Study Title Test Item Study Director Sponsor Study Monitor Test Facility Acute oral toxicity study in Wistar rats Nualgi Nano Nutrients Mr. S. Haribabu, B Tech (Biotech), MSc Nualgi Nano Biotech Co 651, 11 th Main Road V Block, Jayanagar Bengaluru 560041 Mr. Sunil Nanda 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 17 th December, 2001). Report Issued 01 October 2015 Total Number of Pages 18...committed to scientific and service excellence Page 1
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 - Page 2 of 18 -
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 Director GLR Laboratories Pvt Ltd Study completion date * With the exception of the identity and composition of the test item, which were the responsibilities of the sponsor. - Page 3 of 18 -
QUALITY ASSURANCE STATEMENT Final Report 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: S. No Type of Inspection Date of Inspection Phase(s) of Study Inspected Date of Reporting to Management, Study Director (Inspection Report No.) 1 Study Based Inspection 06 August 2015 Draft Study Plan 06 August 2015 (SBI/161/001/001) 2 Study Based Inspection 07 August 2015 Definitive Study Plan 07 August 2015 (SBI/161/001/002) 3 Study Based Inspection 18 August 2015 Test Item Administration 18 August 2015 (SBI/161/001/003) 4 Study Based Inspection 15 September 2015 Definitive Study Plan Amendment 15 September 2015 (SBI/161/001/004) 5 Study Based Inspection 30 September 2015 Draft Report 30 September 2015 (SBI/161/001/005) 6 Study Based Inspection 01 October 2015 Final Report 01 October 2015 (SBI/161/001/006) Dr. G. Velmani, M Pharm, PhD Executive-Quality Assurance GLR Laboratories Pvt Ltd Date - Page 4 of 18 -
TEST FACILITY MANAGEMENT STATEMENT Final Report 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 Test Facility Management GLR Laboratories Pvt Ltd Date - Page 5 of 18 -
PEER REVIEW STATEMENT Final Report 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 American Board Certified and UK Registered Toxicologist GLR Laboratories Pvt Ltd Date - Page 6 of 18 -
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: Step Date Dose (mg/kg) Number of animals Number of moribund or dead animals Subsequent action 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 LD 50 value is greater than 5000 mg/kg. - Page 7 of 18 -
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 17 th December, 2001). - Page 8 of 18 -
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 Appearance Colour Nualgi Nano Nutrients Liquid Greenish Batch No. 60453 Formulation/Composition Nano nutrients mixture of primary, Secondary, Micro CO, Si Manufacture Date 03 July 2015 Expiry Date Chemical Name CAS No Molecular Formula Molecular Weight Solubility ph 8.01 Photo-Stability 3 years from manufacturing Nano nutrients Not Applicable Not Applicable Not Applicable Soluble in water Yes - Page 9 of 18 -
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 Strain Age Sex Source Rattus norvegicus (rat) Wistar 8-12 weeks Female 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 of experiment (g) Number of animals used Step 1 160.0-167.5 Step 2 163.6-170.3 Step 3 161.1-170.4 Step 4 168.9-173.8 Twelve (Three animals/step) Acclimatization period Step 1 6 days Justification for animal use Step 2 Step 3 Step 4 8 days 8 days 12 days 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). - Page 10 of 18 -
ANIMAL HUSBANDRY Final Report Location Test room 2 Test room temperature 19.1-22.7 C Relative humidity 35-68% Housing Method of identification Diet Water Bedding material Photo period Contaminants Personnel Selection of animals Animals were housed in groups of three in standard polypropylene cages with stainless steel top grill. 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. Rat pellet feed (Amrut) Purified drinking water was provided ad libitum Sterilized paddy husk 12 h light and 12 h dark cycle Contaminants reasonably expected in feed and water supplied was not believed to influence the outcome of the study. Associates involved in this study were appropriately qualified and trained. 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: Date Dose Concentration (mg/kg b.w.) a Test item (mg) Volume of vehicle made up to (ml) Concentration of dosing solution (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 - Page 11 of 18 -
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. - Page 12 of 18 -
DATA EVALUATION Based on the observations the test item were categorised as per the Globally Harmonized Classification System and appropriate cut off LD 50 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 LD 50 value is greater than 5000 mg/kg. - Page 13 of 18 -
REFERENCES 1. Organization for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals (Sec. 4, No.423, Acute Toxic Class Method, Adopted 17 th 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. - Page 14 of 18 -
Table 1: Results of Mortality & Morbidity; Clinical signs and Gross pathology Steps Date Dose concentration Animal No. Clinical Signs Mortality & Morbidity Day of Necropsy Gross Pathology 1 18 August 2015 300 mg/kg 2 20 August 2015 300 mg/kg 1 NAD All animals were in somnolence condition with Nil 2 decreased motor activity following test item 01 September 2015 NAD administration on day 0. 3 NAD 4 NAD All animals were in somnolence condition with 5 decreased motor activity following test item Nil 03 September 2015 NAD administration on day 0. 6 NAD 3 03 September 2015 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 4 07 September 2015 2000 mg/kg 11 All animals were in somnolence condition with decreased motor activity following test item Nil 21 September 2015 NAD administration up to day 2. 12 NAD NAD - No Abnormality Detected; - Page 15 of 18 -
Table 2: Individual body weights (g) Step (mg/kg b.w.) 1 (300) 2 (300) 3 (2000) 4 (2000) Animal No. Day 0 Day 7 Day 14 Increase in body weight at the end of the experiment 1 160.0 168.4 175.8 15.8 2 167.5 174.0 180.2 12.7 3 164.2 171.1 178.4 14.2 4 170.3 176.9 183.9 13.6 5 164.8 171.0 177.0 12.2 6 163.6 170.8 178.6 15.0 7 161.1 168.3 175.4 14.3 8 165.7 173.4 180.1 14.4 9 170.4 176.9 183.7 13.3 10 173.8 180.2 186.9 13.1 11 170.2 177.8 183.4 13.2 12 168.9 175.2 182.9 14.0 Table 3: Dosing time and volume administered Step Date Animal No. Dosing Time Total Dose Volume (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 - Page 16 of 18 -
RESPONSIBLE PERSONNEL Mr. S. Haribabu, B Tech (Biotech), MSc Dr. R. M. Balaje, MVSc Mr. M. Vasanthan, M Tech (Biotech) Study Director Animal House In-charge 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 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. - Page 17 of 18 -
APPENDIX 1 Taken from OECD Guidelines for Testing of Chemicals, 423 (adopted on 17 th December, 2001) - Page 18 of 18 -