858 0.021 0.669 0.146 1.270 0.489

41 0.820 0.317 3.818 0.800 0.020 0.581 0.049 1.316 0.496

42 0.656 0.264 2.913 0.625 0.026 0.622 0.054 1.112 0.414

Female


43 1.126 0.458 4.260 0.805 0.039 0.609 0.098 0.046 0.288

44 1.185 0.478 4.058 0.844 0.033 0.596 0.216 0.065 0.550

45 0.972 0.403 3.958 0.863 0.032 0.671 0.160 0.045 0.153

46 1.155 0.435 4.177 0.738 0.038 0.549 0.170 0.057 0.284

47 1.110 0.432 3.387 0.753 0.031 0.432 0.142 0.043 0.185

48 1.023 0.470 4.019 0.927 0.030 0.529 0.131 0.071 0.256

D- Dead




APPENDIX – H

Gross and Microscopic Findings of Individual Animals

Group: G1 (Control) Sex : Male

Animal N°

Mode of Death

Gross Findings Microscopic Findings

External Internal

1 TS NAD Lung Abscess NAD

2 TS NAD NAD NAD

3 TS NAD NAD NAD

4 TS NAD NAD NAD


6 TS NAD Lung Abscess NAD Group : G1 (Control) Sex : Female

Animal N°

Mode of Death


External Internal

7 TS NAD Foci in Lungs NAD

8 TS NAD Patches in lungs NAD

9 TS NAD NAD NAD

10 TS NAD NAD NAD

11 TS NAD NAD NAD

12 TS NAD NAD NAD Key: TS = Terminal Sacrifice; NAD = No Abnormalities Detected



APPENDIX – H (Continued)


Group : G2 (Therapeutic Dose) Sex : Male

Animal N°

Mode of Death


External Internal

13 TS NAD NAD X

14 TS NAD NAD X

15 TS NAD NAD X

16 TS NAD NAD X

17 TS NAD NAD X

18 TS NAD NAD X Group : G2 (Therapeutic Dose) Sex : Female

Animal N°

Mode of Death


External Internal

19 TS NAD NAD X

20 TS NAD NAD X

21 TS NAD Liver Cyst X

22 TS NAD NAD X

23 TS NAD NAD X

24 TS NAD Patches in Lungs X

Key: TS = Terminal Sacrifice; NAD = No Abnormalities Detected Note: “X” organs not examined as per protocol, except where gross lesions were recorded





Group : G3 (Average Dose) Sex : Male

Animal N°

Mode of Death


External Internal

25 TS NAD NAD X


27 TS NAD NAD X

28 TS NAD Lung Abscess X

29 TS NAD NAD X

30 TS NAD NAD X Group : G3 (Average Dose) Sex : Female

Animal N°

Mode of Death


External Internal


32 TS NAD White patches in lungs X

33 TS NAD NAD X

34 TS NAD Foci in Lung X

35 TS NAD NAD X

36 TS NAD Liver Cyst X Key: TS = Terminal Sacrifice; NAD = No Abnormalities Detected Note: “X” organs not examined as per protocol, except where gross lesions were recorded





Group : G4 (High Dose) Sex : Male

Animal N°

Mode of Death


External Internal

37 TS NAD NAD NAD

38 TS NAD Lung

Abscess, liver cyst

NAD

39 Dead NAD Lung Abscess Dead

40 TS NAD NAD NAD


42 TS NAD Lung Abscess NAD Group : G4 (High Dose) Sex : Female

Animal N°

Mode of Death


External Internal

43 TS NAD NAD NAD

44 TS NAD NAD NAD


46 TS NAD NAD NAD

47 TS NAD Liver Cyst NAD

48 TS NAD NAD NAD Key: TS = Terminal Sacrifice; NAD = No Abnormalities Detected




APPENDIX – I

Feed Analysis Report




APPENDIX – J

Water Analysis Report




APPENDIX – L

Certificate – Department of Science and Industrial Research

Appendix 4: Glycemic Index Measurement of Diabliss Sugar

121

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