View
228
Download
0
Category
Tags:
Preview:
DESCRIPTION
For the first time in the world, Diabliss has been successful in making cane sugar diabetic friendly by blending cane sugar with a proprietary herbal extract. The herbal extract is prepared from Black Pepper, Fenugreek, Cinnamon, Ginger, Turmeric, Gooseberry and Pomagranate using water and no other chemicals or solvents. The propriatary herbal extract is rich in antioxidants, polyphenols, soluble fibres and other nutrients. Diabliss Sugar is a drop in replacement to cane sugar and diabetics can now safely consume a limitless choice of foods. This report summarises product performance trials including extensive analysis, acute & sub acture toxicity trials, glycermic index tests, short and long term studies on type 1 and type 2 diabetics for periods ranging from 3 months to 4 years. More details on diabliss may be found at www.diabliss.com
Citation preview
1 | P a g e
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.
2 | P a g e
TABLE OF CONTENTS
Section Sub
Section
Topic Page
Number
1 1.1 Executive Summary 3
1.2 Company Background 4
2 Diabetes Background 6
2.1 Diabetes Trends 6
2.2 Diabetes - Lifestyle Issues 9
3 DiaBliss Herbally Blended Products: An Integrated Food System for
Sugar, Carbohydrate and Fructose Management
9
3.1 DiaBliss Product Versatility & Competitive Differentiation 11
4 DiaBliss Product Performance 12
4.1 Product Characterisation at SGS 12
4.2 Animal Trials at Sugen Life Sciences 13
4.2a Summary of Acute Toxicity Trials 13
4.2b Summary of Sub-Acute Toxicity Trials 14
4.3
4.4
Glycemic Index Studies of DiaBliss Herbal Treated Sugar
GTT Test with Diabliss based sweets on diabetics
15
16
4.5 Short Term Glucose Tolerance Tests 17
4.5a GTT Blood Sugar Levels 18
4.5b Extended GTT Tests – Urine Glucose (Glycosuria) Levels 19
4.6 Long Term Test Results 20
4.7 Fasting and Post Prandial Response – Type 1 and Type 2 Diabetics 23
4.8 Fasting, Post Prandial and HbA1c Correlations: Type 1 & Type 2
Diabetics
23
4.9 DiaBliss Sugar Long Term Use in Juvenile Diabetic 24
5 References 25
A1 Appendix 1A: Metals Analysis of Diabliss Sugar analyzed at SGS
Laboratories
Appendix 1B: Summary of Herbicide Analysis of Herbal Solution
analyzed at SGS Laboratories
26
29
A2 Appendix 2: ACUTE ORAL TOXICITY STUDY OF DIABLISS
SUGAR IN WISTAR RATS
34
A3 Appendix 3: SUB-ACUTE ORAL TOXICITY STUDY OF
DIA BLISS SUGAR IN WISTAR RATS
60
A4
Appendix 4: Glycemic Index Measurement of Diabliss Sugar 121
3 | P a g e
Section 1:
1.1 Executive Summary:
DIABLISS HERBAL CANE SUGAR – SUMMARY OF PRODUCT PERFORMANCE
For the first time in the world we are able to render pure cane diabetic friendly sugar by blending
sugar with a proprietary blend of herbal extracts using techniques from traditional Indian medicine
system. The herbal extract is derived from several herbs and spices (black pepper, turmeric,
fenugreek, ginger, pomegranate, etc) using water and no other chemicals or solvents. The herbal
extract is colorless, odorless and tasteless and thereby restores the original taste factor of cane sugar
& derivative products.
We have successfully completed acute and sub-acute oral toxicity tests in mice. The product has
been extensively analyzed for heavy metals, pesticides and insecticides and proven to be fit for
human consumption. The product has been tested in thousands of people from periods ranging from
3 to 36 months and data to date indicates that the product is safe and in fact lowers long term blood
sugar levels as measured by HbA1c. The product has been tested in subjects ranging in age from 7 to
82 and is effective in Type 1 & Type 2 diabetics.
The attached report summarizes product performance test results.
We have also developed and tested a range of products for carbohydrate & fructose
management. These products enable diabetics to consume an unlimited choice of foods without
compromising on taste factors and thereby restore serious lifestyle constraints that diabetics face
every day with limited food choices. We are the only product that provides sugar, carbohydrate and
fructose management system thereby providing a complete and integrated approach to diabetic
friendly foods. Over 20 products have been formulated and tested. They include salt, rice, multi
grain porridges, instant beverages (lemon tea, masala tea, coffee, lime juice mix), chocolates,
premixed sweet mixes (kheer mix, kesari bath mix, etc). These products provide comprehensive
carbohydrate, fructose and glucose management.
4 | P a g e
1.2 Company Background:
DiaBliss Consumer Products Pvt Ltd is a private limited company formed to commercialize
DiaBlissTM
brand consumer products to meet the extensive and unmet needs of diabetics all over the
world. The company has developed herbal treated sugar and a variety of derivative products that
allow diabetics to consume a limitless variety of foods without compromising taste factors normally
associated with artificial sugar substitutes. DiaBliss Consumer Products is committed to improving
the lifestyle of diabetics all over the world by providing safe, holistic and integrated sugar and sugar
based foods.
Management Team & Board of Directors:
V R Ramanathan is CEO & Board Member of DiaBliss Consumer Products. He also serves as the CEO of Electron India and has been involved in manufacturing since1974. He has been involved in manufacture of photoelectric cells, electronic head for fuel pumps at retail outlets. For the last 12 years he is involved in manufacture of RFID related products for physical access, logical access, vehicle access & traking, asset tracking, e-cash solution on smart cards. Ramanathan has been working on Diabliss herbal treated sugar for over four years in all aspects of animal & human safety testing, long term subject trials, product testing, and derivative products development. He holds a BE degree in Electronics Engineering from Guindy College of Engineering.
Siva Vallabhaneni is COO & Board Member of DiaBliss Consumer Products Pvt Ltd. He served as a senior executive in several MNCs including DuPont, Tronox, Cristal Global. Most recently he served as Chief, Global Intelligence at Cristal Global serving as an advisor to Chairman, President and Senior Executives in the areas of business transformation and strategic growth. He has 25 years’ experience in strategic planning, supply chain management, operational excellence, and general business management, M&A, Manufacturing, R&D in Chemicals, FMCG industries. He is holder of two worldwide patents. He holds a B Tech in Chemical Engineering from Andhra University and M. S. in Chemical Engineering from Clarkson University, USA
5 | P a g e
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).
6 | P a g e
Section 2. Diabetes Background1
Diabetes, also known as Diabetes Mellitus, is a chronic health condition where the body is unable to
produce enough insulin and properly break down sugar (glucose) in the blood. Glucose comes from
food and is used by the cells for energy. Glucose is also made in the liver. Insulin is a hormone
produced by the pancreas, a large gland behind the stomach. Insulin is needed to move sugar into the
cells where it can be used for energy needed for bodily processes.
After digestion of food, glucose passes into the bloodstream. For glucose to get into cells, insulin
must be present. Throughout the pancreas are clusters of cells called the islets of Langerhans. Islets
are made up of several types of cells, including beta cells that make insulin. When normal individuals
eat, beta cells in the pancreas automatically produce the right amount of insulin to move glucose from
blood into the cells of the body. In individuals with diabetes, however, the pancreas either produces
little or no insulin, or the cells do not respond appropriately to the insulin that is produced. Glucose
builds up in the blood, overflows into the urine, and passes out of the body in the urine. Thus, the
body loses its main source of fuel even though the blood contains large amounts of glucose. Glucose
may also interact with cells, especially those in very narrow blood vessels. This process may lead to
neuropathies and decreased immune function.
With Type 1 diabetes, the body does not make any insulin. With Type 2 diabetes, the more common
type, the body does not make or use insulin properly. Without enough insulin, glucose stays in the
blood and causes a condition called hyperglycemia, or high blood sugar levels.
Diabetes is associated with long-term complications that affect almost every part of the body. The
disease often leads to blindness, heart and blood vessel disease, stroke, kidney failure, amputations,
and nerve damage. Uncontrolled diabetes can complicate pregnancy, and birth defects are more
common in babies born to women with diabetes. Pregnant women can temporarily develop
gestational diabetes, a type of diabetes that begins late in pregnancy.
2.1 Diabetes Trends2,3
:
Although increases in both the prevalence and incidence of type 2 diabetes have occurred globally,
they have been especially dramatic in societies in economic transition in much of the newly
industrialized world and in developing countries. Worldwide, the number of cases of diabetes is
currently estimated to be around 150 million as per the World Health Organization. This number is
predicted to double by 2025, with the greatest number of cases being expected in China and India.
These numbers may represent an underestimate and there are likely to be many undiagnosed cases.
Previously a disease of the middle aged and elderly, type 2 diabetes has recently escalated in all age
groups and is now being identified in younger age groups, including adolescents and children,
especially in high-risk populations.
India is now being called the diabetes capital of the world. India accounts for the largest number of
the diabetic population in the world: 62.4 million live with diabetes (5.4% of Indian population), 77.2
million people (6.4% of Indian population) live with pre-diabetes with high propensity to acquire the
7 | P a g e
disease. This is followed by China (43.2 million) and the United States (26.8 million), as per figures
released by the International Diabetes Federation (IDF).
Another major region where diabetes is expected to reach epidemic levels is the Middle East and
Northern Africa (MENA) Region. IDF estimates 32.6 million or 9.1% of the population now have
diabetes and this number is expected to double in less than 20 years. In most parts of the world, for
every diabetic there is a pre diabetic with propensity to acquire the disease. For example India’s
diabetic population is estimated at 7.1% of the population and pre diabetic levels are estimated at 7%
of the population.
IDF estimates that 347 million people worldwide have diabetes. By 2030, nearly 9 per cent of the
country’s population is likely to be affected from the disease, warns the fourth edition of the World
Diabetes Atlas launched by the IDF at the 20th World Diabetes Congress in Montreal, Canada. New
data projected that the number of people with diabetes will rise to 552 million by 20304.
In fact the prevalence of diabetes in India has almost doubled in a period of 11 years as per ICMR-
INDIAB study3. This alarming increase in diabetes could be partially related to better diagnostic
infrastructure, but largely related to life style factors combined with genetic propensity for the
disease. Diabetes in India is also no longer the disease of the middle aged. Like developed countries,
the disease has become prevalent among people in their 20s, 30s and 40s. In fact we are also
beginning to see an alarming increase in diabetes among teenagers, following similar trends being
observed in United States and other western countries.
.
8 | P a g e
In India, genetic disposition, coupled with lifestyle factors such as stress and eating habits are largely
contributing towards the rapid spread of the disease
Data on sugar consumption has shown significant increase in per capita sugar consumption across the
world. The following data indicates a 350% increase in per capita sugar consumption in India in the
last 50 years.
Per Capita Sugar Consumption in India is 18-20 kg/year and about 5 kg/yr of Gur & Khandasari
bringing total consumption to approximately 25 kg/yr/per person (67g/person/day)
US per capita sugar consumption is 71 kg/yr/person (194g/person/day). As we are moving towards a
more hectic life style, there has been a corresponding multi fold increase in per capita sugar
consumption as there is sugar in almost every prepared food to impart the desired taste factor. If we
only study the urban population in tier 1 cities in India, we can expect per capital sugar consumption
approaching the same levels in the US where the per capital consumption is almost 300% higher than
the average per capital consumption in India. We are following similar eating patterns of fast foods
and prepared foods, all of which contain sugar.
Currently, up to 11 per cent of India’s urban population and 3 per cent of rural population, above the
age of 15 years, have diabetes. Diabetes affects all people in the society, not just those who live with
it. The World Health Organization estimates that mortality from diabetes and heart disease cost India
about $210 billion every year and is expected to increase to $335 billion in the next ten years. These
estimates are based on lost productivity, resulting primarily from premature death.
9 | P a g e
2.2 Diabetes – Lifestyle Issues:
Most diabetics undergo a significant change in lifestyle after being diagnosed with the disease:
i. They cannot consume sugar or sweets
ii. They cannot consume foods rich in carbohydrates
As a result, their life style is severely impeded.
Diabetics typically use sugar substitutes such as Aspertame, Sucralose, Saccharin, Stevia, Levulose,
etc. that go by various trade names. These products are either derived from chemical routes or
extracts from fruits. Some diabetics consume sugar in limited quantities for their coffee or tea every
day, as they are used to the natural cane sugar taste and are not able to be satisfied by the sweeteners.
Products made with these substitutes have a serious after taste, but diabetics normally do not have an
option but to consume these products. In some cases diabetics occasionally consume sugar based
sweets to quench their craving for sugar. All these sweets or product preparations only address the
sugar portion of the diet, but when a diabetic consumes foods rich in carbohydrates or fruits, they are
not able to address carbohydrate and fructose management issues as these materials are next in line
with respect to breakdown into blood sugars due to their rapid breakdown and conversion into
glucose which becomes available in the blood stream.
Section 3: DiaBliss Herbal Blended Products: An Integrated Food System for Sugar,
Carbohydrate and Fructose Management
In order to address the diabetes epidemic, the inventors of DiaBliss turned back to the classical
Indian medical sciences system to understand the disease. After 10 years of R&D and 4 years of
product testing, for the first time in the world, we are able to render sugar diabetic friendly by
blending with herbal extracts. The herbal extracts are derived from herbs and spices (Black Pepper,
Turmeric, Fenugreek, Ginger, Gooseberry, Cinnamon and Pomegranate) using water and no other
chemicals or solvents and retains the original taste of cane sugar.
Herbs have been used as food and for medicinal purposes for centuries. Herbal medicine is based on
the premise that plants contain natural substances that can promote health, wellness and alleviate
illness. In different herbs, a wide variety of active phytochemicals, including the flavonoids,
terpenoids, lignans, sulfides, polyphenolics, carotenoids, coumarins, saponins, plant sterols,
curcumins, and phthalides have been identified. Many of these herbs contain potent antioxidant
compounds that provide significant protection against chronic diseases. These compounds may
protect LDL cholesterol from oxidation, inhibit cyclooxygenase and lipoxygenase enzymes, improve
metabolism, enhance insulin sensitivity, inhibit lipid peroxidation, or have antiviral or antitumor
activity. Today we are witnessing a great deal of public interest in the use of herbal remedies and
ingredients in foods. There has been considerable research in the efficacy of the various components
used in the Diabliss herbal extract. The following summarizes some of the key findings from
research into efficacy of these herbs and spices.
10 | P a g e
1. Black Pepper has anti-inflammatory and antioxidant properties, and so helps to prevent the
risks of cancers. It also may have anti-microbial properties, and it contains vanadium which is
being researched for its potential benefits in improving insulin sensitivity and blood sugar
levels in people with Type -2 diabetes.
2. Turmeric – Curcumin, an ingredient derived from turmeric, combined with extracts from
black pepper and onion skin has been found to be effective against obesity, diabetes and
abnormal cholesterol levels. The combination which goes by acronym CPQ - curcumin,
piperine (derived from black pepper) and quercetin (found in onion and skinks of some fruits)
- could control diet-induced changes in body weight, blood glucose, triglyceride, cholesterol,
and low-density lipoprotein (LDL).
3. Fenugreek – Trigonella foenum graecum – The seeds of this plant contain an alkaloid
trigonelline and another compound known as choline. These seeds have been reported to be
diuretic, anti-tussive and hypoglycaemic in nature. Research studies show that fenugreek
seeds decrease blood glucose and triglyceride levels and have significant effect on LDL or
HDL cholesterol when consumed at higher doses.
4. Ginger - The active ingredient in ginger, the pungent phenolic gingerol constituents were
identified as the major active compounds in the ginger extract enhancing glucose uptake.
According to researchers, extracts from ginger are able to increase the uptake of glucose into
muscle cells independently of insulin.
5. Indian gooseberry (Amla) has been used widely in ayurvedic medicine for thousands of
years. It has been shown to be effective in lowering cholesterol, “hardening of the arteries”
(atherosclerosis), diabetes, pain and swelling of the pancreas (pancreatitis), cancer, upset
stomach, eye problems, joint pain, diarrhea, dysentery, obesity, and “organ restoration.” Some
research studies also indicate that amla may prevent age-related hyperlipidaemia through
attenuating oxidative stress in the ageing process.
6. Cinnamon – Polyphenols from cinnamon impacts glucose metabolism through helping in the
following five pathways: 1. Increase in glucose metabolism about 20-fold, which
significantly improves blood sugar regulation. 2. Polyphenols in Cinnamon has been found to
have "insulin-like effects" due to a bioactive compound, qualifying it as a candidate for an
insulin substitute. 3. Cinnamon slows the emptying of your stomach to reduce sharp rises in
blood sugar following meals, and improves the effectiveness, or sensitivity, of insulin. 4.
Cinnamon actually enhances antioxidant defenses. Several studies have shown that daily
consumption of cinnamon lowers serum glucose, triglyceride, LDL cholesterol, and total
cholesterol in people with type 2 diabetes and suggest that the inclusion of cinnamon in the
diet of people with type 2 diabetes will reduce risk factors associated with diabetes and
cardiovascular diseases.
7. Pomagranate - Consumption of pomegranate (Punica granatum, Punicaceae) fruit and/or
juice has been reported to reduce the risk of CVD and prostate cancer. Each pomegranate
contains 600–800 seeds and when purified, oil from these seeds (POMo) is a rich naturally
occurring source of the bioactive compound 9-cis, 11-trans conjugate linolenic acid (CLA).
Studies have shown that Pomogranate Seed Oils showed improvement of insulin sensitivity
which is associated with a decrease in the risk of developing type 2 diabetes.
11 | P a g e
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.
12 | P a g e
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
13 | P a g e
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
14 | P a g e
DiaBliss Sugar is being classified as follows :
Globally Harmonised System (GHS) : Low Toxicity
Appendix 2 summmarises Acute Oral Toxicity trial report of DiaBliss Herbal Cane Sugar in Wistart
rats.
4.2b Summary of Sub-Acute Toxicity Trials
In the sub acute toxicity trials, 28 Day Repeated Dose Oral Toxicity Study of DiaBliss Sugar in
Wistar Rats was conducted. This study was conducted to assess the toxic effects of DiaBliss Sugar in
Wistar Rats, when administered orally for a period of 28 consecutive days.
Three groups of Rats comprising of 6 males and 6 females per group were treated with DiaBliss
Sugar at 180.0, 540.0 and 1800.0 mg/kg bd. wt. for a period of 28 consecutive days. A concurrent
control group, comprising of the same number of animals was also maintained in the study.
Rats were observed individually for visible signs of reaction to the treatment twice a day throughout
the study period. Individual body weight and feed consumption were monitored weekly.
Haematological and biochemical analyses of blood samples were performed on all animals at the end
of the treatment period. All the surviving rats were sacrificed and subjected to a gross pathological
examination at the end of the treatment period. Absolute organ weights were recorded and relative
organ weights were calculated for the organs viz., liver, kidneys, adrenals, testis/ovary,
epididymides/uterus, thymus, spleen, brain and heart in all rats.
The animals exposed to test item did not show any specific treatment related clinical changes in all
the treated groups. No significant changes were observed in the mean body weight and food
consumption in any of the treated group animals.
Haematological analysis and clinical chemistry analysis did not reveal any treatment related changes
in any of the parameters at various dose levels studied in either sex.
The absolute and relative organ weights did not reveal any significant difference in the treated
groups’ animals when compared with the control group animals in either sex. Gross pathological
examination of the animals did not reveal any treatment related effects in any of the dose group.
Histopathological examination of the high dose group animals did not reveal any treatment related
effects compared to control group animals. There was no test item related toxicity recorded in any of
the treatment groups under the experimental conditions.
Appendix 3 summmarises Sub-Acute Oral Toxicity trial report of DiaBliss Herbal Cane Sugar in
Wistart rats.
15 | P a g e
4.3 Glycemic Index Studies of DiaBliss Herbal Cane Sugar
The effect of Natural Sugar on the alterations in the blood glucose levels as measured by Oral
Glucose Tolerance Test (OGTT) was compared against a glucose standard in a non-blind, repeated
measure, crossover design trial. Twelve healthy subjects with a mean age of 30.25 (SD 4.55) years
were recruited. Subjects were served with either 75 grams of Natural Sugar (Test food) or a standard
substance glucose weighing 75 grams, on two different days. Blood glucose was determined from
venous blood samples in fasted subjects (0 min) and at every 15 min interval in first one hour and
every 30 minute intervals in second hour after the consumption of Natural Sugar or glucose. The GI
value was calculated geometrically by expressing the incremental area under the blood glucose curve
(iAUC) as a percentage of each subject’s average iAUC for the standard food. The GI value of the
Natural Sugar was found to be 53.93 (range 26.04-77.60), and can be classified under low glycemic
food.
Results
Table1: Calculation of iAUC after supplementation with glucose and DiaBliss Herbal Cane Sugar
(Data as Mean±S.D)
--------------------------------------------------------------------------------------------------------------------------
Blood glucose concentration (mg %) Mean iAUC GI
------------------------------------------------------------------------------------------------
Time periods N 0 15 30 45 60 90 120
(min)
Glucose 12 68.00 107.42 140.08 136.67 120.50 121.17 102.00 86.33
Bal ±11.40 ±13.15 ±16.34 ±21.56 ±25.72 ±21.94 ±25.25
DiaBliss 12 67.83 96.25 110.75 102.83 96.67 91.58 85.08 53.93
Sugar ±11.42 ±13.80 ±11.93 ±14.66 ±15.84 ±10.63 ±13.61 47.08
--------------------------------------------------------------------------------------------------
N = Sample size, iAUC = Incremental Area under the Curve, GI=Glycemic Index, min=minutes
Inference: The GI value of the DiaBliss Herbal Cane Sugar tested in the present study was found to be 53.93.
Therefore it is classified as a food / nutrient with low GI.
As per the Food and Agriculture Organization GI cut-off values are as follows:
Low <55; Medium 56–69 inclusive; High >70 (Brand-Miller et al. 2003).
GI values of other foods: Glucose: 99; Fructose: 19 ; Sucrose: 69-80 (Table sugar); Maltose:
105; Lactose: 46
16 | P a g e
Detailed test report on Glycemic Index studies on Diabliss Sugar conducted at Sugen Life Sciences
may be found in Appendix
4.5 GTT Test with Untreated Sugar & Diabliss Sugar based sweets on diabetics
A similar curve was observed when a GTT test was conducted on four diabetcis using an Indian
sweet meat (Ravva laddu).
In the first testing diabetics were given four ravva laddus containing 60g of ravva mix along with 60g
of untreated sugar. The blood sugar levels were measured with a strip glucometer every ½ hour for a
period of two hours. The following day, the same ravva laddu was made with 60g of Diabliss Sugar.
Also, the carbohydrate in the ravva was addressed by treating it with herbal solution at the rate of
20ml/kg.
The average blood sugar levels were normalized and data in Figure below clearly demonstrates the
increased rate of absorption of the Diabliss based sweets.
17 | P a g e
4.5 Short Term Glucose Tolerance Tests
We initiated a short term glucose tolerance tests with 11 subjects, 9 of whom were known diabetics.
Also, two of the subjects were regular consumers of alcohol. Each subject was given 50 grams of
DiaBliss Herbal Cane Sugar solution dissolved in water. Blood samples were collected prior to
intake (i.e., fasting level) and at 1, 2 and 3 hours. Blood samples were collected by an independent
laboratory and blood sugar levels were measured by colorimetric technique.
Table 1 summarizes the details of the test candidates. In order to preserve the confidentiality, we
have not shown the names of the subjects. Please note, subjects 3 & 6 are regular consumers of
alcohol whose liver function may have already been impeded and alcohol load in the system may
already be high.
Table 1: Details of Subjects for extended GTT Test:
ID Age Sex How long
diagnosed
with
diabetes?
(yrs)
Did you
consume
Medication?
If yes, Specify
Type 1 Type 2 Salt Sugar
1 58 M x 8 Yes Amaril, 1000mg, 1-0-1 Yes Yes
2 54 M x 15 None No No
3 49 M No No
4 48 M x No No
5 65 M x 3 Yes Yes Yes
6 49 M x 17 Yes Metformin 500mg 1-0-1 Yes Yes
7 45 M x 3 Yes Yes Yes
8 80 M x 7 Yes No No
What Diabiss
Products are you
cosuming
Type of Diabestes
Not Diabetic
18 | P a g e
All subjects who consumed 50g of the DiaBliss sugar did not complain of any discomfort after
consuming this quantity of sugar. In addition, subject 9, a 45 year old male consumed both normal
cane sugar and DiaBliss sugar on two separate occasions.
4.5a GTT Blood Sugar Levels
The blood sugar profile of the subjects is summarized in Figure 1 and Table 2 below:
Table 2: Blood Sugar Profiles of Subjects consuming 50g of DiaBliss Herbal Blended Sugar:
We can see from the data above that even after consuming 50g (over 200% of daily sugar
consumption in a single dose), all subjects, with the exception of the two alcoholics, have shown that
0
50
100
150
200
250
300
0 20 40 60 80 100 120 140 160 180 200
Blo
od
Su
gar
Re
adin
gs, m
g/d
l
Time, Minutes
Fig 1: Extended GTT Tests with 50g intake of Diabliss Sugar Among Group of Diabetic & Non-diabetic Subjects
1 2 3 4 5 6 7 (Reg Sugar) 7 (Diabliss) 8
Subject
Number
1 2 3 4 5 6 7 (Reg
Sugar)
7
(Diabliss)
8
Diabetic Diabetic Non-Diabetic Diabetic Diabetic Diabetic Diabetic
Time, min
0 116 142 104 156 135 121 142 81 140
60 221 233 154 261 224 179 255 203 266
120 164 158 97 217 142 191 226 134 189
180 123 118 90 164 112 103 108 70 171
Diabetic
Blood Sugar Readings with Calorimeter, mg/dl
19 | P a g e
the blood sugar levels have dropped below or very close to fasting sugar levels confirming the
excellent absorption characteristics of DiaBliss Herbal treated sugar.
4.5b Extended GTT Tests – Urine Glucose (Glycosuria) Levels
The data summarized in table 3 shows urine glucose (glycosuria) levels of the subjects during the
tests. When the blood sugar levels typically exceed 180-200 mg/l level, the kidneys start to excrete
glucose from the urine as glycosuria5.
Table 3: Urine Analysis from Extended GTT Test: Glycosuria Profiles in Test Subjects:
The above data on the ranges of glycosuria characterized by the measurements indicate interesting
trends:
1. Urine glucose levels of all subjects, with the exception of the two chronic alcoholics in terms
of not seeing major spikes, as well as returning back to levels at fasting is a further indication
of excellent sugar absorption from the herbal treated sugar.
2. Subject number 7 shows an interesting glycisuria level. This is the subject who participated in
two extended GTT tests: Data labeled 7 (Regular Sugar) and 9(Diabliss) characterizes
glycosuria profiles with regular cane sugar and DiaBliss herbal treated sugar respectively. It
is interesting to note a lower glycosuria profile with herbal treated sugar suggesting good
blood sugar control which therefore does not require triggering of the body safety mechanism
to reject excess blood glucose through urine as glucosuria.
4.6 Long Term Test Results
DiaBliss herbal cane sugar has been consumed by hundreds of diabetics over the course of last four
years. We have also followed their blood sugar readings over the years and the following sections
summarize a sampling of the results among type 1 and type 2 diabetics. In the current data summary,
the consumption time line varied from 3 months to 34 months. Subjects varied in age from 7 to 72,
type 1 and type 2 diabetics.
Subject Note:
Number Fasting 60 min 120 min 180 min Reading
1 Nil + Trace Nil Trace
3 Nil Trace Nil Nil ++
4 Trace ++ + Trace +++
5 Nil + Nil Nil
6 Nil Trace + Nil
7 (Regular Sugar) Nil ++ + Nil
7 (DiaBliss) Nil Trace Trace Nil
8 Nil Trace Nil Nil
9 Nil + Trace
500-1,000 mg/dl
Urine Analysis
Up to 250 mg/dl
Urine Glucose (Glycosuria)
1,000-1,500 mg/dl
20 | P a g e
We have measured fasting blood sugar, post prandial blood sugar and HbA1c (long term, i.e., 3
month blood sugar average). We have found the following from this long term study:
Product lowered HbA1c levels within 60-90 days from consumption of DiaBliss products
Fasting & PP levels showed decreasing trends immediately
Subjects report more energetic feeling; Major lifestyle improvements were observed,
especially with juvenile diabetics
HbA1c or glycated hemoglobin or glycosylated hemoglobin is a form of hemoglobin that is measured
primarily to identify the average plasma glucose concentration over prolonged periods of time,
typically two to three months. Normal levels of glucose produce a normal amount of glycated
hemoglobin. As the average amount of plasma glucose increases, the fraction of glycated hemoglobin
increases in a predictable way. This serves as a marker for average blood glucose levels over the
previous months prior to the measurement.
Recommended HbA1c readings fall within the reference range of 6.5 to 7%. This implies that for
every 100 red blood cells, 6-7 cells have glucose attached to them. Mean blood sugar levels can be
better understood from the following table7.
Table 4 summarises fasting, post prandial (2 hour readings) and HbA1C levels of various subjects
who are continuing to consume DiaBliss Herbal treated sugar for periods ranging from 3 months to
36 months. These subjects ranged in age between 7 years and 82 years. We also selected data to
illustrate product performance among type 1 and type 2 diabetic
% mmol/L mg/dL Inference
3-4 2-4 36-72 Extremely low to low blood sugar
4-5 5.5 99-100 Perfect!
4.5-5 5-6 90-108 Normal range for non-diabetics (pre-prandial)
5.5 - 6 7 – 8 126 - 144 Normal post-prandial in non-diabetics
6.5 – 7 9-10 162 - 180 Maximum post-prandial in non-diabetics
6.5 – 7.5 9 – 11 162 - 198 High, even for diabetics
7.5 – 9.5 11 – 15 198 - 270 Indicates to poorly controlled BG
12.5 - 25 20 - 25 360 - 450 Extremely high
> 19 33 or > 33 > 594 High possibility of serious electrolyte imbalance!!
HbA1c-Average Blood Glucose Reading
21 | P a g e
The above data is presented in the following plots to give us a better graphical characterization of
DiaBliss product performance among a vastly different population. Figure 2 compares HbA1c levels
between type 1 and type 2 diabetics. Data plotted below show profiles prior to start of consumption
of DiaBliss herbal treated sugar and after commencement of regular consumption of sugar.
The following are details of the subjects:
1. Male, Type 2 diabetic, Age 51, diabetic since 1 year. He has been consuming Diabliss since
10 months. HbA1c levels have dropped from 7.4 to 6.2 or 16% reduction within three months
from start of consumption of DiaBliss.
2. Male, Type 2 diabetic, Age 50, diabetic since 14 years. DiaBliss consumption since 30
months. HbA1C levels varied between 7.0 and 8.2 pror to consumption of DiaBliss Sugar.
His current levels have stabilized around 6.7 currently. In the graph, there is a step change
reduction in HbA1c levels since November 2011. The subject reports increasing DiaBliss
consumption since this period and a step reduction in HbA1c has been recorded. Over the
course of 30 months, average reduction in HbA1c is from about 7.5 to 6.7 or 11%
3. Male, age 8 years, Type 1 Juvenile diabetic, consuming DiaBliss Sugar since 18 months.
HbA1c levels have dropped from 11.5 to 6.5 or about 45%.
4. Female, age 59 years, Type 1 dabetic, consuming DiaBliss sugar since 9 months. HbA1c
levels have dropped from 8.9 to 6.1 or about 31%.
Figure 2: HbA1C Profiles: Type 1 vs. Type 2 Diabetics
Table 4: Representative Sampling of Fasting, Post Prandial and HbA1c levels of long term consumers of DiaBlissNo Age Sex Profession Type of
Diabetic
Diabetic
Since
How Long
Consuming
Herbal Sugar
Before
Herbal
Sugar
After
Herbal
Sugar
Latest
Reading
HbA1c HbA1c HbA1c Fasting
Average
PP
Average
Fasting
Average
PP
Average
Medication Details
1 54 M 1 Many
Years
12 months 5.4 5.4 210 231 98 137 Insulin 25 units Morning &
Evening before herbal sugar, 15
units morning & evening
2 51 M Consulting
Engineer
2 1 Year 10 months 6.8 6.3 6.2 136 185 122 141 Continuing medication Glycomet
500mg 1-0-1
3 45 M Traveling
Sales
2 3 Years 34 months 8.8 6.7 6.9 Amryl m - 2 mg
4 61 F House Wife 2 8 Years 36 months 7.0 7.0 107 137
5 50 M Engineer 14 Years 30 months 7.6 7.0 6.7 107 177 101 157 Iscept Fort 1- 0 -1 and Pioglit
6 58 M Catering
Business
2 25 months 7.2 6.3 6.3 121 182 114 140 Glycomet 500mg 1-0-1 lowered
to 1/2-0-1/2
7 7.5 M School 1 22 months 18 months 11.5 8.2 7.9 204 281 Child started going to school
after starting herbal treated
sugar; Weight Gain of 2.5 kg
(+10%), Insulin dose increased
8 59 F Housewife 1 7 months 8.9 7.0 6.1 140 200 90 139
9 66 M 2 3 Years 6.6 6.6 101 121
10 58 M 2 2 Years 18 months 7.2 7.2 124 177 No Medication
11 82 M Retired 20 Years 3 Months 8.4 228 372 93 139 Started noticing significant
increase in energy levels
Before Herbal
Sugar
After Herbal Sugar
22 | P a g e
So, in the four subjects we have seen HbA1c levels dropped by 11-16% for type 2 diabetics and about
45% for type 1 diabetics over DiaBliss consumption period ranging from 9 months to 30 months
4.7 Fasting and Post Prandial Response – Type 1 and Type 2 Diabetics
Fasting and Post Prandial (PP) response has the potential to immediately show the impact of
consuming DiaBliss sugar. However, both Fasting and PP are subject to considerable variability. For
example if the subject, the previous night may have indulged in eating foods high in sugar or
carbohydrates, the fasting readings could be skewed.
Figure 3 compares typical fasting and PP plots for type 1 and type 2 diabetics. We can clearly see
immediate responses for both the subjects. In the case of the 82 year old type 2 diabetic, his blood
sugar levels were not responding to medications. Within a short period of consuming DiaBliss Sugar,
he reported feeling more energetic. The other subject, a 53 year old type 1 diabetic who has been
consuming DiaBliss sugar for over 12 months, has lowered Insulin intake from 25 units to 18 units, or
a 28% reduction in Insulin intake.
This response is important as the product seems to have a very visible short term benefit that can be
readily measured. This can be augmented by HbA1c measurements which will start to show longer
term benefit, within 60-90 days from the start of consumption of the DiaBliss product.
23 | P a g e
Figure 3: Fasting & Postprandial response of type 1 & type 2 diabetics
4.8 Fasting, Post Prandial and HbA1c Correlations: Type 1 & Type 2 Diabetics
Figure 4 below shows the plot of Fasting, PP and HbA1c for the type 1 diabetic. We can clearly see
correlation between Fasting, PP and HbA1c levels for this type 1 diabetic. Similarly, Figure 5 shows
Fasting, PP and HbA1c level plots are shown for a type 2 diabetic, a 58 year old who has been
consuming Diabliss Sugar since 15 months. He also shows a similar correlation. In case of this
subject, his diabetes medication level has been lowered by 50%, in close consultation with his
physician.
Figure 4: Fasting, PP, HbA1c Profiles for Type 1 diabetic
Figure 5: Fasting, PP, HbA1c Profiles for Type 2 diabetic
0
50
100
150
200
250
300
350
9.9.06 22.1.07 21.9.07 28.6.09 13.11.2011
Blo
od
Su
gar,
mg
/dl
Type 1 Diabetic, Male Age 52 - Fasting & PP Profiles
Fasting
PP
Started consuming DiaBlissHerbal Cane Sugar15 months consumption
Insulin 25 Units Morning & EveningGemerfort 1-0-1
Insulin 18 Units Morning & Evening Gemerfort 1-0-1HbA1c (4/6/12) 5.4
0
50
100
150
200
250
300
350
400
16/6/2012 5/7/2012 25/7/2012 22/8/2012
Blo
od
Su
gar,
mg/
dl
Type 2 Diabetic, Male Age 82 - Fasting & PP profiles
Fasting
PP
Started Consuming DiaBliss Herbal Cane Sugarin early/mid-June 2012; 4 months; Baseline HbA1c = 8.4
Note: January 2013 HbA1c = 6.3
0
50
100
150
200
250
4/3/2012 14/8/2012 15/9/2012 17/9/2012
Blo
od
Su
gar
Leve
ls, m
g/d
l
Type 1 Diabetic, Female, age 58 - Fasting & PP Profiles
Fasting
PP
Started consuming DiaBliss Herbal Cane Sugarfor Tea & Coffee
6
6.5
7
7.5
8
8.5
9
4/3/2012 6/6/2012 14/8/2012 17/9/2012
Hb
A1
c
Type 1 Diabetic, Female, age 58 - HbA1c Profile
Started consuming DiaBliss Herbal Cane Sugar for Tea & Coffee
24 | P a g e
As we can see from the above charts there is a direct correlation (barring some day to day variability
due to lifestyle or diet factors) between Fasting/Post Prandial blood sugar levels and HbA1c readings
for both type 1 and type 2 diabetics.
4.9 DiaBliss Sugar Long Term Use in Juvenile Diabetic
We wanted to share the impact of DiaBliss product on a Juvenile diabetic. The subject is a 7.5 year
Juvenile diabetic whose life style has significantly improved since consuming DiaBliss. Figure 6
summarises his HbA1c profile. The juvenile diabetic is now able to go to school on a full time basis
(which was not the case prior to use of DiaBliss), gain weight and able to enjoy all kinds of food
preparations at home. During this period the child has gained 2.5kg of body weight (10% increase).
Figure 6: Data from Juvenile Diabetic
100
110
120
130
140
150
160
170
180
190
11.9.2011 19.12.2011 20.4.2012 20.04.2012 2.10.2012
Blo
od
Su
gar,
mg/
dl
Type 2 Diabetic, Male, Age 51: Fasting & PP Profiles
Fasting
PP
Started DiaBliss Herbal Cane Sugar26 months consumption
6
6.2
6.4
6.6
6.8
7
7.2
7.4
7.6
11.9.2011 19.12.2011 20.04.2012 2.10.2012
Hb
A1
c
Type 2 Diabetic, Male, Age 51: HbA1c Profile
Started DiaBliss Herbal Cane Sugar26 months consumption
8 year old juvenile
diabetic
Two years on
DiaBliss Sugar
Major lifestyle
improvement
Able to go to
school full-time
Able to enjoy all
kinds of foods &
sweets
Gaining body
weight without
significant increase
in insulin dosage
45% reduction in
HbA1c levels
25 | P a g e
5: References
1. National Standard, The Authority on Integrative Medicine, taken
fromhttp://www.naturalstandard.com/demo/demo-mc-diabetes.asp
2. WHO Report Series 913, “DIET, NUTRITION AND THE PREVENTION OF CHRONIC
DISEASES”, Section on Diabetes – Pages 72-78, 2003
3. Diabetologia 2011
4. International Diabetic Foundation Annual Report 2011. Web Site Address
http://www.idf.org/sites/default/files/attachments/IDF-AR2011-EN.pdf
5. Glycosuria, Wikepedia, http://en.wikipedia.org/wiki/Glycosuria
6. Comparison of Accuracy of Five Glucometers in South Africa, JEMDSA 2009;14(2):102-105
7. Decoding HbA1c Test for Blood Sugar - Normal reading for the HbA1c Calculator
Medindia http://www.medindia.net/patients/patientinfo/hba1c-blood-sugar-test-normal-
reading.htm#ixzz2BhvlSsjy
26 | P a g e
Appendix 1: Comprehensive Analysis at SGS Laboratories:
APPENDIX 1A: METALS ANALYSIS OF DIABLISS CANE SUGAR
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 1 of 26
CONFIDENTIAL TITLE PAGE
REPORT
ACUTE ORAL TOXICITY STUDY OF DIA BLISS SUGAR IN WISTAR RATS
DATA REQUIREMENT GUIDELINE: OECD N° 423
Study Director: Syed Nasir Ahamed, M.Sc
Report Submission: February 07, 2012
SPONSOR
En-Joy Consumer Products Pvt Ltd 1/283, Shripuram Street
Shripuram, Chennai- 600 097
Tamil Nadu
TESTING FACILITY
Sugen Life Sciences Pvt. Ltd #4/86, S.V. Nagar
Perumalla palli (post) Tirupati – 517 505
Andhra Pradesh India
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 2 of 26
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
Sugen Life Sciences Pvt Ltd Page 3 of 26
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
Sugen Life Sciences Pvt Ltd Page 4 of 26
STATEMENT OF QUALITY ASSURANCE UNIT
Study Title : Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats This study has been audited and the final report is examined with respect to the study plan, standard operating procedures and raw data. The report is a true reflection of the raw data. The audits were carried out according to the standard operating procedures of the Quality Assurance Unit of Sugen Life Sciences Pvt Ltd (SLS). Findings resulting from the audits were reported to the Study Director and the Management on the dates specified below. These reports are kept in the Archives at SLS. During the course of the study, the following areas were audited:
Audit
No
Details
Dates of Audit
Dates Reported to the
Study Director
Management
1 Draft Study Plan 29/12/2011 29/12/2011 29/12/2011
2 Dosing Activity 18/01/2012 18/01/2012 18/01/2012
3 Gross Pathology and Necropsy
01/02/2012 01/02/2012 01/02/2012
4 Draft Report 04/02/2012 04/02/2012 04/02/2012
K.S.V.P. Ratnam Quality Assurance Unit Date:
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 5 of 26
TABLE OF CONTENTS
TITLE PAGE ............................................................................................................................. 1 STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS ................................................. 2 STATEMENT OF COMPLIANCE ............................................................................................ 3 STATEMENT OF QUALITY ASSURANCE UNIT .................................................................. 4 TABLE OF CONTENTS............................................................................................................ 5 PERSONNEL INVOLVED IN THE STUDY ............................................................................ 7 LIST OF ABBREVIATIONS ..................................................................................................... 8 SUMMARY ............................................................................................................................... 9
1.0 INTRODUCTION ...................................................................................................... 10 1.1 Study Objective ....................................................................................................... 10 1.2 Study Guidelines ..................................................................................................... 10 1.3 Justification for Selection of Test System ................................................................ 10 1.4 Testing Facility and Study Period ............................................................................ 10 1.5 Archives .................................................................................................................. 10
2.0 EXPERIMENTAL PROCEDURE ............................................................................. 11 2.1 Test Item................................................................................................................. 11 2.2 Instruments and Equipments .................................................................................... 11 2.3 Solvent and Chemical .............................................................................................. 11 2.4 Animals ................................................................................................................... 11 2.5 Acclimatization ....................................................................................................... 12 2.6 Husbandry Practices ................................................................................................ 12 2.7 Animal Identification ............................................................................................... 12 2.8 Feed and Water ....................................................................................................... 12 2.9 Environmental Conditions ....................................................................................... 12 2.10 Experimental Activities............................................................................................ 14 2.11 Preparation of Dosing Solution................................................................................ 14 2.12 Dose Administration ................................................................................................ 14 2.13 Experimental Design ............................................................................................... 14 2.14 Observations ........................................................................................................... 14
3.0 RESULTS .................................................................................................................. 15 3.1 Mortalities ............................................................................................................... 15 3.2 Clinical Observations ............................................................................................... 15 3.3 Body Weights ......................................................................................................... 15 3.4 Terminal Studies ..................................................................................................... 15
4.0 CONCLUSION .......................................................................................................... 15 5.0 REFERENCES ........................................................................................................... 16
TABLE 1 - Mortality ................................................................................................................ 17 TABLE 2 - Mean Body Weights ............................................................................................... 18 APPENDIX 1 ........................................................................................................................... 19 Clinical Observations of Individual Animal ................................................................................ 19 APPENDIX 2 ........................................................................................................................... 20
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 6 of 26
Individual Animal Body Weight Data ........................................................................................ 20 APPENDIX 3 ........................................................................................................................... 21 Necropsy Findings of Individual Animal .................................................................................... 21 APPENDIX 4 ........................................................................................................................... 22 Classification ............................................................................................................................ 22 APPENDIX 5 ........................................................................................................................... 23 Record of Deviation from the Study Protocol............................................................................ 23 APPENDIX 6 ........................................................................................................................... 24 Feed Analysis Report…………………………………………………………………. ............... 26 APPENDIX 7……………………………………………………………………………………...25 Water Analysis Report………………………………………………………………………….....25 APPENDIX ………………………………………………………………………………….…...26 Certificate - Department of Science and Industrial Research……………………………………..26
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 7 of 26
PERSONNEL INVOLVED IN THE STUDY
Signature Study Director Syed Nasir Ahamed, M.Sc _________________
Study Personnel O. Lokanatha, M.Sc _________________
REPORT APPROVAL
This study report is approved by:
C. Sreenivasulu Reddy, B.Com Test Facility Management
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 8 of 26
LIST OF ABBREVIATIONS
CPCSEA Committee for Purpose of Control and Supervision of Experiments on Animals
IAEC Institutional Animal Ethics Committee
Mg Milligram
Kg Kilogram
B.wt. Body weight
GHS Globally Harmonised System
SLS Sugen Life Sciences Pvt Ltd
NA Not Available
NAD No Abnormalities Detected
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 9 of 26
SUMMARY
This study was performed to assess the acute oral toxicity of Dia Bliss Sugar supplied by Nityananda Sweets and Snacks in Wistar Rats. The method followed was as per the guideline of
OECD N° 423, "Acute Oral Toxicity–Acute Toxic Class Method”. The Organisation for Economic Co-operation and Development (OECD) guidelines for the Testing of Chemicals, adopted by the council on 17th December, 2001.
Wistar rats fasted overnight, were dosed with Dia Bliss Sugar dissolved in sterile distilled water as single oral gavage, using intubation cannula. The food was withheld until 4 hours of post dosing. The first group of three female rats was given a single dose of 2000 mg of Dia Bliss Sugar per kg body weight. No mortality was observed at this dose level; hence the Group II was administered at the same dose level. No mortality was observed at this dose level also. Hence, further testing was not essential (Appendix 4). The animal’s din’t show any clinical signs of toxicity when treated with 2000 mg of Dia Bliss Sugar per kg body weight. (Appendix1). At the end of 14 days observation period, all the rats were subjected to gross pathological examination (Appendix 1) and the symptoms were physiological variation in nature, hence there were no test item related changes. So, the test item is considered to be safe.
The acute oral median lethal dose LD50 (cut off value) of Dia Bliss Sugar in Wistar rats was found to be > 2000-5000 mg/kg body weight Dia Bliss Sugar is being classified as follows: Globally Harmonised System (GHS) : “Category 5”
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 10 of 26
1.0 INTRODUCTION
1.1 Study Objective
This objective of this study was to assess the acute oral toxicity (LD50) as a result of single dose administration of Dia Bliss Sugar in Wistar rats. The study was conducted in compliance with the OECD Principles of GLP (1998).
1.2 Study Guidelines
The present study was conducted following the guideline:
The Organisation for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, N° 423 (Adopted by the Council on 17th December, 2001) "Acute Oral Toxicity - Acute Toxic Class Method".
1.3 Justification for Selection of Test System
The rat (Rattus norvegicus) was selected as test system because it is a readily available rodent species. It has been historically shown to be a suitable model for acute oral toxicity assessment and is recommended by the OECD and other regulatory authorities. The results of the study are expected to be of value in predicting the acute toxicity of the test item in humans beings.
1.4 Testing Facility and Study Period
The study was performed at Sugen Life Sciences, Tirupati – 517 505, Andhra Pradesh, India, and all data generated and recorded during this study will be stored in the Archives at SLS
Study Initiation : December 29, 2011 Acclimatization Start : January 11, 2012 Experiment Start : January 18, 2012 Experiment Termination : February 02, 2012 Study Completion : February 07, 2012
1.5 Archives
All original raw data including any storage medium for electronically recorded data, documentation, the signed study plan, the draft report, a copy of the final report and a one gram sample of the test substance will be retained in the Archives at Sugen Life Sciences Pvt Ltd for a period of five years. At the end of this period, the Sponsor's instructions will be
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 11 of 26
sought for archiving period.
2.0 EXPERIMENTAL PROCEDURE
2.1 Test Item
Details of the test substance provided by the Sponsor
Test Item Name : Dia Bliss Sugar C.A. Name : NA CAS N° : NA Analysed Concentration : NA Batch/Lot N° : NA Supplied by : Nityananda Sweets and Snacks Date of Manufacture : NA Date of Expiry : NA Appearance : Crystal (White color) Storage Condition : Preserve in well closed container and away from moisture
2.2 Instruments and Equipments
Balance : Electronic Weighing Balance, TP4101DE (Capable of measuring 0.1 g to 4.1 kg) Electronic Balance, GE7101 (2.0g to 7.1Kg) Ohaus Micro Balance, PO214C (Capable of measuring 0.1 mg to 200.0 g) Intubation Cannula : Metal Cannula, Size 18 G x 5 cm, Syringe : “Top” Hypodermic syringe, Boro-silicate hard glass, Size 5 ml
2.3 Solvent and Chemical
Distilled water : Glass apparatus Disinfectant Solution : Labolene
2.4 Animals
Test Species : Rat (Rattus norvegicus) Strain : Wistar Animal Source : Sri Venkateswara Enterprises, Bangalore.
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 12 of 26
Total Number of Animals Used : 06 Females (nulliparous and non-pregnant) Body Weight (g) before Dosing : Minimum: 171.6, Maximum: 175.5. Age of the Animals : 8 to 10 weeks at the time of dosing.
2.5 Acclimatization
Rats were allowed to acclimatize to the experimental room conditions for a period of five days prior to randomization for group I and group-II prior to commencement of dosing. During the acclimatization period, the rats were observed daily twice for clinical signs of disease. Prior to randomization, a detailed physical examination was performed on all animals.
2.6 Husbandry Practices
Caging : Polypropylene rat cages covered with stainless steel grid top were used. Autoclaved clean paddy husk was used as the bedding material.
Water Bottle : Each cage was supplied with a polypropylene water bottle with a stainless steel nozzle
Housing : Three rats per cage Room Sanitation : Each day, the floor of the experiment room was swept and all work
tops and the floor were mopped with a disinfectant solution.
2.7 Animal Identification
Individual animal was identified with number marked on the tail using permanent marker pen and coloured cage label showing study number, study code, test item code, group number, number of animals, sex, species, strain, dose, cage number and animal number.
2.8 Feed and Water
The animals were provided ad libitum laboratory rodent pellet feed supplied by Provimi Animal Nutrition India Pvt. Ltd., Bangalore and charcoal filtered, UV sterilized water (Aqua guard water filter system). Fresh feed was supplied at least once a week and water bottles were refilled daily or whenever required.
2.9 Environmental Conditions
Rats were maintained in an environment-controlled room. The experimental room temperature and humidity were recorded daily. The temperatures recorded were in the range of 23.12 – 24.15 °C respectively. The relative humidity recorded was in the range of 54.43 – 61.69%. In the experimental room, 12 hours of artificial lighting and 12 hours darkness were
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 13 of 26
maintained, light hours being 6.00 to 18.00h.
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 14 of 26
2.10 Experimental Activities
The following observations/activities were performed during the experimental period:
Daily - animal observations - providing feed and water - floor sweeping and mopping - rack cleaned - recording room temperature and relative humidity Weekly - recording body weight
- recording photoperiod
2.11 Preparation of Dosing Solution
The test item was found to be soluble in sterile distilled water, so the actual dosing solution was prepared using sterile distilled water as vehicle. Required quantity of Dia Bliss Sugar was mixed in distilled water and the final volume was made up to 10 ml/kg. Gavage solutions were prepared freshly prior to dosing on all the occasions.
2.12 Dose Administration
A dose volume of 10 ml/kg body weight was maintained for each rat. All rats were dosed by gavage (day 0) using a metal cannula attached to a Boro-silicate hard glass syringe, which was graduated up to 5 ml. Rats were fasted overnight prior to dosing till four hours post-dosing.
2.13 Experimental Design
The Group I of three female rats was given a single dose of 2000 mg of Dia Bliss Sugar per kg body weight. No mortality was observed at this dose level, hence the group II was administered at the same dose level, no mortality was observed at this dose level hence, further testing was not essential (Table 1 and Appendix 4).
2.14 Observations
The rats were observed for signs of toxicity and mortality at specific time points for 30 minutes, 1, 2, 3, and 4 hours post dosing on the day of dosing (Appendix 1). Subsequently, the rats were observed twice a day for morbidity and mortality for a period of 14 days following oral dosing (Table 1). The clinical signs were recorded twice a day. Individual body
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 15 of 26
weights were recorded prior to dosing on days 0, 7 and 14 (Appendix 2).
3.0 RESULTS
3.1 Mortalities
No mortality was observed in the rats from group I and from Group II treated at 2000mg of Dia Bliss Sugar per kg B.wt. (Table 1).
3.2 Clinical Observations
No behavioral changes were observed as clinical sign in the rats from group I and group II treated with Dia Bliss Sugar per kg B.wt between day 0 to day 14. All rats survived and appeared normal from post dosing till to the termination of the experiment (Appendix 1).
3.3 Body Weights
Positive gain in body weights were recorded in all the rats treated with Dia Bliss Sugar at the dose level of 2000 mg /kg body weight (Appendix 2).
3.4 Terminal Studies
External
External examination of rats sacrificed terminally did not reveal any lesion of pathological significance (Appendix 3).
Internal
Visceral examination of rats sacrificed terminally did not show any lesions when treated with Dia Bliss Sugar at the dose level of 2000 mg/kg b.wt.
4.0 CONCLUSION
The acute oral median lethal dose LD50 (cut off value) of Dia Bliss Sugar in Wistar rats was found to be > 2000 - 5000 mg/kg body weight
Dia Bliss Sugar is being classified as follows:
Globally Harmonised System (GHS) : “Category 5”
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 16 of 26
5.0 REFERENCES
5.1 OECD, 1998 : OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, Number 1, “OECD Principles on Good Laboratory Practice” ENV/MC/CHEM(98)17 (as revised in 1997).
5.2 OECD, 2001 : OECD N° 423, "Acute Oral Toxicity – Acute Toxic Class Method”. The Organisation for Economic Co-operation and Development (OECD) Guidelines for the Testing of Chemicals, Adopted by the Council on 17th December 2001.
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 17 of 26
Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
TABLE 1 - Mortality
F- Female h- Hours
0- No Mortality
Group Dose
(mg/kg)
Animal No
& Sex 1 h 2 h 3 h 4 h 24 h Day 1-7 Day 8-14
I
II
2000.0 1F-3F 0 0 0 0 0 0 0
2000.0 4F-6F 0 0 0 0 0 0 0
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 18 of 26
Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
TABLE 2 - Mean Body Weights
Group Dose (mg/kg)
Animal No & Sex
Mean body weight (g)
Day 0 Day 7 Day 14
I
II
2000.0 1F-3F 172.90±1.12 178.73±0.83 185.96±1.26
2000.0 4F-6F 173.47±1.79 180.67±1.72 187.10±2.23
Percent Mean Body Weight Changes
Group Dose (mg/kg)
Animal No & Sex
Percentage Mean body weight Change on
Day 7 Day 14
I 300.0 1F-3F 3.4±0.2 7.6±0.2
II 50.0 4F-6F 4.2±0.1 7.9±0.7
F- Female
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 19 of 26
Acute Oral Toxicity Study of Dia Bliss Sugar in Rats
APPENDIX 1
Clinical Observations of Individual Animal
Group – I
Animal N° & Sex
Dose Level
(mg/kg)
Clinical Signs Observed Post Dosing
At Hour (Day 0) On Day
0 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14
1F
2000.0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2F 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
3F 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Group – II
Animal N° & Sex
Dose Level
(mg/kg)
Clinical Signs Observed Post Dosing
At Hour (Day 0) On Day
0 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14
1F
2000.0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2F 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
3F 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 = Normal
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 20 of 26
Acute Oral Toxicity Study of Dia Bliss Sugar in Rats
APPENDIX 2
Individual Animal Body Weight Data
Group Animal No & Sex
Dose (mg/kg)
Body weight (g)
% Body Weight Change on Day
% Body Weight Change on Day
Day 0 Day 7 Day 14 0 – 7 7 – 14
I
1F
2000.0
171.6 177.8 184.6 3.6 7.6
2F 173.5 179 186.2 3.2 7.3
3F 173.6 179.4 187.1 3.3 7.8
II
4F
2000.0
172.1 179.3 184.6 4.2 7.3
5F 172.8 180.1 187.8 4.2 8.7
6F 175.5 182.6 188.9 4.0 7.6
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 21 of 26
Acute Oral Toxicity Study of Dia Bliss Sugar in Rats
APPENDIX 3
Necropsy Findings of Individual Animal
Group N°
Animal No & Sex
Dose (mg/kg)
Mode of Death External Internal
I
II
1F
2000.0
TS NAD NAD
2F TS NAD NAD
3F TS NAD NAD
4F
2000.0
TS NAD NAD
5F TS NAD NAD
6F TS NAD NAD
NAD- No Abnormalities Detected; TS: Terminal Sacrifice
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 22 of 26
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)
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
Sugen Life Sciences Pvt Ltd Page 24 of 26
Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX 6
Feed Analysis Report
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 25 of 26
Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX 7
Water Analysis Report
SLS Study Number : SLS/043-11
Sugen Life Sciences Pvt Ltd Page 26 of 26
Acute Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX 9
Certificate – Department of Science and Industrial Research
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 1 of 61
CONFIDENTIAL TITLE PAGE
REPORT
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
DATA REQUIREMENT GUIDELINE: OECD N° 407
Study Director: Syed Nasir Ahamed, M.Sc
Report Submission: 01/12/2011
SPONSOR
En-Joy Consumer Products Pvt Ltd
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
Sugen Life Sciences Pvt Ltd Page 2 of 61
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
Sugen Life Sciences Pvt Ltd Page 3 of 61
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
Sugen Life Sciences Pvt Ltd Page 4 of 61
STATEMENT OF QUALITY ASSURANCE UNIT
Study Title : 28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in
Wistar Rats This study has been audited and the final report was examined with respect to the study plan, standard operating procedures and raw data. The report is a true reflection of the raw data. The audits were carried out according to the standard operating procedures of the Quality Assurance Unit of Sugen Life Sciences (SLS). Findings resulting from the audits were reported to the Study Director and the Management on the dates specified below. These reports are kept in the Archives at SLS. During the course of the study, the following areas were audited:
Audit
No Details Dates of Audit
Dates Reported to the
Study Director
Management
1 Randomization, Body Weight, Dosing 18/10/2011 18/10/2011 18/10/2011
2 Dosing, Body Weight and Food Consumption
24/10/2011 24/10/2011 24/10/2011
3 Necropsy, Clinical Chemistry 15/11/2011 15/11/2011 15/11/2011
4 Draft Report 30/11/2011 30/11/2011 30/11/2011
K.S.V.P. Ratnam Quality Assurance Unit Date:
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 5 of 61
TABLE OF CONTENTS
TITLE PAGE........................................................................................................................................ 1 STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS ..................................................... 2 STATEMENT OF COMPLIANCE .................................................................................................... 3 STATEMENT OF QUALITY ASSURANCE UNIT........................................................................ 4 Table of Contents ................................................................................................................................. 5 PERSONNEL INVOLVED IN THE STUDY ................................................................................... 7 LIST OF ABBREVIATIONS ............................................................................................................. 8 SUMMARY........................................................................................................................................ 10
1.0 INTRODUCTION ................................................................................................................ 11 1.1 Study Objective ............................................................................................................... 11 1.2 Guideline ......................................................................................................................... 11 1.3 Justification for Selection of the Test System ............................................................... 11 1.4 Testing Facility and Study Period .................................................................................. 11
2.0 EXPERIMENTAL PROCEDURE ...................................................................................... 11 2.1 Test Item .......................................................................................................................... 11 2.2 Animals ............................................................................................................................ 12 2.3 Acclimatization ............................................................................................................... 12 2.4 Environmental Conditions .............................................................................................. 12 2.5 Housing ............................................................................................................................ 12 2.6 Feed and Water................................................................................................................ 12 2.7 Grouping .......................................................................................................................... 13 2.8 Animal Identification ...................................................................................................... 13 2.9 Experimental Outline ...................................................................................................... 13 2.10 Route of Administration ................................................................................................. 13 2.11 Duration of Treatment .................................................................................................... 13 2.12 Selection of Vehicle ........................................................................................................ 13
3.0 OBSERVATIONS .............................................................................................................. 14 3.1 Clinical Signs .................................................................................................................. 14 3.2 Body Weight.................................................................................................................... 14 3.3 Food Consumption .......................................................................................................... 14 3.4 Clinical Pathology Observations .................................................................................... 14 3.5 Gross Pathology .............................................................................................................. 15 3.6 Evaluation of Results ...................................................................................................... 15
4.0 ARCHIVES ......................................................................................................................... 16 5.0 RESULTS AND DISCUSSION ........................................................................................ 16
5.1 Mortalities ........................................................................................................................ 16 5.2 Clinical Observations ...................................................................................................... 16 5.3 Body Weight.................................................................................................................... 16 5.4 Food Consumption .......................................................................................................... 16 5.5 Haematology ................................................................................................................... 16 5.6 Clinical Chemistry .......................................................................................................... 16 5.7 Organ Weight .................................................................................................................. 17 5.8 Relative Organ Weight ................................................................................................... 17 5.9 Gross Pathological Findings ........................................................................................... 17 5.10 Histopathology ................................................................................................................ 17
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 6 of 61
6.0 CONCLUSION .................................................................................................................. 17 7.0 REFERENCES .................................................................................................................... 18 TABLE 1 - Clinical Signs – Summary by Group ........................................................................ 19 TABLE 2 - Body Weight (g) - Group Mean Values .................................................................... 20 TABLE 3 - Food Consumption (g) - Group Mean Values .......................................................... 22 TABLE 4 - Hematology - Group Mean Values ........................................................................... 24 TABLE 5 - Clinical Chemistry - Group Mean Values ................................................................ 26 TABLE 6 - Organ Weights (g) - Group Mean Values ................................................................. 28 TABLE 7 - Relative Organ Weights (%) - Group Mean Values ................................................ 30 TABLE 8 - Gross Pathology Findings – Summary by Group ..................................................... 32
APPENDIX – A ................................................................................................................................. 33 Clinical Signs of Individual Animals ........................................................................................ 33
APPENDIX – B ................................................................................................................................. 35 Individual Animal Body Weight (g) ......................................................................................... 35
APPENDIX – C ................................................................................................................................. 37 Food Consumption (g/cage)....................................................................................................... 37
APPENDIX – D ................................................................................................................................. 39 Individual Animal Haematological Values ............................................................................... 39
APPENDIX – E .................................................................................................................................. 43 Individual Animal Clinical Chemistry Values ......................................................................... 43
APPENDIX – F .................................................................................................................................. 47 Individual Animal Organ Weights (g) ...................................................................................... 47
APPENDIX – G ................................................................................................................................. 51 Individual Animal Relative Organ Weights (%) ...................................................................... 51
APPENDIX – H ................................................................................................................................. 55 Gross and Microscopic Findings of Individual Animals ......................................................... 55
APPENDIX – I ................................................................................................................................... 59 Feed Analysis Report ................................................................................................................. 59
APPENDIX – J ................................................................................................................................... 60 Water Analysis Report ............................................................................................................... 60
APPENDIX – L .................................................................................................................................. 61 Certificate – Department of Science and Industrial Research ................................................. 61
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 7 of 61
PERSONNEL INVOLVED IN THE STUDY
Signature
Study Director Syed Nasir Ahamed, M.Sc _________________
Study Personnel O. Lokanatha, M.Sc _________________
Pathology K. Poorani, M.V.Sc _________________
Clinical Chemistry S. Mamatha, M.Sc _________________
Statistical Evaluation I. Nagaraju, M.Sc _________________
REPORT APPROVAL
This study report is approved by:
C. Sreenivasulu Reddy, B.Com. Test Facility Management
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 8 of 61
LIST OF ABBREVIATIONS
AD - Average Dose ALB - Albumin ALP - Alkaline Phosphatase B.wt - Body weight Cfu - colony forming units CPCSEA - Committee for Purpose of Control and Supervision of Experiments on
Animals CR - Creatinine dl - Deciliter EDTA - Ethylene Diamine Tetra Acetic Acid F - Female F.C - Food Consumption fl - Femtolitre g - Grams Hb - Hemoglobin HCT - Haematocrit HD - High Dose IAEC - Institutional Animal Ethics Committee IU/l - International Unit per Litre kg - Kilogram M - Male MCH - Mean Corpuscular Hemoglobin MCHC - Mean Corpuscular Hemoglobin Concentration MCV - Mean Corpuscular Volume mg - Milligrams mm3 - cubic millimeter N° - Number NAD - No Abnormalities Detected pg - Pictogram PLT - Platelet Count ppb - parts per billion RBC - Red Blood Cells SD - Standard Deviation SGOT - Serum Glutamate Oxaloacetate Transaminase SGPT - Serum Glutamate Pyruvate Transaminase SLS - Sugen Life Sciences TABC - Total Aerobic Bacterial Count TD - Therapeutic Dose
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 9 of 61
LIST OF ABBREVIATIONS (Continued) TI - Test Item TLC - Thin Layer Chromatography TP - Total Protein VC - Vehicle Control WBC - White Blood Cells % - Percent µ - Micron
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 10 of 61
SUMMARY This study was conducted to assess the toxic effects of Dia Bliss Sugar in Wistar Rats, when administered orally for a period of 28 consecutive days. The methods followed were as per the guideline:
The Organization for Economic Co-operation and Development (OECD) for Testing of Chemicals No 407, entitled "Repeated Dose 28-day Oral Toxicity Study in Rodents", adopted on July 27, 1995.
Three groups of Rats comprising 6 male and 6 females per group were treated with Dia Bliss Sugar at 180.0, 540.0 and 1800.0 mg/kg bd. wt. for a period of 28 consecutive days. A concurrent control group, comprising the same number of animals was also maintained in the study.
Rats were observed individually for visible signs of reaction to the treatment twice a day throughout the study period. Individual body weight and feed consumption were monitored weekly. Haematological and biochemical analyses of blood samples were performed on all animals at the end of the treatment period. All the surviving rats were sacrificed and subjected to a gross pathological examination at the end of treatment period. Absolute organ weights were recorded and relative organ weights were calculated for the organs viz., liver, kidneys, adrenals, testis/ovary, epididymides/uterus, thymus, spleen, brain and heart in all Rats.
The animals exposed to test item did not show any specific treatment related clinical changes in all the treated groups. No significant changes were observed in the mean body weight and food consumption in any of the treated group animals.
Haematological analysis and clinical chemistry analysis did not reveal any treatment related changes in any of the parameter at various dose levels studied in either sex.
The absolute and relative organ weights did not reveal any significant difference in the treated groups’ animals when compared with the control group animals in either sex. Gross pathological examination of the animals did not reveal any treatment related effects in any of the dose group. Histopathological examination of the high dose group animals did not reveal any treatment related effects compared to control group animals. There was no test item related toxicity recorded in any of the treatment group under the experimental conditions.
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 11 of 61
1.0 INTRODUCTION
1.1 Study Objective
The objective of this study was to determine the adverse effects occurring as a result of the repeated oral administration of Dia Bliss Sugar in Wistar Rats for a period of 28 consecutive days.
1.2 Guideline
The study was conducted following the guideline:
The Organisation for Economic Co-operation and Development (OECD) for Testing of Chemicals No 407, entitled "Repeated Dose 28-day Oral Toxicity Study in Rodents", adopted on July 27, 1995.
1.3 Justification for Selection of the Test System
Rats were selected as test system because it is a readily available laboratory rodent species. It has been historically shown to be a suitable model for repeated dose toxicity assessment and is recommended by the OECD and other regulatory authorities. The results of the study are believed to be of value in predicting the toxicity of the test item in human beings.
1.4 Testing Facility and Study Period
The study was performed at Sugen Life Sciences, Tirupathi – 517 505, Andhra Pradesh, India, and all data generated and recorded during this study will be stored SLS Archives.
Study Initiation : 08 October, 2011 Acclimatization Start : 13 October, 2011 Experiment Start : 18 October, 2011 Date of Sacrifice : 15 November, 2011 Study Completion : 01 December, 2011
2.0 EXPERIMENTAL PROCEDURE
2.1 Test Item
Details of the test item as provided by the sponsor are: Test Item Name : Dia Bliss Sugar Supplied by : Nityananda Sweets and Snacks, Shripuram Manufactured by : Nityananda Sweets and Snacks, Shripuram Date of Manufacture : NA Appearance : Small white crystals.
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 12 of 61
Solubility : Slightly soluble in water Storage Condition : At room Temperature
2.2 Animals
24 male and 24 female healthy, young Rats (Rattus norvegicus) of Wistar strain, obtained from CPCSEA approved vendor, were used for the study. Female Rats were nulliparous and non-pregnant. All animals were examined for good health at the time of receipt. Age of the animals at the start of treatment was approximately 9 weeks.
2.3 Acclimatization
Rats were allowed to acclimatize to the experimental room conditions for a period of five days prior to randomization. During the acclimatization period, the rats were observed daily twice for clinical signs of disease. Prior to randomization, a detailed physical examination was performed on all animals.
2.4 Environmental Conditions
Rats were maintained in an environment-controlled room. The experimental room temperature and humidity were recorded daily. The temperatures recorded were in the range of 22.0 – 25.0 °C respectively. The relative humidity recorded was in the range of 42.6 – 56.73%. In the experimental room, 12 hours of artificial lighting and 12 hours darkness were maintained, light hours being 6.00 to 18.00h. The experimental room was cleaned and mopped with a disinfectant daily.
2.5 Housing
The animals were housed in group of two of same sex per cage in solid floor polypropylene Rats cages. Each cage was fitted with a stainless steel top grill and a polypropylene water bottle with stainless steel drinking nozzle. The bottom of the cage was layered with clean, sterilized paddy husk. The cages were kept on 5 tier racks and their positions were rotated weekly.
2.6 Feed and Water
The animals were provided laboratory rodent pellet feed supplied by Provimi Animal Nutrition India Pvt. Ltd., Bangalore and charcoal filtered, UV sterilized water (Aqua guard water filter system) ad libitum. Fresh feed was supplied at least once a week and water bottles were refilled daily or whenever required. Feed, drinking water and paddy husk were analyzed for microbial contaminants at six-month intervals and the results of the recent analyses are furnished in the appendix (Appendices I and J).
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 13 of 61
2.7 Grouping
The animals were allocated to different groups using the principle of randomization. The animals were equally distributed to four groups viz., control (G1), therapeutic dose (G2), average dose (G3) and high dose (G4). Each group was comprised of 6 male and 6 female rats. At the time of treatment commencement, the body weight variation among the animals was within ± 20% of the mean body weight for each sex. Individual body weights of the animals ranged between 164.8 to 204.9 g for males and152.0 to 168.0 g for females at the time of treatment commencement.
2.8 Animal Identification
Individual animal was identified with number marked on the tail using permanent marker pen and coloured cage label showing study N°, study code, test item code, group N°, N° of animals, sex, species, strain, dose, cage N° and animal N°.
2.9 Experimental Outline
Group N° Group
Dose (mg/kg
b.wt./day)
Number of Rats (Males + Females)
Treated Clinical Pathology Histo-pathology
G1
G2
G3
G4
Control
Therapeutic dose
Average dose
High dose
0.0
180.0
540.0
1800.0
6 +6
6 +6
6 +6
6 +6
6 +6
6 +6
6 + 6
6 + 6
6 + 6
-
-
6 + 6
2.10 Route of Administration
The route of administration was oral through oral gavage. The oral administration represents a possible route of human exposure and was agreed by the study sponsor.
2.11 Duration of Treatment
Animals were administered with Dia Bliss Sugar mixed with sterile distilled water for a period of 28 days.
2.12 Selection of Vehicle
Vehicle was selected based on the solubility of the test item. Dia Bliss Sugar being a water soluble test item, water was used as vehicle.
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 14 of 61
3.0 OBSERVATIONS
3.1 Clinical Signs
Animals were observed for mortality and morbidity twice a day. All visible signs and symptoms such as skin and fur changes, eye and mucous membrane changes, respiratory, behavioral pattern and general changes were recorded twice a day.
3.2 Body Weight
Individual body weight was recorded for all animals on the day of commencement of treatment and at weekly intervals thereafter.
3.3 Food Consumption
The weekly food consumption of the animals was calculated through out the study.
3.4 Clinical Pathology Observations
Clinical pathological tests were conducted on blood samples collected from all the surviving animals at the end of the treatment period. Animals were deprived of food overnight and blood samples were collected by puncturing the orbital sinus plexus with the help of a fine capillary tube.
Around 0.8 ml of blood was collected in vials containing EDTA for haematology analysis.
Two to three ml blood was collected from each animal in clean centrifuge tubes. The blood was allowed to clot at room temperature and the serum was separated by centrifugation at low speed. The serum thus separated was used for all clinical chemistry analyses.
Clinical pathology parameters studied and instrument used are given below: ---------------------------------------------------------------------------------------------------------------- Parameter Sample Type Instrument Used ----------------------------------------------------------------------------------------------------------------
Haematology Leucocyte count (WBC) Whole blood Mindray BC-2800Vet Erythrocyte count (RBC) Whole blood Mindray BC-2800 Vet Haemoglobin (Hb) Whole blood Mindray BC-2800 Vet Haematocrit (HCT) Whole blood Mindray BC-2800 Vet Mean Corpuscular Volume (MCV) Whole blood Mindray BC-2800 Vet Mean Corpuscular Haemoglobin (MCH) Whole blood Mindray BC-2800 Vet Mean Corpuscular Haemoglobin Concentration Whole blood Mindray BC-2800 Vet (MCHC) Platelet (Plt) Whole blood Mindray BC-2800 Vet ----------------------------------------------------------------------------------------------------------------
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 15 of 61
---------------------------------------------------------------------------------------------------------------- Parameter Sample Type Instrument Used ---------------------------------------------------------------------------------------------------------------- Clinical Chemistry
Alkaline Phosphatase (ALP) Serum Merck – Microlab 300 Albumin Serum Merck – Microlab 300 Triglycerides Serum Merck – Microlab 300 Creatinine Serum Merck – Microlab 300 Glucose Serum Merck – Microlab 300 Serum Glutamate Oxaloacetate Transaminase Serum Merck – Microlab 300 (SGOT) Serum Glutamate Pyruvate Transaminase Serum Merck – Microlab 300 (SGPT) Total protein Serum Merck – Microlab300 Urea Serum Merck – Microlab 300
---------------------------------------------------------------------------------------------------------------- 3.5 Gross Pathology
All the animals were euthanized using anesthetic ether and subjected to a complete necropsy under the direct supervision of the veterinary pathologist at the end of the treatment period. The animals were examined carefully for external abnormalities before the necropsy. The thoracic, abdominal and cranial cavities were then cut open and thorough examinations of the organs were carried out to detect changes or abnormalities, if any. Absolute weights of adrenals, brain, uterus, ovaries, testes, epididymides, heart, kidneys, liver, spleen and thymus were recorded immediately after dissection of all animals. Paired organs were weighed together. Relative weights of these organs were calculated later.
3.6 Evaluation of Results
Raw data were processed by the Department of Statistics and Systems Management, Sugen Life Sciences to give group means and standard deviations for comparison between the control and treated groups, using SPSS statistical software. All the parameters characterized as continuous data such as, body weight, feed consumption, organ weight, relative organ weight, hematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. Where the data did not meet the homogeneity of variance, Kruskal-Wallis test was performed to calculate the significance.
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 16 of 61
4.0 ARCHIVES
All raw data, the signed study plan, study plan amendment, a copy of the final report, slides and a proportionate amount of the test item will be retained in the Archives at Sugen Life Sciences for a period of five years. At the end of this period, the sponsor's instructions will be sought to either extend the archiving period or return the archived material to the sponsor or for the material to be disposed off.
5.0 RESULTS AND DISCUSSION
5.1 Mortalities
No mortality was observed in rats exposed to test item at 180.0, 540.0 and there was mortality observed at 1800.0 mg/kg bd. wt. orally for a period of 28 days , but the mortality was not due to test item toxicity.
5.2 Clinical Observations
No treatment related clinical signs were recorded in any of the animal administered with different doses of Dia Bliss Sugar as compared to control group animals in either sex. (Table 1, Appendix A)
5.3 Body Weight
No statistical significant variation was observed in the average weekly body weight of animals treated with Dia Bliss Sugar as compared to control animals while in female animals also, no such variation was observed. (Table 2; Appendix B)
5.4 Food Consumption
No significant difference was observed in the average food consumption in treated group animals of either sex when compared to the control group animals. (Table 3; Appendix C)
5.5 Haematology
No significant difference was observed in all the heamatological parameters of the animals administered with different doses of Dia Bliss Sugar as compared to the control group animals in either sex. (Table 4; Appendix D)
5.6 Clinical Chemistry
No significant difference was observed in all the clinical chemistry parameters of the animals treated with different doses of Dia Bliss Sugar as compared to the control group animals in either sex. All the clinical chemistry parameters were found to be within the normal range. (Table 5; Appendix E)
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 17 of 61
5.7 Organ Weight
No significant difference was observed in the absolute organ weights of the animals treated with different doses of Dia Bliss Sugar as compared to the control group animals in either sex. (Table 6; Appendix F)
5.8 Relative Organ Weight
No significant difference was observed in the relative organ weights of the animals treated with different doses of Dia Bliss Sugar as compared to the control group animals in females while a low variation was observed in Heart and Thymus in males. (Table 7; Appendix G)
5.9 Gross Pathological Findings
5.9.1 External
The external examination of the carcasses of all animals did not reveal any changes.
5.9.2 Internal
After the external examination, the carcass was cut open and examined for gross pathology of visceral organs. The lobes of the liver were examined for changes and collected. The spleen, pancreas were examined for changes. The small intestine and large intestine were observed and samples of jejunum, ileum, cecum, colon and rectum were collected. The kidneys were removed and examined for changes. The larynx, trachea, esophagus, thymus and thyroids were collected along with lungs. The brain was collected and examined. No significant lesions or abnormalities like changes in size, color, congestion, hemorrhage, inflammation and necrosis were detected in all the organs. Gross examination at necropsy did not reveal any Dia Bliss Sugar treatment related abnormalities.
5.10 Histopathology
The animals treated with Dia Bliss Sugar at dose level of 1800.0 mg/kg bd. wt did not reveal any histopathological changes in either sex as compared to control group animals. Results of the histopathological examination are shown in Table 8 and Appendix H.
6.0 CONCLUSION
Wistar Rats treated with Dia Bliss Sugar orally for 28 days at various dose levels did not reveal any treatment related adverse/toxic effects under the experimental conditions.
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 18 of 61
7.0 REFERENCES
7.1 Physician’s Desk Reference, 1999, 53rd ed., 1667-76
7.2 Martindale, 32nd ed., K. Parfitt ed., 44.
7.3 Paget and Barnes (1964): Evaluation of drug activities Pharma Eds. Laurance and Bacharach, Vol.1, Academic Press, New York.
7.4 OECD Guidelines for Testing of Chemicals, No 407 entitled, "Repeated Dose 28-day Oral Toxicity Study in Rodents", adopted on July 27, 1995.
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 19 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
TABLE 1 - Clinical Signs – Summary by Group
Normal Day of Observation
0 7 14 21 28
Male
G1 6 6 6 6 6
G2 6 6 6 6 6
G3 6 6 6 6 6
G4 6 6 5 5 5
Female
G1 6 6 6 6 6
G2 6 6 6 6 6
G3 6 6 6 6 6
G4 6 6 6 6 6 Note: Number of animals with clinical sign at least once during the week interval.
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 20 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
TABLE 2 - Body Weight (g) - Group Mean Values
Sex: Male
Day N°
G1 G2 G3 G4
Mean SD N Mean SD N Mean SD N Mean SD N
0 174.18 17.41 6 166.28 13.41 6 176.08 10.19 6 177.93 17.43 6
7 208.32 18.10 6 202.47 17.88 6 199.07 19.53 6 198.13 27.71 6
14 226.82 24.15 6 224.55 19.53 6 218.25 16.54 6 227.54 20.10 5
21 238.23 23.96 6 233.30 20.08 6 226.98 17.22 6 237.52 21.41 5
28 247.08 25.32 6 243.45 20.34 6 235.47 17.77 6 245.96 21.92 5
Average Body Weight - Male
Average Body Weight- Male
140.00
160.00
180.00
200.00
220.00
240.00
260.00
280.00
300.00
Day 0 Day 7 Day 14 Day 21 Day 28Days
Ave
rage
Foo
d C
onsu
mpt
ion
(g)
G1G2G3G4
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 21 of 61
Table 2 - Body Weight (g) - Group Mean Values (Continued)
Sex: Female
Day N°
G1 G2 G3 G4
Mean SD N Mean SD N Mean SD N Mean SD N
0 159.57 5.20 6 156.52 3.52 6 158.02 6.51 6 158.47 5.04 6
7 183.55 6.67 6 179.88 6.33 6 188.75 12.57 6 182.60 5.83 6
14 191.02 7.23 6 186.22 7.69 6 196.93 17.20 6 189.37 5.77 6
21 195.73 7.95 6 190.43 8.40 6 202.77 21.52 6 194.43 6.14 6
28 204.27 7.87 6 197.73 8.23 6 210.35 21.55 6 201.73 8.97 6
Average Body Weight – Female
Average Food Consumption- Females
120.00
140.00
160.00
180.00
200.00
220.00
240.00
260.00
280.00
Day 0 Day 7 Day 14 Day 21 Day 28
Days
Ave
rage
Foo
d Co
nsum
ptio
n (g
)
G1G2G3G4
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 22 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
TABLE 3 - Food Consumption (g) - Group Mean Values
Sex: Male
Day N°
G1 G2 G3 G4
Mean SD N Mean SD N Mean SD N Mean SD N
7 206.17 8.84 6 215.37 12.51 6 222.07 12.09 6 223.87 11.39 6
14 258.80 35.51 6 247.20 36.03 6 247.63 20.08 6 220.30 11.59 5
21 237.93 23.48 6 231.87 17.22 6 253.20 5.66 6 214.60 12.10 5
28 246.23 25.52 6 239.20 19.79 6 261.07 6.30 6 217.90 15.34 5
Average Food Consumption – Male
Average Food Consumption- Males
150.00
190.00
230.00
270.00
310.00
7 14 21 28Days
Aver
age
Food
Con
sum
ptio
n (g
)
G1G2G3G4
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 23 of 61
TABLE 3 - Food Consumption (g) - Group Mean Values (Continued)
Sex: Female
Day N°
G1 G2 G3 G4
Mean SD N Mean SD N Mean SD N Mean SD N
7 165.07 3.70 6 165.53 7.71 6 162.47 1.88 6 149.33 4.09 6
14 180.97 1.46 6 179.10 9.81 6 176.80 1.44 6 172.83 6.62 6
21 195.37 3.39 6 188.20 5.80 6 187.83 3.39 6 181.17 6.67 6
28 204.00 2.21 6 200.97 5.06 6 197.83 6.31 6 193.93 7.26 6
Average Feed Consumption – Female
Average Food Consumption-Females
100.00
120.00
140.00
160.00
180.00
200.00
220.00
240.00
260.00
7 14 21 28Days
Ave
rage
Foo
d C
onsu
mpt
ion
(g)
G1G2G3G4
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 24 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
TABLE 4 - Hematology - Group Mean Values
Sex: Male
Parameter G1 G2
Mean SD N Mean SD N
RBC (1x106/mm3) 7.29 0.46 6 7.12 0.92 6
WBC (1x103/mm3) 12.77 1.23 6 14.00 1.37 6
Hb (g/dl) 16.55 1.72 6 16.93 1.15 6
HCT (%) 38.40 5.38 6 40.37 1.28 6
MCV (um3) 50.02 1.17 6 50.65 2.02 6
MCH (pg) 25.65 1.34 6 25.17 1.44 6
MCHC (g/dl) 54.17 1.42 6 54.33 1.35 6
Plt (1x103/mm3) 393.15 35.44 6 401.63 73.40 6
Parameter G3 G4
Mean SD N Mean SD N
RBC (1x106/mm3) 6.90 0.53 6 7.79 0.52 5
WBC (1x103/mm3) 13.40 1.24 6 13.96 1.39 5
Hb (g/dl) 17.78 0.90 6 16.49 0.71 5
HCT (%) 40.57 1.52 6 40.38 1.25 5
MCV (um3) 51.42 1.04 6 49.56 1.25 5
MCH (pg) 24.73 0.89 6 25.50 0.95 5
MCHC (g/dl) 55.53 1.59 6 54.20 1.91 5
Plt (1x103/mm3) 373.58 72.48 6 387.72 54.34 5
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 25 of 61
TABLE 4 - Hematology - Group Mean Values (Continued)
Sex: Female
Parameter G1 G2
Mean SD N Mean SD N
RBC (1x106/mm3) 6.65 0.64 6 6.60 0.74 6
WBC (1x103/mm3) 12.84 0.94 6 14.31 1.53 6
Hb (g/dl) 16.00 1.49 6 16.08 1.47 6
HCT (%) 40.35 1.26 6 39.57 1.13 6
MCV (um3) 49.77 1.19 6 50.18 1.55 6
MCH (pg) 25.52 1.08 6 24.93 0.97 6
MCHC (g/dl) 55.20 1.06 6 55.47 1.08 6
Plt (1x103/mm3) 401.13 34.44 6 370.38 67.16 6
Parameter G3 G4
Mean SD N Mean SD N
RBC (1x106/mm3) 6.89 0.84 6 6.87 0.77 6
WBC (1x103/mm3) 15.23 2.03 6 14.35 1.11 6
Hb (g/dl) 15.93 1.25 6 16.48 1.27 6
HCT (%) 39.77 1.73 6 40.28 1.19 6
MCV (um3) 50.05 1.57 6 50.52 1.26 6
MCH (pg) 25.58 1.03 6 25.30 0.76 6
MCHC (g/dl) 54.15 0.72 6 55.35 1.68 6
Plt (1x103/mm3) 385.20 26.49 6 390.27 44.32 6
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 26 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
TABLE 5 - Clinical Chemistry - Group Mean Values
Sex: Male
Parameter G1 G2
Mean SD N Mean SD N
Glucose (mg/dl) 98.00 11.87 6 79.00 11.51 6
SGOT (IU/l) 48.50 1.87 6 49.00 2.00 6
SGPT (IU/l) 31.17 3.82 6 30.00 4.05 6
ALP (IU/l) 312.58 12.89 6 292.00 10.47 6
Total Protein (g/dl) 6.00 0.41 6 6.13 0.37 6
Albumin (g/dl) 4.45 0.39 6 4.65 0.43 6
Triglycerides (mg/dl) 65.00 9.61 6 62.83 5.31 6
Creatinine (mg/dl) 0.57 0.24 6 0.57 0.24 6
Urea (mg/dl) 42.17 2.56 6 42.67 3.33 6 .
Parameter G3 G4
Mean SD N Mean SD N
Glucose (mg/dl) 98.80 8.01 6 64.20 20.73 5
SGOT (IU/l) 48.33 3.01 6 48.00 3.54 5
SGPT (IU/l) 33.83 5.38 6 36.40 3.36 5
ALP (IU/l) 302.92 17.40 6 322.52 15.93 5
Total Protein (g/dl) 6.33 0.27 6 6.26 0.40 5
Albumin (g/dl) 4.43 0.23 6 4.42 0.18 5
Triglycerides (mg/dl) 63.33 10.82 6 67.33 9.97 5
Creatinine (mg/dl) 0.53 0.25 6 0.54 0.24 5
Urea (mg/dl) 42.33 2.07 6 43.60 3.51 5
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 27 of 61
TABLE 5 - Clinical Chemistry - Group Mean Values (Continued)
Sex: Female
Parameter G1 G2
Mean SD N Mean SD N
Glucose (mg/dl) 91.50 8.38 6 93.17 13.70 6
SGOT (IU/l) 42.67 2.58 6 47.83 2.79 6
SGPT (IU/l) 40.50 3.56 6 38.33 3.44 6
ALP (IU/l) 306.80 26.77 6 304.48 12.33 6
Total Protein (g/dl) 5.98 0.34 6 5.97 0.35 6
Albumin (g/dl) 4.40 0.24 6 4.20 0.23 6
Triglycerides (mg/dl) 62.83 8.70 6 62.33 5.05 6
Creatinine (mg/dl) 0.43 0.18 6 0.43 0.20 6
Urea (mg/dl) 37.67 4.08 6 39.17 2.64 6
Parameter G3 G4
Mean SD N Mean SD N
Glucose (mg/dl) 97.17 11.43 6 99.67 21.65 6
SGOT (IU/l) 47.50 3.27 6 45.67 4.27 6
SGPT (IU/l) 41.17 3.19 6 40.83 3.25 6
ALP (IU/l) 313.02 17.32 6 313.87 11.21 6
Total Protein (g/dl) 5.92 0.35 6 5.80 0.29 6
Albumin (g/dl) 4.33 0.23 6 4.50 0.21 6
Triglycerides (mg/dl) 59.83 5.74 6 62.33 11.06 6
Creatinine (mg/dl) 0.45 0.16 6 0.67 0.28 6
Urea (mg/dl) 37.83 3.82 6 39.17 2.79 6
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 28 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
TABLE 6 - Organ Weights (g) - Group Mean Values
Sex: Male
Parameter G1 G2
Mean SD N Mean SD N
Brain 1.760 0.100 6 1.727 0.067 6
Heart 0.822 0.071 6 0.750 0.093 6
Liver 8.362 0.939 6 7.903 0.622 6
Kidneys 1.758 0.175 6 1.728 0.109 6
Adrenals 0.037 0.019 6 0.037 0.033 6
Spleen 1.310 0.197 6 1.310 0.162 6
Thymus 0.218 0.086 6 0.247 0.049 6
Testis 3.122 0.277 6 3.047 0.218 6
Epididymis 1.110 0.068 6 1.131 0.058 6
Parameter G3 G4
Mean SD N Mean SD N
Brain 1.645 0.148 6 1.912 0.190 5
Heart 0.707 0.018 6 0.810 0.155 5
Liver 7.327 0.769 6 9.062 1.148 5
Kidneys 1.675 0.096 6 1.940 0.274 5
Adrenals 0.037 0.014 6 0.046 0.017 5
Spleen 1.348 0.121 6 1.438 0.209 5
Thymus 0.242 0.058 6 0.202 0.089 5
Testis 2.853 0.260 6 2.958 0.188 5
Epididymis 1.112 0.084 6 1.138 0.048 5
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 29 of 61
TABLE 6 - Organ Weights (g) - Group Mean Values (Continued)
Sex: Female
Parameter G1 G2
Mean SD N Mean SD N
Brain 1.790 0.097 6 1.660 0.043 6
Heart 0.593 0.088 6 0.537 0.057 6
Liver 6.305 1.034 6 6.246 0.181 6
Kidneys 1.337 0.101 6 1.317 0.073 6
Adrenals 0.052 0.016 6 0.035 0.010 6
Spleen 0.822 0.208 6 0.737 0.078 6
Thymus 0.209 0.058 6 0.238 0.045 6
Ovaries 0.098 0.020 6 0.123 0.026 6
Uterus 0.523 0.211 6 0.324 0.086 6
Parameter G3 G4
Mean SD N Mean SD N
Brain 1.637 0.054 6 1.733 0.114 6
Heart 0.582 0.048 6 0.707 0.052 6
Liver 6.118 0.113 6 6.295 0.455 6
Kidneys 1.285 0.029 6 1.303 0.140 6
Adrenals 0.052 0.022 6 0.053 0.005 6
Spleen 0.820 0.069 6 0.892 0.113 6
Thymus 0.269 0.033 6 0.242 0.059 6
Ovaries 0.125 0.012 6 0.087 0.021 6
Uterus 0.275 0.022 6 0.450 0.209 6
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 30 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
TABLE 7 - Relative Organ Weights (%) - Group Mean Values
Sex: Male
Parameter G1 G2
Mean SD N Mean SD N
Brain 0.723 0.070 6 0.718 0.045 6
Heart 0.336 0.015 6 0.312 0.043 6
Liver 3.425 0.385 6 3.273 0.066 6
Kidneys 0.718 0.041 6 0.717 0.032 6
Adrenals 0.015 0.008 6 0.016 0.015 6
Spleen 0.541 0.109 6 0.542 0.036 6
Thymus 0.089 0.031 6 0.101 0.012 6
Testis 1.281 0.138 6 1.263 0.055 6
Epididymis 0.455 0.038 6 0.469 0.020 6
Parameter G3 G4
Mean SD N Mean SD N
Brain 0.704 0.026 6 0.787 0.078 5
Heart 0.304 0.021 6 0.332 0.053 5
Liver 3.137 0.207 6 3.732 0.479 5
Kidneys 0.719 0.046 6 0.798 0.104 5
Adrenals 0.015 0.005 6 0.019 0.006 5
Spleen 0.577 0.012 6 0.589 0.059 5
Thymus 0.103 0.018 6 0.084 0.039 5
Testis 1.222 0.067 6 1.218 0.097 5
Epididymis 0.477 0.036 6 0.470 0.047 5
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 31 of 61
TABLE 7 - Relative Organ Weights (%) - Group Mean Values (Continued)
Sex: Female
Parameter G1 G2
Mean SD N Mean SD N
Brain 1.122 0.057 6 1.061 0.017 6
Heart 0.373 0.066 6 0.342 0.030 6
Liver 3.964 0.747 6 3.993 0.164 6
Kidneys 0.838 0.069 6 0.842 0.061 6
Adrenals 0.032 0.011 6 0.022 0.006 6
Spleen 0.554 0.135 6 0.470 0.047 6
Thymus 0.127 0.033 6 0.152 0.026 6
Ovaries 0.554 0.135 6 0.079 0.016 6
Uterus 0.330 0.135 6 0.208 0.058 6
Parameter G3 G4
Mean SD N Mean SD N
Brain 1.037 0.038 6 1.095 0.081 6
Heart 0.368 0.031 6 0.446 0.028 6
Liver 3.876 0.148 6 3.976 0.309 6
Kidneys 0.814 0.041 6 0.822 0.071 6
Adrenals 0.033 0.015 6 0.034 0.004 6
Spleen 0.520 0.057 6 0.564 0.082 6
Thymus 0.170 0.017 6 0.153 0.040 6
Ovaries 0.079 0.009 6 0.055 0.012 6
Uterus 0.174 0.014 6 0.286 0.140 6
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 32 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
TABLE 8 - Gross Pathology Findings – Summary by Group
Organs &
Lesions
Group G1 G2 G3 G4
Sex M F M F M F M F N° of Animals 6 6 6 6 6 6 5 6
Brain NAD NAD NAD NAD NAD NAD NAD NAD
Liver NAD NAD NAD NAD NAD NAD NAD NAD
Kidney NAD NAD NAD NAD NAD NAD NAD NAD
Heart NAD NAD NAD NAD NAD NAD NAD NAD
Thymus NAD NAD NAD NAD NAD NAD NAD NAD
Adrenals NAD NAD NAD NAD NAD NAD NAD NAD
Testis NAD X NAD X NAD X NAD X
Epididymis NAD X NAD X NAD X NAD X
Ovaries X NAD X NAD X NAD X NAD
Oviduct & Uterus X NAD X NAD X NAD X NAD
Pancreas NAD NAD NAD NAD NAD NAD NAD NAD
Key: M= Male, F= Female, NAD = No Abnormality Detected Note: “X” organs not examined as per sex difference.
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 33 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX – A
Clinical Signs of Individual Animals
Group: G1 (Control) Animal
N° Day of Observation
0 7 14 21 28 Male
1 1 1 1 1 1 2 1 1 1 1 1 3 1 1 1 1 1 4 1 1 1 1 1 5 1 1 1 1 1 6 1 1 1 1 1
Female 7 1 1 1 1 1 8 1 1 1 1 1 9 1 1 1 1 1 10 1 1 1 1 1 11 1 1 1 1 1 12 1 1 1 1 1
Group: G2 (Therapeutic Dose)
Animal N°
Day of Observation 0 7 14 21 28
Male 13 1 1 1 1 1 14 1 1 1 1 1 15 1 1 1 1 1 16 1 1 1 1 1 17 1 1 1 1 1 18 1 1 1 1 1
Female 19 1 1 1 1 1 20 1 1 1 1 1 21 1 1 1 1 1 22 1 1 1 1 1 23 1 1 1 1 1 24 1 1 1 1 1
Key: 1 – Normal
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 34 of 61
APPENDIX – A (Continued)
Clinical Signs of Individual Animals Group : G3 (Average Dose)
Animal N°
Day of Observation 0 7 14 21 28
Male 25 1 1 1 1 1 26 1 1 1 1 1 27 1 1 1 1 1 28 1 1 1 1 1 29 1 1 1 1 1 30 1 1 1 1 1
Female 31 1 1 1 1 1 32 1 1 1 1 1 33 1 1 1 1 1 34 1 1 1 1 1 35 1 1 1 1 1 36 1 1 1 1 1
Group : G4 (High Dose)
Animal N°
Day of Observation 0 7 14 21 28
Male 37 1 1 1 1 1 38 1 1 1 1 1 39 1 1 2 2 2 40 1 1 1 1 1 41 1 1 1 1 1 42 1 1 1 1 1
Female 43 1 1 1 1 1 44 1 1 1 1 1 45 1 1 1 1 1 46 1 1 1 1 1 47 1 1 1 1 1 48 1 1 1 1 1
Key: 1 – Normal, 2 - Dead
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 35 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX – B
Individual Animal Body Weight (g)
Group : G1 (Control)
Animal N°
Day of Observation Terminal 0 7 14 21 28 Male
1 161.8 194.7 214.6 228.4 236.2 234.1 2 160.5 192.2 206.6 214.5 221.6 220.1 3 169.9 217.3 238.6 251.2 260.3 258.3 4 172.6 207.3 211.7 223.5 231.2 229.6 5 172.2 198.3 218.7 231.4 241.6 239.5 6 208.1 240.1 270.7 280.4 291.6 288.7
Female 7 152.0 179.3 187.1 193.5 201.4 200.1 8 156.1 177.0 183.2 188.0 196.2 194.2 9 159.4 186.7 193.3 196.0 206.4 205.1 10 162.5 183.9 194.6 197.5 204.9 202.9 11 160.3 195.1 202.6 210.1 218.3 216.4 12 167.1 179.3 185.3 189.3 198.4 196.2
Group : G2 (Therapeutic Dose) Animal
N° Day of Observation Terminal 0 7 14 21 28
Male 13 150.1 178.3 200.4 208.6 219.2 217.1 14 152.8 182.3 204.4 212.3 221.1 219.5 15 164.0 205.4 220.0 228.4 239.6 237.6 16 171.1 220.3 233.3 243.6 252.1 250.6 17 174.4 212.8 241.7 250.1 261.3 259.7 18 185.3 215.7 247.5 256.8 267.4 265.2
Female 19 150.7 174.8 180.7 184.3 189.2 188.4 20 155.1 177.7 182.1 186.1 196.3 197.9 21 155.5 171.7 177.2 181.0 188.2 187.4 22 158.8 188.9 197.6 203.5 208.9 206.4 23 158.5 182.6 192.2 196.4 204.5 202.3 24 160.5 183.6 187.5 191.3 199.3 197.8
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 36 of 61
APPENDIX – B (Continued)
Individual Animal Body Weight (g) Group : G3 (Average Dose)
Animal N°
Day of Observation Terminal 0 7 14 21 28 Male
25 165.0 170.4 195.4 203.4 213.1 211.4 26 179.8 183.0 223.5 234.6 241.6 239.4 27 162.8 200.7 222.8 231.2 239.8 237.2 28 177.8 222.3 243.5 252.7 263.5 261.9 29 182.1 215.6 218.7 226.8 234.7 232.6 30 189.0 202.4 205.6 213.2 220.1 218.8
Female 31 153.7 184.5 185.9 189.3 195.6 193.4 32 156.0 172.7 175.5 178.0 186.4 184.3 33 154.2 179.0 188.2 191.0 199.1 198.1 34 159.7 189.9 202.1 208.5 216.2 214.9 35 154.0 203.2 206.4 211.3 218.3 217.6 36 170.5 203.2 223.5 238.5 246.5 244.7
Group : G4 (High Dose)
Animal N°
Day of Observation Terminal 0 7 14 21 28 Male
37 153.7 180.6 196.6 206.1 214.6 212.1 38 176.7 202.4 244.1 256.7 264.7 262.4 39 166.3 153.7 FD FD FD FD 40 180.2 210.6 219.9 227.5 234.6 233.1 41 185.8 207.3 232.1 240.6 248.7 246.2 42 204.9 234.2 245.0 256.7 267.2 265.4
Female 43 152.8 179.3 185.3 189.6 196.4 195.1 44 152.8 179.5 186.2 190.0 194.7 199.7 45 156.4 178.4 185.6 191.5 198.7 206.7 46 158.5 179.5 190.3 196.0 204.6 210.6 47 162.1 185.7 188.3 193.5 201.3 209.5 48 168.2 193.2 200.5 206.0 214.7 220.7
Key: FD : Found Dead
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 37 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX – C
Food Consumption (g/cage)
Group : G1 (Control) Animal
N° Day of Observation
7 14 21 28 Male
1 196 217.8 213.4 218.6 2 3 210.5 279 260.2 268.9 4 5
212 279.6 240.2 251.2 6 Female
7 161.3 179.8 191.5 201.7 8
9 168.7 182.6 197.8 206.1 10
11 165.2 180.5 196.8 204.2 12
Group : G2 (Therapeutic Dose) Animal
N° Day of Observation
7 14 21 28 Male
13 208.6 284 251.1 261.9 14
15 207.7 212 217.9 225.6 16 17 229.8 245.6 226.6 230.1 18
Female 19 171.4 185.4 192.3 200.1 20 21 156.8 167.8 184.1 196.4 22 23
168.4 184.1 195..9 206.4 24
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 38 of 61
APPENDIX – C (Continued)
Food Consumption (g/cage) Group : G3 (Average Dose)
Animal N°
Day of Observation 7 14 21 28
Male 25 219.3 234.1 259 267.5 26 27 235.3 238.1 247.7 254.9 28 29 211.6 270.7 252.9 260.8 30
Female 31 163.4 175.6 184.7 192.3 32 33 160.3 178.4 187.1 196.5 34 35 163.7 176.4 191.4 204.7 36
Group : G4 (High Dose)
Animal N°
Day of Observation 7 14 21 28
Male 37 233 217.9 208.9 209.6 38 39 227.5 210.1 206.4 208.5 40 41
211.1 232.9 228.5 235.6 42 Female
43 145.6 165.6 173.8 185.7 44 45
153.7 178.6 182.9 196.7 46 47 148.7 174.3 186.8 199.4 48
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 39 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX – D
Individual Animal Haematological Values
Group : G1 (Control)
Animal No
Haematological Parameters
RBC (1x106/mm3)
WBC (1x103/mm3)
Hb (g/dl)
HCT (%)
MCV (um3)
MCH (pg)
MCHC (g/dl)
Plt (1x103/mm
3)
Male
1 7.73 14.60 17.50 38.60 51.30 23.80 53.20 365.40
2 6.59 12.50 14.60 42.30 48.60 26.40 52.60 389.40
3 6.84 13.70 18.60 41.70 50.20 25.40 54.60 351.20
4 7.53 12.80 16.30 38.60 48.70 25.10 55.30 389.70
5 7.51 11.70 14.50 41.30 50.10 27.80 53.10 451.20
6 7.56 11.30 17.80 27.90 51.20 25.40 56.20 412.00
Female
7 6.45 12.36 16.50 42.30 48.90 25.10 56.50 365.30
8 6.82 12.78 14.60 41.00 50.30 24.60 54.60 421.30
9 6.91 13.45 18.40 39.40 51.40 26.40 55.30 421.60
10 7.63 11.63 16.70 38.70 50.70 27.10 55.00 435.60
11 5.69 12.48 14.60 40.10 48.70 24.30 53.60 412.00
12 6.42 14.32 15.20 40.60 48.60 25.60 56.20 351.00
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 40 of 61
APPENDIX – D (Continued)
Individual Animal Haematological Values
Group : G2 (Therapeutic Dose)
Animal No
Haematological Parameters
RBC (1x106/mm3)
WBC (1x103/mm3)
Hb (g/dl)
HCT (%)
MCV (um3)
MCH (pg)
MCHC (g/dl)
Plt (1x103/mm3)
Male
13 7.48 12.40 15.60 41.30 52.40 26.40 54.60 356.70
14 7.63 16.30 17.60 42.10 53.10 23.50 55.50 402.30
15 8.54 14.50 15.90 38.40 51.40 24.10 52.30 465.30
16 6.35 14.30 17.80 39.70 50.20 27.30 53.20 512.30
17 6.48 13.40 16.30 40.50 48.90 25.10 54.60 345.60
18 6.21 13.10 18.40 40.20 47.90 24.60 55.80 327.60
Female
19 6.47 12.45 18.60 38.50 47.90 26.10 54.60 346.50
20 6.93 15.30 14.70 40.20 50.20 24.60 56.30 256.70
21 7.65 14.80 16.30 41.20 51.30 24.50 55.40 347.90
22 5.39 16.40 14.60 38.40 50.70 25.40 57.10 421.60
23 6.42 12.70 15.80 40.20 52.10 23.40 55.20 423.10
24 6.73 14.20 16.50 38.90 48.90 25.60 54.20 426.50
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 41 of 61
APPENDIX – D (Continued)
Individual Animal Haematological Values
Group : G3 (Average Dose)
Animal No
Haematological Parameters
RBC (1x106/mm3)
WBC (1x103/mm3)
Hb (g/dl)
HCT (%)
MCV (um3)
MCH (pg)
MCHC (g/dl)
Plt (1x103/mm3)
Male
25 6.41 14.50 18.40 39.40 52.10 24.10 55.10 456.30
26 6.21 12.70 17.60 42.60 53.10 23.60 57.60 356.20
27 6.89 12.50 18.40 40.10 51.20 24.70 53.60 285.60
28 7.32 12.70 18.60 38.70 50.20 26.20 54.40 294.70
29 7.63 12.60 17.50 42.10 51.20 24.70 55.20 423.10
30 6.92 15.40 16.20 40.50 50.70 25.10 57.30 425.60
Female
31 6.49 14.50 17.40 37.90 50.30 24.60 54.60 347.60
32 6.43 16.70 15.60 42.10 51.40 24.80 52.80 396.10
33 5.69 18.40 14.50 40.30 51.80 26.50 54.70 378.90
34 7.21 14.30 14.60 41.10 50.30 25.10 53.90 363.80
35 7.63 14.90 16.30 39.40 48.90 25.30 54.60 412.50
36 7.91 12.60 17.20 37.80 47.60 27.20 54.30 412.30
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 42 of 61
APPENDIX – D (Continued)
Individual Animal Haematological Values
Group : G4 (High Dose)
Animal No
Haematological Parameters
RBC (1x106/mm3)
WBC (1x103/mm3)
Hb (g/dl)
HCT (%)
MCV (um3)
MCH (pg)
MCHC (g/dl)
Plt (1x103/mm3)
Male
37 8.64 14.30 15.40 41.70 50.20 24.10 56.30 347.90
38 7.89 14.70 16.40 40.30 50.90 26.30 54.00 421.30
39 D D D D D D D D
40 7.42 15.70 17.20 38.50 47.90 25.60 54.20 312.40
41 7.63 12.40 17.07 40.10 48.60 26.40 55.30 435.60
42 7.35 12.70 16.40 41.30 50.20 25.10 51.20 421.40
Female
43 6.27 14.70 17.60 41.20 48.70 24.60 55.40 326.40
44 6.37 14.90 15.30 42.10 50.30 24.80 55.30 357.80
45 5.94 15.80 14.60 40.30 51.20 26.10 55.60 396.40
46 7.62 12.70 17.40 38.90 52.40 24.60 56.10 402.30
47 7.73 13.40 16.50 39.40 50.70 25.40 52.30 402.40
48 7.26 14.60 17.50 39.80 49.80 26.30 57.40 456.30
D- Dead
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 43 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX – E
Individual Animal Clinical Chemistry Values
Group: G1 (Control)
Animal No
Clinical Chemistry Parameters
Glucose (mg/dl)
SGOT (IU/l)
SGPT (IU/l)
Total Protein (g/dl)
Creatinine (mg/dl)
Albumin (g/dl)
ALP (IU/l)
Urea (mg/dl) Triglycerides
Male
1 119.00 46.00 34.00 5.50 0.80 4.20 296.50 38.00 62
2 86.00 49.00 33.00 5.90 0.60 4.30 297.50 42.00 59
3 93.00 51.00 26.00 6.20 0.40 3.90 312.40 46.00 71
4 104.00 50.00 28.00 6.70 0.90 4.60 321.40 42.00 63
5 95.00 47.00 36.00 5.80 0.40 4.70 326.40 43.00 54
6 91.00 48.00 30.00 5.90 0.30 5.00 321.30 42.00 81
Female
7 88.00 43.00 36.00 5.80 0.30 4.60 268.90 39.00 56
8 88.00 42.00 39.00 6.20 0.30 4.20 312.60 31.00 69
9 84.00 46.00 42.00 5.40 0.40 4.30 325.80 35.00 71
10 97.00 41.00 46.00 6.20 0.60 4.20 343.60 41.00 53
11 86.00 39.00 42.00 6.30 0.30 4.80 287.30 38.00 72
12 106.00 45.00 38.00 6.00 0.70 4.30 302.60 42.00 56
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 44 of 61
APPENDIX – E (Continued)
Individual Animal Clinical Chemistry Values Group: G2 (Therapeutic Dose)
Animal No
Clinical Chemistry Parameters
Glucose (mg/dl)
SGOT (IU/l)
SGPT (IU/l)
Total Protein (g/dl)
Creatinine (mg/dl)
Albumin (g/dl)
ALP (IU/l)
Urea (mg/dl) Triglycerides
Male
13 74.00 49.00 27.00 5.70 0.50 5.20 285.40 38.00 63
14 71.00 48.00 36.00 5.90 0.70 4.90 294.60 42.00 57
15 77.00 52.00 28.00 6.70 0.40 4.70 302.10 46.00 61
16 91.00 46.00 29.00 6.20 0.80 4.60 305.70 42.00 72
17 95.00 50.00 34.00 6.40 0.20 3.90 284.60 41.00 59
18 66.00 49.00 26.00 5.90 0.80 4.60 279.60 47.00 65
Female
19 102.00 48.00 33.00 5.90 0.60 4.10 289.60 41.00 59
20 85.00 43.00 37.00 5.40 0.50 4.00 312.70 40.00 63
21 100.00 48.00 38.00 6.20 0.70 4.60 324.60 35.00 71
22 98.00 51.00 43.00 5.80 0.30 4.20 301.40 37.00 56
23 105.00 50.00 41.00 6.40 0.20 4.30 300.50 42.00 63
24 69.00 47.00 38.00 6.10 0.30 4.00 298.10 40.00 62
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 45 of 61
APPENDIX – E (Continued)
Individual Animal Clinical Chemistry Values Group: G3 (Average Dose)
Animal No
Clinical Chemistry Parameters
Glucose (mg/dl)
SGOT (IU/l)
SGPT (IU/l)
Total Protein (g/dl)
Creatinine (mg/dl)
Albumin (g/dl)
ALP (IU/l)
Urea (mg/dl) Triglycerides
Male
25 89.00 49.00 34.00 5.90 0.60 4.30 281.50 42.00 63
26 94.00 43.00 40.00 6.20 0.70 4.70 283.60 46.00 71
27 93.00 48.00 38.00 6.30 0.20 4.50 325.40 41.00 56
28 93.00 52.00 36.00 6.70 0.40 4.70 302.40 43.00 81
29 103.00 50.00 29.00 6.50 0.90 4.20 312.20 42.00 53
30 111.00 48.00 26.00 6.40 0.40 4.20 312.40 40.00 56
Female
31 85.00 46.00 37.00 6.20 0.40 4.50 345.60 32.00 53
32 103.00 42.00 46.00 6.10 0.30 4.30 312.40 37.00 69
33 96.00 49.00 43.00 6.30 0.60 4.70 302.40 38.00 56
34 91.00 51.00 42.00 5.90 0.30 4.30 298.70 42.00 57
35 91.00 50.00 39.00 5.40 0.40 4.10 302.60 36.00 63
36 117.00 47.00 40.00 5.60 0.70 4.10 316.40 42.00 61
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 46 of 61
APPENDIX – E (Continued)
Individual Animal Clinical Chemistry Values Group: G4 (High Dose)
Animal No
Clinical Chemistry Parameters
Glucose (mg/dl)
SGOT (IU/l)
SGPT (IU/l)
Total Protein (g/dl)
Creatinine (mg/dl)
Albumin (g/dl)
ALP (IU/l)
Urea (mg/dl) Triglycerides
Male
37 78.00 46.00 34.00 5.60 0.60 4.60 302.40 46.00 72
38 89.00 43.00 42.00 6.30 0.70 4.20 326.40 42.00 83
39 D D D D D D D D D
40 43.00 50.00 34.00 6.70 0.40 4.30 314.70 43.00 63
41 43.00 49.00 35.00 6.40 0.80 4.40 345.60 48.00 59
42 68.00 52.00 37.00 6.30 0.20 4.60 323.50 39.00 71
Female
43 139.00 42.00 37.00 5.40 0.40 4.70 312.40 37.00 56
44 99.00 50.00 46.00 6.00 0.30 4.60 306.40 42.00 59
45 81.00 51.00 42.00 6.20 0.80 4.20 296.40 40.00 61
46 105.00 43.00 38.00 5.90 0.90 4.50 326.10 39.00 84
47 80.00 47.00 42.00 5.70 1.00 4.70 318.20 35.00 53
48 94.00 41.00 40.00 5.60 0.60 4.30 323.70 42.00 61
D- Dead
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 47 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX – F
Individual Animal Organ Weights (g)
Group: G1 (Control) Animal
No Organ Weights (g)
Male
Brain Heart Liver Kidneys Adrenals Spleen Thymus Testis Epididymis
1 1.750 0.820 9.460 1.830 0.020 1.240 0.090 3.340 1.150
2 1.800 0.750 7.550 1.470 0.020 1.200 0.220 3.180 1..118
3 1.930 0.820 8.370 1.870 0.030 1.250 0.160 3.130 1.170
4 1.640 0.750 6.970 1.650 0.060 1.710 0.240 3.020 1.020
5 1.690 0.850 8.850 1.770 0.060 1.240 0.260 2.640 1.030
6 1.750 0.940 8.970 1.960 0.030 1.220 0.340 3.420 1.170
Female
Brain Heart Liver Kidneys Adrenals Spleen Thymus Ovaries Uterus
7 1.750 0.740 8.150 1.420 0.070 0.950 0.220 0.12 0.460
8 1.870 0.610 6.000 1.330 0.030 0.950 0.160 0.12 0.750
9 1.720 0.580 6.010 1.340 0.070 1.030 0.174 0.11 0.440
10 1.800 0.620 6.460 1.300 0.040 1.070 0.230 0.08 0.350
11 1.670 0.500 4.990 1.170 0.050 0.510 0.160 0.08 0.820
12 1.930 0.510 6.220 1.460 0.050 0.780 0.310 0.08 0.320
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 48 of 61
APPENDIX – F (Continued)
Individual Animal Organ Weights (g) Group: G2 (Therapeutic Dose) Animal
No Organ Weights (g)
Male
Brain Heart Liver Kidneys Adrenals Spleen Thymus Testis Epididymis
13 1.650 0.630 7.340 1.650 0.100 1.120 0.180 2.860 1.054
14 1.670 0.780 7.020 1.560 0.020 1.232 0.210 2.890 1.062
15 1.780 0.860 7.860 1.720 0.040 1.240 0.230 2.954 1.148
16 1.690 0.720 8.210 1.860 0.010 1.260 0.260 3.010 1.186
17 1.820 0.840 8.350 1.780 0.020 1.560 0.290 3.120 1.169
18 1.750 0.670 8.640 1.800 0.030 1.450 0.310 3.450 1.167
Female
Brain Heart Liver Kidneys Adrenals Spleen Thymus Ovaries Uterus
19 1.620 0.480 6.340 1.430 0.020 0.640 0.178 0.130 0.345
20 1.630 0.520 6.480 1.360 0.030 0.710 0.189 0.140 0.470
21 1.640 0.510 6.020 1.240 0.040 0.810 0.260 0.090 0.360
22 1.710 0.560 6.047 1.260 0.030 0.690 0.240 0.100 0.270
23 1.640 0.510 6.241 1.270 0.050 0.850 0.270 0.120 0.240
24 1.720 0.640 6.345 1.340 0.040 0.720 0.290 0.160 0.260
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 49 of 61
APPENDIX – F (Continued)
Individual Animal Organ Weights (g) Group : G3 (Average Dose) Animal
No Organ Weights (g)
Male
Brain Heart Liver Kidneys Adrenals Spleen Thymus Testis Epididymis
25 1.530 0.710 6.850 1.680 0.020 1.210 0.190 2.410 1.027
26 1.620 0.720 7.540 1.710 0.040 1.340 0.220 2.890 1.096
27 1.670 0.690 6.480 1.560 0.030 1.380 0.230 2.880 1.078
28 1.930 0.720 8.640 1.840 0.060 1.560 0.340 3.210 1.154
29 1.560 0.720 7.560 1.640 0.040 1.340 0.280 2.790 1.260
30 1.560 0.680 6.890 1.620 0.030 1.260 0.194 2.940 1.055
Female
Brain Heart Liver Kidneys Adrenals Spleen Thymus Ovaries Uterus
31 1.540 0.640 6.210 1.270 0.060 0.890 0.211 0.120 0.260
32 1.690 0.620 6.240 1.290 0.080 0.730 0.270 0.110 0.270
33 1.620 0.530 6.024 1.340 0.070 0.860 0.260 0.140 0.250
34 1.670 0.560 6.024 1.280 0.040 0.760 0.280 0.140 0.310
35 1.630 0.530 6.000 1.260 0.040 0.890 0.280 0.120 0.290
36 1.670 0.610 6.210 1.270 0.020 0.790 0.310 0.120 0.270
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 50 of 61
APPENDIX – F (Continued)
Individual Animal Organ Weights (g) Group : G4 (High Dose) Animal
No Organ Weights (g)
Male
Brain Heart Liver Kidneys Adrenals Spleen Thymus Testis Epididymis
37 1.680 0.670 8.050 1.720 0.030 1.100 0.170 2.710 1.110
38 2.110 1.060 10.480 2.350 0.030 1.450 0.240 2.930 1.120
39 D D D D D D D D D
40 2.011 0.841 9.650 2.000 0.050 1.560 0.340 2.960 1.140
41 2.020 0.780 9.400 1.970 0.050 1.430 0.120 3.240 1.220
42 1.740 0.700 7.730 1.660 0.070 1.650 0.142 2.950 1.100
Female
Brain Heart Liver Kidneys Adrenals Spleen Thymus Ovaries Uterus
43 1.720 0.730 6.510 1.230 0..060 0.930 0.150 0.070 0.44
44 1.810 0.730 6.200 1.290 0.050 0.910 0.330 0.100 0.840
45 1.520 0.630 6.190 1.350 0.050 1.050 0.250 0.070 0.240
46 1.830 0.690 6.620 1.170 0.060 0.870 0.270 0.090 0.450
47 1.800 0.700 5.490 1.220 0.050 0.700 0.230 0.070 0.300
48 1.720 0.790 6.760 1.560 0.050 0.890 0.220 0.120 0.430
D- Dead
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 51 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX – G
Individual Animal Relative Organ Weights (%)
Group : G1 (Control) Animal
No Relative Organ Weights (%)
Male
Brain Heart Liver Kidneys Adrenals Spleen Thymus Testis Epididymis
1 0.748 0.350 4.041 0.782 0.009 0.530 0.038 1.427 0.491
2 0.818 0.341 3.430 0.668 0.009 0.545 0.100 1.445 0.508
3 0.747 0.317 3.240 0.724 0.012 0.484 0.062 1.212 0.453
4 0.714 0.327 3.036 0.719 0.026 0.745 0.105 1.315 0.444
5 0.706 0.355 3.695 0.739 0.025 0.518 0.109 1.102 0.430
6 0.606 0.326 3.107 0.679 0.010 0.423 0.118 1.185 0.405
Female
Brain Heart Liver Kidneys Adrenals Spleen Thymus Ovaries Uterus
7 1.151 0.487 5.362 0.934 0.046 0.625 0.124 0.625 0.303
8 1.198 0.391 3.844 0.852 0.019 0.609 0.102 0.609 0.480
9 1.079 0.364 3.770 0.841 0.044 0.646 0.109 0.646 0.276
10 1.108 0.382 3.975 0.800 0.025 0.658 0.142 0.658 0.215
11 1.042 0.312 3.113 0.730 0.031 0.318 0.100 0.318 0.512
12 1.155 0.305 3.722 0.874 0.030 0.467 0.186 0.467 0.192
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 52 of 61
APPENDIX – G (Continued)
Individual Animal Relative Organ Weights (%)
Group: G2 (Therapeutic Dose) Animal
No Relative Organ Weights (%)
Male
Brain Heart Liver Kidneys Adrenals Spleen Thymus Testis Epididymis
13 0.760 0.290 3.381 0.760 0.046 0.516 0.083 1.317 0.485
14 0.761 0.355 3.198 0.711 0.009 0.561 0.096 1.317 0.484
15 0.749 0.362 3.308 0.724 0.017 0.522 0.097 1.243 0.483
16 0.674 0.287 3.276 0.742 0.004 0.503 0.104 1.201 0.473
17 0.701 0.323 3.215 0.685 0.008 0.601 0.112 1.201 0.450
18 0.660 0.253 3.258 0.679 0.011 0.547 0.117 1.301 0.440
Female
Brain Heart Liver Kidneys Adrenals Spleen Thymus Ovaries Uterus
19 1.075 0.319 4.207 0.949 0.013 0.425 0.118 0.086 0.229
20 1.051 0.335 4.178 0.877 0.019 0.458 0.122 0.090 0.303
21 1.055 0.328 3.871 0.797 0.026 0.521 0.167 0.058 0.232
22 1.077 0.353 3.808 0.793 0.019 0.435 0.151 0.063 0.170
23 1.035 0.322 3.938 0.801 0.032 0.536 0.170 0.076 0.151
24 1.072 0.399 3.953 0.835 0.025 0.449 0.181 0.100 0.162
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 53 of 61
APPENDIX – G (Continued)
Individual Animal Relative Organ Weights (%) Group: G3 (Average Dose) Animal
No Relative Organ Weights (%)
Male
Brain Heart Liver Kidneys Adrenals Spleen Thymus Testis Epididymis
25 0.724 0.336 3.240 0.795 0.009 0.572 0.090 1.140 0.486
26 0.677 0.301 3.150 0.714 0.017 0.560 0.092 1.207 0.458
27 0.704 0.291 2.732 0.658 0.013 0.582 0.097 1.214 0.454
28 0.737 0.275 3.299 0.703 0.023 0.596 0.130 1.226 0.441
29 0.671 0.310 3.250 0.705 0.017 0.576 0.120 1.199 0.542
30 0.713 0.311 3.149 0.740 0.014 0.576 0.089 1.344 0.482
Female
Brain Heart Liver Kidneys Adrenals Spleen Thymus Ovaries Uterus
31 1.002 0.416 4.040 0.826 0.039 0.579 0.137 0.078 0.169
32 1.083 0.397 4.000 0.827 0.051 0.468 0.173 0.071 0.173
33 1.051 0.344 3.907 0.869 0.045 0.558 0.169 0.091 0.162
34 1.046 0.351 3.772 0.802 0.025 0.476 0.175 0.088 0.194
35 1.058 0.344 3.896 0.818 0.026 0.578 0.182 0.078 0.188
36 0.979 0.358 3.642 0.745 0.012 0.463 0.182 0.070 0.158
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 54 of 61
APPENDIX – G (Continued)
Individual Animal Relative Organ Weights (%) Group: G4 (High Dose) Animal
No Relative Organ Weights (%)
Male
Brain Heart Liver Kidneys Adrenals Spleen Thymus Testis Epididymis
37 0.792 0.316 3.795 0.811 0.014 0.519 0.080 1.278 0.523
38 0.804 0.404 3.994 0.896 0.011 0.553 0.091 1.117 0.427
39 D D D D D D D D D
40 0.863 0.361 4.140 0.858 0.021 0.669 0.146 1.270 0.489
41 0.820 0.317 3.818 0.800 0.020 0.581 0.049 1.316 0.496
42 0.656 0.264 2.913 0.625 0.026 0.622 0.054 1.112 0.414
Female
Brain Heart Liver Kidneys Adrenals Spleen Thymus Ovaries Uterus
43 1.126 0.458 4.260 0.805 0.039 0.609 0.098 0.046 0.288
44 1.185 0.478 4.058 0.844 0.033 0.596 0.216 0.065 0.550
45 0.972 0.403 3.958 0.863 0.032 0.671 0.160 0.045 0.153
46 1.155 0.435 4.177 0.738 0.038 0.549 0.170 0.057 0.284
47 1.110 0.432 3.387 0.753 0.031 0.432 0.142 0.043 0.185
48 1.023 0.470 4.019 0.927 0.030 0.529 0.131 0.071 0.256
D- Dead
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 55 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX – H
Gross and Microscopic Findings of Individual Animals
Group: G1 (Control) Sex : Male
Animal N°
Mode of Death
Gross Findings Microscopic Findings
External Internal
1 TS NAD Lung Abscess NAD
2 TS NAD NAD NAD
3 TS NAD NAD NAD
4 TS NAD NAD NAD
5 TS NAD Lung Abscess NAD
6 TS NAD Lung Abscess NAD Group : G1 (Control) Sex : Female
Animal N°
Mode of Death
Gross Findings Microscopic Findings
External Internal
7 TS NAD Foci in Lungs NAD
8 TS NAD Patches in lungs NAD
9 TS NAD NAD NAD
10 TS NAD NAD NAD
11 TS NAD NAD NAD
12 TS NAD NAD NAD Key: TS = Terminal Sacrifice; NAD = No Abnormalities Detected
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 56 of 61
APPENDIX – H (Continued)
Gross and Microscopic Findings of Individual Animals
Group : G2 (Therapeutic Dose) Sex : Male
Animal N°
Mode of Death
Gross Findings Microscopic Findings
External Internal
13 TS NAD NAD X
14 TS NAD NAD X
15 TS NAD NAD X
16 TS NAD NAD X
17 TS NAD NAD X
18 TS NAD NAD X Group : G2 (Therapeutic Dose) Sex : Female
Animal N°
Mode of Death
Gross Findings Microscopic Findings
External Internal
19 TS NAD NAD X
20 TS NAD NAD X
21 TS NAD Liver Cyst X
22 TS NAD NAD X
23 TS NAD NAD X
24 TS NAD Patches in Lungs X
Key: TS = Terminal Sacrifice; NAD = No Abnormalities Detected Note: “X” organs not examined as per protocol, except where gross lesions were recorded
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 57 of 61
APPENDIX – H (Continued)
Gross and Microscopic Findings of Individual Animals
Group : G3 (Average Dose) Sex : Male
Animal N°
Mode of Death
Gross Findings Microscopic Findings
External Internal
25 TS NAD NAD X
26 TS NAD Liver Cyst X
27 TS NAD NAD X
28 TS NAD Lung Abscess X
29 TS NAD NAD X
30 TS NAD NAD X Group : G3 (Average Dose) Sex : Female
Animal N°
Mode of Death
Gross Findings Microscopic Findings
External Internal
31 TS NAD Liver Cyst X
32 TS NAD White patches in lungs X
33 TS NAD NAD X
34 TS NAD Foci in Lung X
35 TS NAD NAD X
36 TS NAD Liver Cyst X Key: TS = Terminal Sacrifice; NAD = No Abnormalities Detected Note: “X” organs not examined as per protocol, except where gross lesions were recorded
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 58 of 61
APPENDIX – H (Continued)
Gross and Microscopic Findings of Individual Animals
Group : G4 (High Dose) Sex : Male
Animal N°
Mode of Death
Gross Findings Microscopic Findings
External Internal
37 TS NAD NAD NAD
38 TS NAD Lung
Abscess, liver cyst
NAD
39 Dead NAD Lung Abscess Dead
40 TS NAD NAD NAD
41 TS NAD Lung Abscess NAD
42 TS NAD Lung Abscess NAD Group : G4 (High Dose) Sex : Female
Animal N°
Mode of Death
Gross Findings Microscopic Findings
External Internal
43 TS NAD NAD NAD
44 TS NAD NAD NAD
45 TS NAD Lung Abscess NAD
46 TS NAD NAD NAD
47 TS NAD Liver Cyst NAD
48 TS NAD NAD NAD Key: TS = Terminal Sacrifice; NAD = No Abnormalities Detected
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 59 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX – I
Feed Analysis Report
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 60 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX – J
Water Analysis Report
SLS Study Number: SLS/042-11
Sugen Life Sciences Pvt Ltd Page 61 of 61
28 Day Repeated Dose Oral Toxicity Study of Dia Bliss Sugar in Wistar Rats
APPENDIX – L
Certificate – Department of Science and Industrial Research
Recommended