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DIABLISS CONSUMER PRODUCTS PVT LTD DiaBliss Herbal Blended Cane Sugar Product Performance Report 8/3/2013 For the

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.

TABLE OF CONTENTS Section Sub Topic Page Section Number 1 Executive Summary 1.1 3 1.2 Company

TABLE OF CONTENTS

Section

Sub

Topic

Page

Section

Number

  • 1 Executive Summary

1.1

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

Glycemic Index Studies of DiaBliss Herbal Treated Sugar

15

4.4

GTT Test with Diabliss based sweets on diabetics

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

A1

A2

A3

A4

25

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

26

Laboratories Appendix 1B: Summary of Herbicide Analysis of Herbal Solution

29

analyzed at SGS Laboratories Appendix 2: ACUTE ORAL TOXICITY STUDY OF DIABLISS

34

SUGAR IN WISTAR RATS Appendix 3: SUB-ACUTE ORAL TOXICITY STUDY OF

60

DIA BLISS SUGAR IN WISTAR RATS Appendix 4: Glycemic Index Measurement of Diabliss Sugar

121

Section 1:

1.1 Executive Summary:

Section 1: 1.1 Executive Summary: DIABLISS HERBAL CANE SUGAR – SUMMARY OF PRODUCT PERFORMANCE For the

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.

1.2 Company Background:

1.2 Company Background: DiaBliss Consumer Products Pvt Ltd is a private limited company formed to commercialize

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

1.2 Company Background: DiaBliss Consumer Products Pvt Ltd is a private limited company formed to commercialize

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

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).

Section 2. Diabetes Background 1

Section 2. Diabetes Background Diabetes, also known as Diabetes Mellitus, is a chronic health condition where

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 Trends 2,3 :

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

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 2030 4 .

In fact the prevalence of diabetes in India has almost doubled in a period of 11 years as per ICMR- INDIAB study 3 . 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.

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

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.

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

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.

2.2 Diabetes Lifestyle Issues:

2.2 Diabetes – Lifestyle Issues: Most diabetics undergo a significant change in lifestyle after being diagnosed

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

  • i. They cannot consume sugar or sweets They cannot consume foods rich in carbohydrates

ii.

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.

1. Black Pepper has anti-inflammatory and antioxidant properties, and so helps to prevent the risks of
  • 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 600800 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

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.

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

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

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

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

DiaBliss Sugar is being classified as follows :

Globally Harmonised System (GHS)

:

Low Toxicity

DiaBliss Sugar is being classified as follows : Globally Harmonised System (GHS) : Low Toxicity Appendix

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.

4.3 Glycemic Index Studies of DiaBliss Herbal Cane Sugar The effect of Natural Sugar on the

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 5669 inclusive; High >70 (Brand-Miller et al. 2003).

GI values of other foods: Glucose: 99; Fructose: 19 ; Sucrose: 69-80 (Table sugar); 105; Lactose: 46

Maltose:

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

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.

4.5 Short Term Glucose Tolerance Tests We initiated a short term glucose tolerance tests with 11
4.5 Short Term Glucose Tolerance Tests We initiated a short term glucose tolerance tests with 11

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

ID

Age

Sex

Type of Diabestes

How long

Did you

If yes, Specify

What Diabiss

What Diabiss

diagnosed

consume

Products are you

with

Medication?

cosuming

diabetes?

(yrs)

     

Type 1

Type 2

     

Salt

Sugar

 

58

  • 1 M

   

x

8

Yes

Amaril, 1000mg, 1-0-1

Yes

Yes

 
  • 2 M

54

   

x

15

None

 

No

No

 

49

M

  • 3 Not Diabetic

     

No

No

 

48

  • 4 M

   

x

     

No

No

 

65

  • 5 M

   

x

3

Yes

 

Yes

Yes

 
  • 6 M

49

   

x

17

Yes

Metformin 500mg 1-0-1

Yes

Yes

 

45

  • 7 M

   

x

 
  • 3 Yes

 

Yes

Yes

 
  • 8 M

80

   

x

 
  • 7 Yes

 

No

No

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

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:

20 40 0 300 250 200 150 100 50 0 8 7 (Diabliss) 7 (Reg Sugar)
20
40
0
300
250
200
150
100
50
0
8
7 (Diabliss)
7 (Reg Sugar)
6
5
4
3
2
1
Fig 1: Extended GTT Tests with 50g intake of Diabliss Sugar Among
Group of Diabetic & Non-diabetic Subjects
Time, Minutes
Blood Sugar Readings, mg/dl
200
180
160
140
120
100
80
60

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

 

Diabetic

2

3

4

5

6

7 (Reg

7

(Diabliss)

8

Diabetic

Non-Diabetic

Diabetic

Sugar)

Diabetic

Diabetic

Diabetic

Subject

Number

1

Diabetic

Time, min

Blood Sugar Readings with Calorimeter, mg/dl

0

  • 116 156

104

  • 142 135

  • 121 140

142

81

60

  • 221 261

154

  • 233 224

  • 179 266

255

203

 
  • 120 97

    • 164 217

      • 158 142

      • 191 189

226

134

 
  • 180 90

    • 123 164

      • 118 112

      • 103 171

108

70

180 90 123 164 118 112 103 171 108 70

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

18 | P a g e

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

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 glycosuria 5 .

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

Subject

Subject

Subject Urine Analysis
Urine Analysis
Urine Analysis

Urine Analysis

 

Number

Fasting

60 min

120 min

180 min

  • 1 +

Nil

 

Trace

Nil

  • 3 Trace

Nil

 

Nil

Nil

  • 4 ++

Trace

 

+

Trace

  • 5 +

Nil

 

Nil

Nil

  • 6 Trace

Nil

 

+

Nil

(Regular Sugar)

  • 7 Nil

 

++

+

Nil

(DiaBliss)

  • 7 Nil

 

Trace

Trace

Nil

  • 8 Trace

Nil

 

Nil

Nil

  • 9 +

Nil

 

Trace

 
Note:
Note:
Note:

Urine Glucose (Glycosuria)

Reading

1,000-1,500 mg/dl

Up to 250 mg/dl

500-1,000 mg/dl

+++

++

Trace

Note: Urine Glucose (Glycosuria) Reading 1,000-1,500 mg/dl Up to 250 mg/dl 500-1,000 mg/dl +++ ++ Trace

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.

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

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 table 7 .

Normal range for non-diabetics (pre-prandial)

Inference

5-6

4.5-5

Perfect!

  • 5.5 99-100

Extremely low to low blood sugar

36-72

2-4

90-108

  • 5.5 - 6

HbA1c-Average Blood Glucose Reading

  • 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!!

11

%

mmol/L

mg/dL

3-4

4-5

  • 6.5 – 7

Normal post-prandial in non-diabetics

  • 126 - 144

7 – 8

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

– 15

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

Table 4: Representative Sampling of Fasting, Post Prandial and HbA1c levels of long term consumers of

Table 4: Representative Sampling of Fasting, Post Prandial and HbA1c levels of long term consumers of DiaBliss

     
       
       

11

82

M

Retired

20

Years

3

Months

8.4

228

372

93

139

Started noticing significant

   

increase in energy levels

increase in energy levels
59 F 200 140 6.1 7.0 8.9 7 months 1 Housewife 90 8 (+10%), Insulin dose
59 F
200
140
6.1
7.0
8.9
7 months
1
Housewife
90
8
(+10%), Insulin dose increased
sugar; Weight Gain of 2.5 kg
after starting herbal treated
281 Child started going to school
7
204
7.9
8.2
11.5
18 months
22 months
1
7.5
to 1/2-0-1/2
140 Glycomet 500mg 1-0-1 lowered
114
182
6.3 121
9
139
units morning & evening
Evening before herbal sugar, 15
137 Insulin 25 units Morning &
98
5.4
5.4
12 months
Years
1 Many
54 M
1
210 231
Before
Sugar
HbA1c
HbA1c
HbA1c
Reading
Latest
Sugar
Herbal
After
Herbal
No
Herbal Sugar
Consuming
How Long
Since
Diabetic
Diabetic
Type of
Profession
Sex
Age
Average
PP
Average
Fasting
Average
PP
After Herbal Sugar
Medication Details
Average
Fasting
Before Herbal
Sugar
M School
7.6
M Catering
6 58
157 Iscept Fort 1- 0 -1 and Pioglit
101
177
107
6.7
7.0
Business
Engineer
136
6.2
6.3
6.8
10 months
1 Year
2
36 months
14 Years
M Engineer
50
5
107 137
61 F
7.0
7.0
M Consulting
8 Years
2
House Wife
6.3
7.2
25 months
2
101
7.2
7.2
18 months
2 Years
2
58 M
10
121
124
6.6
6.6
3 Years
2
M
66
177 No Medication
3 Years
51
2
2
4
6.9 Amryl m - 2 mg
6.7
8.8
34 months
30 months
Sales
M Traveling
45
3
500mg 1-0-1
141 Continuing medication Glycomet
185 122

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

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

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.

Figure 3: Fasting & Postprandial response of type 1 & type 2 diabetics 200 1-0-1 Insulin

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

200 1-0-1 Insulin 18 Units Morning & 150 100 50 0 Insulin 25 Units Morning &
200
1-0-1
Insulin 18 Units Morning &
150
100
50
0
Insulin 25 Units Morning & Evening
Gemerfort 1-0-1
HbA1c (4/6/12) 5.4
Evening Gemerfort
15 months consumption
Cane Sugar
Herbal
Started consuming DiaBliss
PP
Fasting
250
300
Type 1 Diabetic, Male Age 52 - Fasting & PP Profiles
Blood Sugar, mg/dl
13.11.2011
28.6.09
21.9.07
22.1.07
9.9.06
350
16/6/2012 2012; 4 months; Baseline HbA1c = 8.4 in early/mid-June Started Consuming DiaBliss Herbal Cane Sugar
16/6/2012
2012; 4 months; Baseline HbA1c = 8.4
in early/mid-June
Started Consuming DiaBliss Herbal Cane Sugar
Note: January 2013 HbA1c = 6.3
PP
Fasting
Type 2 Diabetic, Male Age 82 - Fasting & PP profiles
Blood Sugar, mg/dl
22/8/2012
25/7/2012
5/7/2012
400
350
250
200
150
100
50
0
300

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

150 PP Fasting Type 1 Diabetic, Female, age 58 - Fasting & PP Profiles Blood Sugar
150
PP
Fasting
Type 1 Diabetic, Female, age 58 - Fasting & PP Profiles
Blood Sugar Levels, mg/dl
17/9/2012
15/9/2012
14/8/2012
4/3/2012
Started
for Tea & Coffee
consuming DiaBliss Herbal
Cane Sugar
100
50
0
250
200
Started consuming DiaBliss Herbal Cane Sugar for Tea & Coffee Type 1 Diabetic, Female, age 58
Started consuming DiaBliss
Herbal Cane Sugar for
Tea & Coffee
Type 1 Diabetic, Female, age 58 - HbA1c Profile
HbA1c
17/9/2012
14/8/2012
6/6/2012
4/3/2012
8.5
8
7.5
7
6.5
6
9

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

110 100 120 130 140 150 160 170 180 190 11.9.2011 20.04.2012 20.4.2012 Started DiaBliss Herbal
110
100
120
130
140
150
160
170
180
190
11.9.2011
20.04.2012
20.4.2012
Started DiaBliss Herbal Cane Sugar
26 months consumption
19.12.2011
2.10.2012
Blood Sugar, mg/dl
Type 2 Diabetic, Male, Age 51: Fasting & PP Profiles
Fasting
PP
110 100 120 130 140 150 160 170 180 190 11.9.2011 20.04.2012 20.4.2012 Started DiaBliss Herbal
Started DiaBliss Herbal Cane Sugar 26 months consumption 6.4 7 Type 2 Diabetic, Male, Age 51:
Started DiaBliss Herbal Cane Sugar
26 months consumption
6.4
7
Type 2 Diabetic, Male, Age 51: HbA1c Profile
HbA1c
2.10.2012
20.04.2012
19.12.2011
11.9.2011
7.6
7.4
7.2
6.8
6.6
6.2
6

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

110 100 120 130 140 150 160 170 180 190 11.9.2011 20.04.2012 20.4.2012 Started DiaBliss Herbal
  • 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

5: References

5: References 1. National Standard, The Authority on Integrative Medicine, taken fromhttp://www.naturalstandard.com/demo/demo-mc-diabetes.asp 2. WHO Report Series7. 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 25 | P a g e " id="pdf-obj-24-4" src="pdf-obj-24-4.jpg">
Appendix 1: Comprehensive Analysis at SGS Laboratories: APPENDIX 1A: METALS ANALYSIS OF DIABLISS CANE SUGAR 26

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 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

Appendix 2: ACUTE ORAL TOXICITY STUDY OF DIABLISS SUGAR IN WISTAR RATS

34

SLS Study Number : SLS/043-11

CONFIDENTIAL

TITLE PAGE

REPORT

SLS Study Number : SLS/043-11 CONFIDENTIAL TITLE PAGE REPORT ACUTE ORAL TOXICITY STUDY OF DIA BLISS

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

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS No claim of confidentiality is

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 Shripuram, Chennai- 600 097 Tamil Nadu

Signature

__________________________

Sponsor/Submitter

Date

 

___________________________

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 STATEMENT OF COMPLIANCE Study Title : Acute Oral Toxicity Study of
STATEMENT OF COMPLIANCE Study Title : Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar
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:
SLS Study Number : SLS/043-11 STATEMENT OF QUALITY ASSURANCE UNIT Study Title : Acute Oral Toxicity
SLS Study Number : SLS/043-11
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:
Dates Reported to the
Audit
Details
Dates of Audit
N o
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:

SLS Study Number : SLS/043-11

TABLE OF CONTENTS

SLS Study Number : SLS/043-11 TABLE OF CONTENTS TITLE PAGE 1 2 STATEMENT OF COMPLIANCE 3

TITLE PAGE

1

2

STATEMENT OF COMPLIANCE

3

4

TABLE OF

5

PERSONNEL INVOLVED IN THE STUDY

7

LIST OF ABBREVIATIONS

...............................................................................................................................

8

SUMMARY

9

  • 1.0 INTRODUCTION ......................................................................................................

10

  • 1.1 Objective .......................................................................................................

Study

10

Study Guidelines

  • 1.2 .....................................................................................................

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 Item.................................................................................................................

Test

11

  • 2.2 and Equipments ....................................................................................

Instruments

11

  • 2.3 Chemical

Solvent and

..............................................................................................

11

  • 2.4 Animals ...................................................................................................................

11

Acclimatization

  • 2.5 .......................................................................................................

12

  • 2.6 Husbandry Practices

12

  • 2.7 Animal Identification

12

Feed and Water

  • 2.8 .......................................................................................................

12

  • 2.9 Environmental Conditions

12

  • 2.10 Activities............................................................................................

Experimental

14

  • 2.11 Preparation of Dosing

14

  • 2.12 Dose Administration

14

  • 2.13 Experimental Design

14

Observations

  • 2.14 ...........................................................................................................

14

  • 3.0 ..................................................................................................................

RESULTS

15

Mortalities

  • 3.1 ...............................................................................................................

15

  • 3.2 Clinical Observations ...............................................................................................

15

Body Weights

  • 3.3 .........................................................................................................

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

19

APPENDIX 2

20

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 Individual Animal Body Weight Data 20 APPENDIX 3 21 Necropsy Findings

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

23

APPENDIX 6

24

Feed Analysis

26

APPENDIX 7……………………………………………………………………………………...25 Water Analysis Report………………………………………………………………………….....25 APPENDIX ………………………………………………………………………………….…...26 Certificate - Department of Science and Industrial Research…………………………………… ..26

SLS Study Number : SLS/043-11 PERSONNEL INVOLVED IN THE STUDY Signature Study Director Syed Nasir Ahamed,
SLS Study Number : SLS/043-11
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

SLS Study Number : SLS/043-11

LIST OF ABBREVIATIONS

SLS Study Number : SLS/043-11 LIST OF ABBREVIATIONS Committee for Purpose of Control and Supervision of

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

SLS Study Number : SLS/043-11

SUMMARY

SLS Study Number : SLS/043-11 SUMMARY This study was performed to assess the acute oral toxicity

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 17 th 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”

SLS Study Number : SLS/043-11

  • 1.0 INTRODUCTION

  • 1.1 Study Objective

SLS Study Number : SLS/043-11 1.0 INTRODUCTION 1.1 Study Objective This objective of this study was

This objective of this study was to assess the acute oral toxicity (LD 50 ) 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 17 th 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

SLS Study Number : SLS/043-11

sought for archiving period.

  • 2.0 EXPERIMENTAL PROCEDURE

  • 2.1 Test Item

SLS Study Number : SLS/043-11 sought for archiving period. 2.0 EXPERIMENTAL PROCEDURE 2.1 Test Item Details

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) Metal Cannula, Size 18 G x 5 cm,

Intubation Cannula 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.

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 Total Number of Animals Used : 06 Females (nulliparous and non-pregnant)

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

:

:

Water Bottle

used. Autoclaved clean paddy husk was used as the bedding material. : Each cage was supplied with a polypropylene water bottle with a

Housing

stainless steel nozzle 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

SLS Study Number : SLS/043-11

maintained, light hours being 6.00 to 18.00h.

SLS Study Number : SLS/043-11 maintained, light hours being 6.00 to 18.00h. Sugen Life Sciences Pvt

SLS Study Number : SLS/043-11

  • 2.10 Experimental Activities

SLS Study Number : SLS/043-11 2.10 Experimental Activities The following observations/activities were performed during the experimental

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

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 weights were recorded prior to dosing on days 0, 7 and

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 LD 50 (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”

SLS Study Number : SLS/043-11

  • 5.0 REFERENCES

SLS Study Number : SLS/043-11 5.0 REFERENCES 5.1 OECD, 1998 : OECD Series on Principles of
  • 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 17 th December 2001.

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

TABLE 1 - Mortality

 

Dose

               

Group

(mg/kg)

Animal N o & Sex

1 h

2 h

3 h

4 h

24 h

Day 1-7

Day 8-14

I

 
  • 2000.0 0

1F-3F

0

0

   

0

  • 0 0

0

 

II

 
  • 2000.0 0

4F-6F

0

0

   

0

  • 0 0

0

 

F- Female

h- Hours

0- No Mortality

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

TABLE 2 - Mean Body Weights

 

Dose

Animal

 

Mean body weight (g)

Group

(mg/kg)

N o & Sex

Day 0

Day 7

Day 14

I

2000.0

1F-3F

172.90±1.12

178.73±0.83

185.96±1.26

II

2000.0

4F-6F

173.47±1.79

180.67±1.72

187.10±2.23

 

Percent Mean Body Weight Changes

 
 

Group

Dose

Percentage Mean body weight Change on

   

Animal

 

(mg/kg)

N o & Sex

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

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 Acute Oral Toxicity Study of Dia Bliss Sugar in Rats APPENDIX

Acute Oral Toxicity Study of Dia Bliss Sugar in Rats

APPENDIX 1

Clinical Observations of Individual Animal

Group – I

Animal

Dose

 

Clinical Signs Observed Post Dosing

 

N° &

Level

At Hour (Day 0)

 

On Day

 

Sex

(mg/kg)

 

0

1

2

3

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

1F

 

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

2F

2000.0

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

Group – II

 

Animal

Dose

 

Clinical Signs Observed Post Dosing

 

N° &

Level

At Hour (Day 0)

 

On Day

 

Sex

(mg/kg)

     
  • 2 4

 
  • 0 2

    • 1 6

      • 3 3

1

   
  • 4 5

   

7

8

9

10

11

12

13

14

1F

       
  • 1 1

 
  • 1 1

    • 1 1

      • 1 1

1

     
  • 1 1

 

1

1

1

1

1

1

  • 1 1

 

2F

2000.0

     
  • 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
3F
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

1 = Normal

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 Acute Oral Toxicity Study of Dia Bliss Sugar in Rats APPENDIX

Acute Oral Toxicity Study of Dia Bliss Sugar in Rats

APPENDIX 2

Individual Animal Body Weight Data

       

% Body

% Body

 

Body weight (g)

Weight

Weight

Animal

Dose

(mg/kg)

Change

Change

Group

N o & Sex

 

on Day

on Day

 

Day 0

Day 7

Day 14

0 – 7

7 – 14

 

1F

 

171.6

177.8

184.6

  • 3.6 7.6

 

I

2F

2000.0

173.5

179

186.2

  • 3.2 7.3

 

3F

173.6

179.4

187.1

  • 3.3 7.8

 
 

4F

 

172.1

179.3

184.6

  • 4.2 7.3

 

II

5F

2000.0

172.8

180.1

187.8

  • 4.2 8.7

 

6F

175.5

182.6

188.9

  • 4.0 7.6

 

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 Acute Oral Toxicity Study of Dia Bliss Sugar in Rats APPENDIX

Acute Oral Toxicity Study of Dia Bliss Sugar in Rats

APPENDIX 3

Necropsy Findings of Individual Animal

Group

Animal

Dose

Mode of

External

Internal

N o & Sex

(mg/kg)

Death

 

1F

 

TS

NAD

NAD

2F

  • 2000.0 TS

 

NAD

NAD

I

3F

TS

NAD

NAD

II

4F

 

TS

NAD

NAD

5F

  • 2000.0 TS

 

NAD

NAD

6F

TS

NAD

NAD

NAD- No Abnormalities Detected; TS: Terminal Sacrifice

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

APPENDIX 4

Classification

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

SLS Study Number : SLS/043-11 Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

APPENDIX 5

Record of Deviation from the Study Protocol

It is declared that there were no study plan deviations during conduct of the study.

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

APPENDIX 6

Feed Analysis Report

SLS Study Number : SLS/043-11 Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

APPENDIX 7

Water Analysis Report

SLS Study Number : SLS/043-11 Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

SLS Study Number : SLS/043-11

SLS Study Number : SLS/043-11 Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats

APPENDIX 9

Certificate – Department of Science and Industrial Research

SLS Study Number : SLS/043-11 Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
Appendix 3: SUB-ACUTE ORAL TOXICITY STUDY OF DIABLISS SUGAR IN WISTAR RATS 60

Appendix 3: SUB-ACUTE ORAL TOXICITY STUDY OF DIABLISS SUGAR IN WISTAR RATS

60

SLS Study Number: SLS/042-11

CONFIDENTIAL

TITLE PAGE

REPORT

SLS Study Number: SLS/042-11 CONFIDENTIAL TITLE PAGE REPORT 28 Day Repeated Dose Oral Toxicity Study of

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

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

SLS Study Number: SLS/042-11

SLS Study Number: SLS/042-11 STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS No claim of confidentiality is made

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

1/283, Shripuram Street Shripuram, Chennai- 600 097 Tamil Nadu

Signature

____________________

Sponsor/Submitter

Date

 

_____________________

SLS Study Number: SLS/042-11 STATEMENT OF COMPLIANCE Study Title : 28 Day Repeated Dose Oral Toxicity
SLS Study Number: SLS/042-11
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

SLS Study Number: SLS/042-11

SLS Study Number: SLS/042-11 STATEMENT OF QUALITY ASSURANCE UNIT Study Title : 28 Day Repeated Dose
STATEMENT OF QUALITY ASSURANCE UNIT Study Title : 28 Day Repeated Dose Oral Toxicity Study of
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:
Dates Reported to the
Audit
Details
Dates of Audit
N o
Study Director
Management
1
Randomization, Body
Weight, Dosing
18/10/2011
18/10/2011
18/10/2011
Dosing, Body Weight
2
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:

SLS Study Number: SLS/042-11

TABLE OF CONTENTS

SLS Study Number: SLS/042-11 TABLE OF CONTENTS TITLE 1 STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS 2

TITLE

1

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

2

STATEMENT

OF

COMPLIANCE

3

STATEMENT OF QUALITY ASSURANCE

4

Table of Contents

5

PERSONNEL INVOLVED IN THE STUDY

7

8

10

  • 1.0 INTRODUCTION

11

Study Objective

  • 1.1 ...............................................................................................................

11

Guideline

  • 1.2 .........................................................................................................................

11

  • 1.3 Selection of the Test System

Justification for

11

  • 1.4 Testing Facility and Study Period

11

  • 2.0 EXPERIMENTAL PROCEDURE

11

  • 2.1 ..........................................................................................................................

Test Item

11

Animals

  • 2.2 ............................................................................................................................

12

Acclimatization

  • 2.3 ...............................................................................................................

12

  • 2.4 Environmental Conditions

12

Housing

  • 2.5 ............................................................................................................................

12

Feed and

  • 2.6 Water................................................................................................................

12

Grouping

  • 2.7 ..........................................................................................................................

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

Clinical Signs

  • 3.1 ..................................................................................................................

14

  • 3.2 Weight....................................................................................................................

Body

14

  • 3.3 Food Consumption

..........................................................................................................

14

  • 3.4 Clinical Pathology Observations

14

Gross Pathology

  • 3.5 ..............................................................................................................

15

  • 3.6 Evaluation of Results

15

 
  • 4.0 ARCHIVES

16

  • 5.0 RESULTS AND DISCUSSION

16

Mortalities

  • 5.1 ........................................................................................................................

16

  • 5.2 Clinical Observations

16

  • 5.3 Weight....................................................................................................................

Body

16

  • 5.4 Food Consumption

16

Haematology

  • 5.5 ...................................................................................................................

16

  • 5.6 Clinical Chemistry

16

Organ Weight

  • 5.7 ..................................................................................................................

17

  • 5.8 Relative Organ Weight

17

  • 5.9 Gross Pathological Findings

17

5.10

Histopathology

17

SLS Study Number: SLS/042-11

SLS Study Number: SLS/042-11 6.0 .................................................................................................................. CONCLUSION 17 7.0 .................................................................................................................... TABLE 1 - Clinical Signs –
  • 6.0 ..................................................................................................................

CONCLUSION

17

  • 7.0 .................................................................................................................... TABLE 1 - Clinical Signs – Summary by Group

REFERENCES

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

....................................................................

18

19

20

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

..........................................................

22

TABLE 4 - Hematology - Group Mean Values

........................................................................... TABLE 5 - Clinical Chemistry - Group Mean Values TABLE 6 - Organ Weights (g) - Group Mean Values

................................................................

.................................................................

24

26

28

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

................................................

TABLE 8 - Gross Pathology Findings – Summary by Group

.....................................................

30

32

APPENDIX – A

................................................................................................................................. Clinical Signs of Individual Animals

........................................................................................

33

33

APPENDIX – B

.................................................................................................................................

35

Individual Animal Body Weight (g)

.........................................................................................

35

APPENDIX – C Food Consumption APPENDIX – D

.................................................................................................................................

(g/cage).......................................................................................................

................................................................................................................................. Individual Animal Haematological Values

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

.................................................................................................................................. Individual Animal Organ Weights (g)

......................................................................................

37

37

39

39

43

43

47

47

APPENDIX – G

................................................................................................................................. Individual Animal Relative Organ Weights (%)

......................................................................

51

51

APPENDIX – H

.................................................................................................................................

55

Gross and Microscopic Findings of Individual Animals APPENDIX – I ...................................................................................................................................

.........................................................

55

59

Feed Analysis Report

.................................................................................................................

59

APPENDIX – J

................................................................................................................................... Water Analysis Report

...............................................................................................................

60

60

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

.................................................

61

61

SLS Study Number: SLS/042-11 PERSONNEL INVOLVED IN THE STUDY Signature Study Director Syed Nasir Ahamed, M.Sc
SLS Study Number: SLS/042-11
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

SLS Study Number: SLS/042-11

LIST OF ABBREVIATIONS

SLS Study Number: SLS/042-11 LIST OF ABBREVIATIONS AD Average Dose ALB Albumin ALP Alkaline Phosphatase B.wt