Final Report

Study Title Acute oral toxicity study in Wistar rats

Test Item Nualgi Nano Nutrients

Study Director Mr. S. Haribabu, B Tech (Biotech), MSc

Sponsor Nualgi Nano Biotech Co

651, 11th Main Road
V Block, Jayanagar
Bengaluru 560041

Study Monitor Mr. Sunil Nanda

Test Facility GLR Laboratories Private Limited

Plot 18-19, Ganesh Nagar, Asisi Nagar
Madhavaram, Chennai - 600 060
Tamil Nadu
India.

Study Number 161/001

Regulatory Guideline OECD Guidelines for Testing of Chemicals, 423

(adopted on 17th December, 2001).

Report Issued 01 October 2015

Total Number of Pages 18

GLR Laboratories Private Limited Page 1

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 Final Report
GLR Study Number: 161/001

CONTENTS

Contents ......................................................................................................................... 2
Study Director Authentication Statement ...................................................................... 3
Quality Assurance Statement ......................................................................................... 4
Test Facility Management Statement............................................................................. 5
Peer Review Statement .................................................................................................. 6
Summary ........................................................................................................................ 7
Introduction .................................................................................................................... 8
Objective ........................................................................................................................ 9
Study Dates .................................................................................................................... 9
Test Item Details ............................................................................................................ 9
Test System .................................................................................................................. 10
Test Method ................................................................................................................. 11
Observations ................................................................................................................ 12
Data Evaluation ............................................................................................................ 13
Results .......................................................................................................................... 13
Conclusion ................................................................................................................... 13
References .................................................................................................................... 14
Responsible Personnel ................................................................................................. 17
Study Plan Amendment ............................................................................................... 17
Study Plan Deviation ................................................................................................... 17
Archive Statement ........................................................................................................ 17
Distribution of Reports ................................................................................................ 17
Appendix 1 ................................................................................................................... 18

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GLR Study Number: 161/001

STUDY DIRECTOR AUTHENTICATION STATEMENT

Nualgi Nano Nutrients: Acute oral toxicity study in Wistar rats

This study was performed in accordance to the agreed Study Plan, One amendment
and with GLR Laboratories Pvt Ltd Standard Operating Procedures, unless otherwise
stated, and the study objective was achieved. I accept responsibility for the work and
generated data, that are scientifically acceptable and valid; and this report provides a
true and accurate record of the results obtained.

This study was performed based on the OECD Principles of Good Laboratory
Practice* ENV/MC/CHEM (98)17 (Revised 1997, issued January 1998).

Mr. S. Haribabu, B Tech (Biotech), MSc Study completion date

Study Director
GLR Laboratories Pvt Ltd

*
With the exception of the identity and composition of the test item, which were the
responsibilities of the sponsor.

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GLR Study Number: 161/001

QUALITY ASSURANCE STATEMENT

This study report has been reviewed by the Quality Assurance Unit of GLR
Laboratories Pvt Ltd, based on the OECD Principles of GLP, Study Plan,
Amendment, Raw Data and applicable Standard Operating Procedures.

This statement confirms that the study report accurately reflects raw data.

The summary of inspections performed during the course of study is as follows:

Date of Reporting to
Management, Study
Type of Phase(s) of Study
S. No Date of Inspection Director
Inspection Inspected
(Inspection Report
No.)
Study Based 06 August 2015
1 06 August 2015 Draft Study Plan
Inspection (SBI/161/001/001)
Study Based Definitive Study 07 August 2015
2 07 August 2015
Inspection Plan (SBI/161/001/002)
Study Based Test Item 18 August 2015
3 18 August 2015
Inspection Administration (SBI/161/001/003)
Study Based Definitive Study 15 September 2015
4 15 September 2015
Inspection Plan Amendment (SBI/161/001/004)
Study Based 30 September 2015
5 30 September 2015 Draft Report
Inspection (SBI/161/001/005)
Study Based 01 October 2015
6 01 October 2015 Final Report
Inspection (SBI/161/001/006)

Dr. G. Velmani, M Pharm, PhD Date

Executive-Quality Assurance
GLR Laboratories Pvt Ltd

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GLR Study Number: 161/001

TEST FACILITY MANAGEMENT STATEMENT

Nualgi Nano Nutrients: Acute oral toxicity study in Wistar rats

This is to certify that the GLR test facility management appointed the Study Director
for this study and provided him with all necessary facilities and resources for proper
conduct of this study, both in terms of GLP and scientific integrity.

Dr. S. S. Murugan, PhD Date

Test Facility Management
GLR Laboratories Pvt Ltd

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GLR Study Number: 161/001

PEER REVIEW STATEMENT

This is to certify that I have reviewed the raw data and report along with the study
director and agree with the scientific conclusions made.

Dr. T S Kumaravel, MD, PhD, DABT Date

American Board Certified and UK Registered Toxicologist
GLR Laboratories Pvt Ltd

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SUMMARY

This study was performed to determine the acute toxicity effect of Nualgi Nano
Nutrients, when administered as a single oral dose to rats, followed by an observation
period of 14 days. A stepwise procedure using 3 female animals per step was used in
this study to assess the acute toxicity of the test item as described in the OECD
guidelines for testing of chemicals, 423. The starting dose was 300 mg/Kg body
weight. Test item was mixed with distilled water to achieve the desired concentration.
All animals received a single dose of the test item by oral route of administration after
being fasted for approximately 15 to 16 hours, but with free access to water. A dose
volume of 10 mL/Kg b.w. was used. Food was supplied approximately 3 h to 4 h after
test item administration. The results are summarised in the table below:

Number of
Step Date Dose Number of
moribund or Subsequent action
(mg/Kg) animals
dead animals
1 18 August 2015 300 3 females 0 Proceeded to Step 2
2 20 August 2015 300 3 females 0 Proceeded to Step 3
3 03 September 2015 2000 3 females 0 Proceeded to Step 4
4 07 September 2015 2000 3 females 0 Stop

No mortality and morbidity were observed in any of the animals administered with
the test item. In all the steps, animals were in somnolence condition with decreased
motor activity. None of the animals exhibit gross lesions related to test item
administration. Increase in body weight was observed in all the treated animals at the
end of the each step of the experiment. Based on the results obtained, it is concluded
that, Nualgi Nano Nutrients, falls under „Category 5 or unclassified‟ according to the
Globally Harmonized System (GHS) for the classification of chemicals. The cut off
LD50 value is greater than 5000 mg/Kg.

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INTRODUCTION

Acute oral toxicity is the study of adverse effects of a chemical that result either from
a single oral exposure or from multiple exposures within 24 hours. Acute oral
toxicity provides general information on health hazards likely to arise from an acute
exposure. An acute toxicity study might be an initial step in establishing a dosage
regimen in sub acute/sub chronic and other studies and may provide information on
the mode of toxic action of a substance by the intended clinical exposure route.

The test selection and methods used in this study are based on the following
guideline:

1. Organization for Economic Co-operation and Development Guidelines for Testing

of Chemicals (Sec. 4, No.423, Acute Toxic Class Method, Adopted 17th
December, 2001).

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GLR Study Number: 161/001

OBJECTIVE

To determine the acute toxic potential of test item when administered by a single oral
dose to rats, followed by an observation period of 14 days.

STUDY DATES

Study Start Date 07 August 2015

Experiment Start Date 18 August 2015
(first dosing date)
Experiment Completion Date 21 September 2015

The study completion date is the date the final report is signed by the Study Director.

This study was performed in line with agreed study plan and one amendment.

TEST ITEM DETAILS

The test item for this study was Nualgi Nano Nutrients. It was received at GLR on
21 July 2015 and stored at Room temperature (20 - 30) °C.

The following test item information provided by the Sponsor, are considered an
adequate description of the characterisation, purity and stability of the test item.

Name Nualgi Nano Nutrients

Appearance Liquid
Colour Greenish
Batch No. 60453
Formulation/Composition Nano nutrients mixture of primary, Secondary, Micro
CO, Si
Manufacture Date 03 July 2015
Expiry Date 3 years from manufacturing
Chemical Name Nano nutrients
CAS No Not Applicable
Molecular Formula Not Applicable
Molecular Weight Not Applicable
Solubility Soluble in water
pH 8.01
Photo-Stability Yes
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Determinations of stability and characteristics of the test item were the responsibility
of the Sponsor. The test item was handled with necessary protective clothing and all
recommended safety measures were followed.

TEST SYSTEM

Species Rattus norvegicus (rat)

Strain Wistar
Age 8 - 12 weeks
Sex Female
Source Sainath Agencies, Hyderabad, India.
This supplier is approved by the Committee for the
Purpose of Control and Supervision of Experiments on
Animals (CPCSEA), Government of India for breeding
laboratory animals.

Weight Range at the start Step 1 160.0 - 167.5

of experiment (g) Step 2 163.6 - 170.3
Step 3 161.1 - 170.4
Step 4 168.9 - 173.8
Number of animals used Twelve (Three animals/Step)
Acclimatization period Step 1 6 days
Step 2 8 days
Step 3 8 days
Step 4 12 days
Justification for animal use Rats were selected because there is a large volume of
background data on this species.
Specified in OECD Guidelines for Testing of
Chemicals, 423 standards, as an appropriate test to
evaluate the oral toxicity of test item and recommended
by various regulatory authorities.

The test system was approved by the GLR Laboratories Pvt Ltd‟s Institutional Animal
Ethics Committee (IAEC).

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ANIMAL HUSBANDRY

Location Test room 2

Test room temperature 19.1 - 22.7 °C
Relative humidity 35 - 68%
Housing Animals were housed in groups of three in standard
polypropylene cages with stainless steel top grill.
Method of identification Animals were identified using cage cards indicating
cage no., study no., species name, strain, animal no.,
sex, age/bodyweight, group, dose, signature and
individual marking.
Diet Rat pellet feed (Amrut)
Water Purified drinking water was provided ad libitum
Bedding material Sterilized paddy husk
Photo period 12 h light and 12 h dark cycle
Contaminants Contaminants reasonably expected in feed and water
supplied was not believed to influence the outcome of
the study.
Personnel Associates involved in this study were appropriately
qualified and trained.
Selection of animals Only healthy, previously unused animals were selected
for this study.

TEST METHOD

Preparation of the test item

The test item was mixed in distilled water to achieve the desired concentrations.
Doses were prepared and administered immediately. The details of the doses and the
concentration of the dosing solutions are given below:

Volume of Concentration of
Dose Concentration Test item
Date vehicle made up dosing solution
(mg/Kg b.w.)a (mg)
to (mL) (mg/mL)b
18 August 2015 300 301.2 10.00 30.12
20 August 2015 300 301.2 10.00 30.12
03 September 2015 2000 2002.1 10.00 200.21
07 September 2015 2000 2001.4 10.00 200.14
b.w., body weight
a
All treatment doses expressed as nominal dose administered
b
All dosing preparations expressed as nominal concentrations

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Test Procedure
A stepwise procedure using 3 female animals per step was used in this study to assess
the acute toxicity of the test item as described in the OECD guidelines for testing of
chemicals, number 423.

In the lack of information, a starting dose of 300 mg/Kg b.w. was selected for Step 1.
The test procedure followed the attached scheme described in Appendix 1.

All the animals received a single dose of the test item by oral route of administration,
after being fasted for approximately 15 to 16 hours, but with free access to water.
Food was supplied approximately 3 h to 4 h after test item administration.

A dose volume of 10 mL/Kg was used. Individual dose volumes are given in
Table 3. Based on the outcomes of the previous step, further steps were carried out.

OBSERVATIONS

Mortality & Morbidity

All the animals were observed for mortality and morbidity for a period of fourteen
days following the test item administration. If no mortality or morbidity is observed at
a particular experimental step at the end of Day 1, the next steps were started based on
the scientific judgement of the Study Director.

Body Weight Recording

Body weights of each animal were recorded prior to the test item administration
(Day 0) and on Days 7 and 14 of the particular experimental step.

Clinical Observation
Clinical observations were performed to look for signs of ill health or overt toxicity
during the first 30 minutes and at approximately 1, 2, 3 and 4 h after dose
administration on Day 0 and daily during days 1-14. Any abnormalities of appearance
or behaviour or other signs of reaction to treatment or ill health were recorded and a
detailed individual record was maintained of the clinical condition of each animal.

Gross Pathology Examination

All the survival animals were necropsied at the end of 14-day observation period and
subjected to gross pathology.

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DATA EVALUATION

Based on the observations the test item were categorised as per the Globally
Harmonized Classification System and appropriate cut off LD50 range determined.

RESULTS

Mortality & Morbidity

The results of mortality and morbidity; and various experimental steps used in this
study are given in Table 1.

Body Weight Recording

Body weights of each animal recorded prior to the test item administration (Day 0)
and on Day 7 and 14 of the experiment are presented in Table 2.

Clinical Observation
Clinical signs were observed in all the steps and the signs are presented in Table 1.

Gross Pathology Examination

Gross pathology observations of the animals at various steps are presented in Table 1.

CONCLUSION

Based on the results obtained, it is concluded that, Nualgi Nano Nutrients, falls under
„Category 5 or unclassified‟ according to the Globally Harmonized System (GHS) for
the classification of chemicals. The cut off LD50 value is greater than 5000 mg/Kg.

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REFERENCES

1. Organization for Economic Co-operation and Development (OECD) Guidelines

for Testing of Chemicals (Sec. 4, No.423, Acute Toxic Class Method, Adopted
17th December, 2001).

2. OECD Principles of Good laboratory Practice. OECD Environmental Health and

Safety Publications, Series on Principles of Good Laboratory Practice and
Compliance Monitoring No. 1. ENV/MC/CHEM (98)17.

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Table 1: Results of Mortality & Morbidity; Clinical signs and Gross pathology

Dose Animal
Steps Date Clinical Signs Mortality & Morbidity Day of Necropsy Gross Pathology
concentration No.

1 18 August 2015
1 NAD
All animals were in somnolence condition with Nil
300 mg/Kg 2 decreased motor activity following test item 01 September 2015 NAD
administration on day 0.
3 NAD

2 20 August 2015
4 NAD
All animals were in somnolence condition with
300 mg/Kg 5 decreased motor activity following test item Nil 03 September 2015 NAD
administration on day 0.
6 NAD

03 September 2015
3 2000 mg/Kg
7 NAD
All animals were in somnolence condition with
8 decreased motor activity following test item Nil 17 September 2015 NAD
administration up to day 2.
9 NAD

10 NAD
All animals were in somnolence condition with
4 07 September 2015 2000 mg/Kg 11 decreased motor activity following test item Nil 21 September 2015 NAD
administration up to day 2.
12 NAD

NAD - No Abnormality Detected;

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Table 2: Individual body weights (g)

Increase in body weight
Step
Animal No. Day 0 Day 7 Day 14 at the end of the
(mg/Kg b.w.)
experiment
1 160.0 168.4 175.8 15.8
1
2 167.5 174.0 180.2 12.7
(300)
3 164.2 171.1 178.4 14.2
4 170.3 176.9 183.9 13.6
2
5 164.8 171.0 177.0 12.2
(300)
6 163.6 170.8 178.6 15.0
7 161.1 168.3 175.4 14.3
3
8 165.7 173.4 180.1 14.4
(2000)
9 170.4 176.9 183.7 13.3
10 173.8 180.2 186.9 13.1
4
11 170.2 177.8 183.4 13.2
(2000)
12 168.9 175.2 182.9 14.0

Table 3: Dosing time and volume administered

Total Dose Volume
Step Date Animal No. Dosing Time
(mL)
1 1.6
1 18 August 2015 2 11.00 am 1.7
3 1.6
4 1.7
2 20 August 2015 5 10.30 am 1.6
6 1.6
7 1.6
3 03 September 2015 8 11.00 am 1.7
9 1.7
10 1.7
4 07 September 2015 11 11.00 am 1.7
12 1.7

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RESPONSIBLE PERSONNEL

Mr. S. Haribabu, B Tech (Biotech), MSc Study Director

Dr. R. M. Balaje, MVSc Animal House In-charge
Mr. M. Vasanthan, M Tech (Biotech) Study Scientist

STUDY PLAN AMENDMENT

One amendment was made to modify the test item name from “Nualgi Foliar Nano
Nutrients” to “Nualgi Nano Nutrients”.

STUDY PLAN DEVIATION

No deviations from the study plan were found during the conduct of the study.

ARCHIVE STATEMENT

All primary data, or authenticated copies thereof, slides (if applicable), tissue
specimens (if applicable), a sample test item and the final report will be retained, for a
period of 9 years, in the GLR Laboratories Private Limited archives after issue of the
final report. At the end of the specified archive period the Sponsor will be contacted
to determine whether the data should be returned, retained or destroyed on their
behalf. Sponsors will be notified of the financial implications of each of these options
at that time.

DISTRIBUTION OF REPORTS

Two originals of the study report are prepared and distributed as mentioned below:
a. One Copy - Sponsor.
b. One Copy - Archive.

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APPENDIX 1

Taken from OECD Guidelines for Testing of Chemicals, 423 (adopted on 17th
December, 2001)

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