Study Title Test Item Study Director Sponsor Study Monitor Test Facility Acute dermal toxicity study in Wistar rats Nualgi Nano Nutrients Mr. M. Vasanthan, M Tech (Biotech) 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 161/003 Regulatory Guideline OECD Guidelines for Testing of Chemicals,402 (adopted on 24 Feb 1987) Report Issued 01 October 2015 Total Number of Pages 15...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... 12 Results... 12 Conclusion... 13 References... 13 Responsible Personnel... 14 Study Plan Amendment... 15 Study Plan Deviation... 15 Archive Statement... 15 Distribution of Reports... 15 - Page 2 of 15 -
STUDY DIRECTOR AUTHENTICATION STATEMENT Nualgi Nano Nutrients: Acute Dermal Toxicity study in Wistar rats This study was performed in accordance to the agreed Study Plan, an 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. M. Vasanthan, M Tech (Biotech) 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 15 -
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, Raw Data, Amendment 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 24 August 2015 Draft Study Plan 24 August 2015 (SBI/161/003/001) 2 Study Based Inspection 24 August 2015 Definitive Study Plan 24 August 2015 (SBI/161/003/002) 3 Study Based Inspection 28 August 2015 Application of Test Item 28 August 2015 (SBI/161/003/003) 4 Study Based Inspection 15 September 2015 Definitive Study Plan Amendment 15 September 2015 (SBI/161/003/004) 5 Study Based Inspection 01 October 2015 Draft Report 01 October 2015 (SBI/161/003/005) 6 Study Based Inspection 01 October 2015 Final Report 01 October 2015 (SBI/161/003/006) Dr. G. Velmani, M Pharm, PhD Executive-Quality Assurance GLR Laboratories Pvt Ltd Date - Page 4 of 15 -
TEST FACILITY MANAGEMENT STATEMENT Nualgi Nano Nutrients: Acute Dermal 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, ERT Test Facility Management GLR Laboratories Pvt Ltd Date - Page 5 of 15 -
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 15 -
SUMMARY This study was performed to determine the acute dermal toxicity potential of Nualgi Nano Nutrients, when administered by a single dermal application to rats, followed by an observation period of 14 days. A range finding study using 1 male and 1 female rat at the dose of 2000 mg/kg b.w. was carried out in order to establish the dose levels for the main study. There was no mortality in the range finding study and therefore, a limit test (2000 mg/kg b.w.) was conducted. The administration of the test item by dermal application at a dose of 2000 mg/kg b.w. to 5 male and 5 female rats produced no mortality in any of the 10 test animals. No signs of ill health or overt toxicity were observed. Furthermore, no gross lesions related to test item application was observed. Based on the results obtained, the acute dermal LD 50 of Nualgi Nano Nutrients is greater than 2000 mg/kg b.w. - Page 7 of 15 -
INTRODUCTION In the assessment and evaluation of the toxic characteristics of a substance, determination of acute dermal toxicity is useful where exposure by the dermal route is likely. It provides information on health hazards likely to arise from a short-term exposure by the dermal route. Data from an acute dermal toxicity study may serve as a basis for classification and labelling. It is an initial step in establishing a dosage regimen in subchronic and other studies and may provide information on dermal absorption and the mode of toxic action of a substance by this 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.402, Adopted 24 Feb 1987). 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 8 of 15 -
OBJECTIVE To determine the acute dermal toxicity in Wistar rats following single dermal application. STUDY DATES Study Start Date 24 August 2015 Experiment Start Date 28 August 2015 (first dosing date) Experiment Completion Date 19 September 2015 The study completion date is the date the final report is signed by the Study Director. This study is performed in line with the agreed study plan and an amendment. TEST ITEM DETAILS The test item, Nualgi Nano Nutrients, 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 Manufacture Date 03 July 2015 Expiry Date Chemical Name CAS No Molecular Formula Molecular Weight Solubility ph 8.01 Photo-Stability Nano nutrients mixture of primary, Secondary, Micro CO, Si 3 years from manufacturing Nano nutrients Not Applicable Not Applicable Not Applicable Soluble in water Yes - Page 9 of 15 -
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 Sex Source Weight Range (at the start of experiment) Rattus norvegicus (rat) Wistar Male and Female NIN, 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 Range finding study Limit Test 210.4 & 240.3 g 207.6-259.1 g Number of animals used Range Finding Two (1 Male + 1 Female) Limit Test Acclimatization period Range Finding 5 Days Justification for animal use Limit Test Ten (5 Male + 5 Female) 5 Days Rats are selected because there is a large volume of background data on this species Specified in OECD Guidelines for Testing of Chemicals, 402 standards, as an appropriate species to evaluate the acute dermal toxicity of the 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). ANIMAL HUSBANDRY Location Test room 2 Test room temperature 19.1-22.7 C Relative humidity 37-63% Housing Animals were housed in groups of three in standard polypropylene cages with stainless steel top grill. - Page 10 of 15 -
Method of identification Diet Water Bedding material Photo period Contaminants Personnel Selection of animals Animals will be 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/or 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 applied as such onto the skin of rats. Test item was prepared under dark conditions. Test Procedure A range finding study using a male and a female rat at dose 2000 mg/kg b.w. was carried out in order to establish the dose levels for the main study. Approximately, 24 hours before the treatment, around 10% dorsal skin area of each rat was clipped free of hair, without any abrasion. The appropriate amount of the test item was applied uniformly over the clipped area of each rat. After the application, the test item was held in contact with the skin for a period of 24 hours, using a porous gauze dressing (Modern Health Care, B. No.: 141, Expiry: October 2017) and bandaged with non-irritating adhesive tape. After 24 hours, the residual test item was wiped gently from the skin using wet cotton, soaked in water. Neck collar was used to prevent the ingestion of the test item from the application site. No mortality was observed for 4 days in the range finding study at 2000 mg/kg b.w. Based on the results from the range finding experiment, limit test was chosen. In the limit test, 5 male and 5 female rats were exposed to 2000 mg/kg b.w. - Page 11 of 15 -
OBSERVATIONS Mortality & Morbidity All the animals were observed for mortality and morbidity for a period of 14 days following the test item application. Body Weight Recording Body weights of each animal were recorded prior to the test item application (Day 0) and on Day 7 and 14 of the experiment. Clinical Observation Clinical observations were performed to look for signs of ill health or overt toxicity after test item application on Day 0 and daily during Days 1-14. Any skin reactions such as erythema, edema and scaling of epidermis at the site of application were specifically looked for. 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 animals were necropsied at the end of 14-day observation period and gross pathology examinations were conducted. DATA EVALUATION The dermal LD 50 value should always be considered in conjunction with the observed toxic effects and the necropsy findings. The LD 50 value is a relatively coarse measurement, useful only as a reference value for classification and labelling purposes, and an expression of the lethal potential of the test substance following dermal exposure. RESULTS Mortality & Morbidity No mortality and morbidity were observed in any of the animals treated with the test item. Body Weight Recording Body weights of each animal recorded prior to the test item application (Day 0) and on Day 7 and 14 of the experiment are presented in Table 1. Clinical Observation No signs of ill health or overt toxicity were observed during the experiment period. - Page 12 of 15 -
Gross Pathology Examination No gross lesions related to test item application were observed and are presented in Table 3. CONCLUSION Based on the results obtained, the acute dermal LD 50 of Nualgi Nano Nutrients is greater than 2000 mg/kg b.w. REFERENCES 1. Organization for Economic Co-operation and Development Guidelines for Testing of Chemicals (Sec.4, No.402, Adopted 24 Feb 1987). 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 13 of 15 -
Table 1: Individual body weights in the limit test (g) Final Report Animal No. Sex Day 0 Day 7 Day 14 Increase in body weight at the end of the experiment (g) 3 M 239.8 242.0 249.4 9.6 4 M 228.9 231.2 238.9 10.0 5 M 259.1 260.7 269.5 10.4 6 M 222.3 224.8 231.6 9.3 7 M 207.6 209.4 217.5 9.9 Mean ± SD 231.5 ± 19.3 233.6 ± 19.2 241.4 ± 19.5 9.8 ± 0.4 8 F 234.7 236.1 244.9 10.2 9 F 217.0 219.9 227.0 10.0 10 F 223.2 225.3 233.7 10.5 11 F 247.1 249.6 256.2 9.1 12 F 230.9 233.0 240.5 9.6 Mean ± SD 230.6 ± 11.5 232.8 ± 11.4 240.5 ± 11.1 9.9 ± 0.5 SD- Standard deviation Table 2: Dosing Time & Volume of test item application Date Animal No. Sex Dosing Time Weight of test item applied (mg) 3 M 479.60 4 M 457.80 05 September 2015 5 M 11:45 am 518.20 6 M 444.60 7 M 415.20 8 F 469.40 9 F 434.00 05 September 2015 10 F 12:25 pm 446.40 11 F 494.20 12 F 461.80 Table 3: Results of Mortality & Morbidity, and Gross pathology Date Dose concentration Animal No. Mortality & Morbidity Gross Pathology 19 September 2015 2000 mg/kg 19 September 2015 2000 mg/kg NAD - No Abnormality Detected 3 NAD 4 NAD 5 Nil NAD 6 NAD 7 NAD 8 NAD 9 NAD 10 Nil NAD 11 NAD 12 NAD - Page 14 of 15 -
RESPONSIBLE PERSONNEL Mr. M. Vasanthan, M Tech (Biotech) Dr. R. M. Balaje, MVSc Mr. S. Haribabu, B Tech (Biotech), MSc Study Director Animal House In-Charge Study Scientist STUDY PLAN AMENDMENT As per the sponsor request, an amendment was raised to change 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 will be prepared and distributed as mentioned below: a. One Copy - Sponsor. b. One Copy - Archive. - Page 15 of 15 -