Sri VENTKATESWARA VETERINARY UNIVERSITY College of Veterinary Science

1. Determination of Fipronil Residues in Bovine Milk from Cows Following Topical Administration of Fipronil Pour-On (1 mg/Kg BW) Sri VENTKATESWARA VETERINARY UNIVERSITY College of Veterinary Science Department of Livestock Production Tirupathi AND Veterinary Medicine Research & DevelopmentPfizer IncKalamazoo, Michigan 49001-0199United States Pfizer Animal Health

2. STUDY LOCATION In-Life Animal Phase The Livestock Production & Management Dairy Farm College of Veterinary Science, Tirupati 517502 Analytical Phase ADVENIUS LABORATORY Bangalore, India

3. SPONSOR Pfizer Pharmaceutical India Pvt. Ltd. Plot No.16, TTC, MIDC Indl. Area Thane Belapur Road, K.U. Bazar Post Turbhe, Navi Mumbai 400705, India

4. Structure Formula, weight: C12H4Cl2F6N4OS; 437.15 g/ml Source OF DRUG Disinfecto Chemical Industries Pvt. Ltd. 569/135 Ka, Bargwa Barabirwa, Kanpur Road, Lucknow-226012, India Telephone: +91522 3297569

5. OBJECTIVE The objective of this study is to measure the Fipronil residues in bovine milk from lactating dairy cows at various withdrawal times following topical administration of Fipronil @1.0 mg/Kg in order to define the residue depletion profile

6. Investigators Team Mr. Girish Kapur, MS Principal Investigator, Analytical Phase VMRD, Pharmaceutical Sciences, Thane, India Dr. Nandakumar D Reddy, B.V.Sc, MVSc, MS, PhD Study Director/Monitor Pfizer Pharmaceuticals India Private Limited, Thane, India Dr. Christophe Derozier, DVM, MBASr. Director, Sponsor / Management Representative ANZ Operations, VMRD, Pfizer Animal Health, Parkville, Australia Dr. Y.Ravindra Reddy, BVSc, PhD Principal Investigator, In-Life Phase Livestock Production & Management Dairy Farm College of Veterinary Science, Tirupati, India Mr. Andrew Hodge, BA, MSc, AStat Biometrics RepresentativeManager, Biometrics, Pfizer VMRD, Australia Dr. Scott A. Brown, DVM., PhDSenior Director, VMRD metabolism and SafetyPfizer Animal Health, Kalamazoo, MI, USA

7. Investigators PrincipalInvestigator: Dr. Dr. Y. Ravindra Reddy Associate Professor Dept. of LPM, C.V.Sc., TPT Co-Investigator : Dr. P.R.S.Moorthy Professor & Head Dept. of LPT C.V.Sc., TPT Co-Investigator : Dr. D. Suresh Babu Assistant Professor Dept. of LPM, C.V.Sc., TPT

8. STANDARD OPERATING PROCEDURE

9. ANIMALS Description Species/Breed/Strain: Dairy Cow- Cross Bred Animals (Sahiwal, Holstein or Jersey) Sex: Females-Lactating Number of Animals: 15 + 5 spares. Animal details will be documented (Form 1). Initial Age: Age is not a study-critical factor. However, the animals should be in the middle part of the lactation cycle and be multiparous with 1, 2, 3 or 4 calvings (parity). Initial Body Weight: Body weight will be documented on day-1 of the study.

14. Video of drug administration

15. The primary enrollment criteria include the following: 1. No signs of illness or infection especially mastitis as determined by veterinarian and milk sample from the animals will be checked for SCC (<400,000 in the composite 4-quarter milk sample, prior to start of the study. 2. Four functioning milking quarters 3. No antibiotics will be administered within 14 days of dose administration. Each cow must not have antibiotics in their milk. 4. Cows should be in mid-lactation (60-250 days in milk) and should be in parity 1-4 (documented for each cow and included in the study report). 5. Each cow must have a milk production of at least 7 kg per day.

16. Pre-study treatment The selected animals will be moved to a location 7 days prior to dose application. Health status of the animals will be checked by a clinical veterinarian and a milk sample from the animals will be checked for somatic cell count (SCC), prior to start of the study

17. Treatment Administration The test formulation will be administered topically in a single dose at a dose level of 1.0 mg of Fipronil/kg body weight as determined in the pre-treatment weighing The correct volume plus 5% to ensure the minimum target dose based on the animals weight will be pulled into a measuring cup that can hold at least >40 mL. The weight of the measuring cup will be recorded before the sample is administered and then again after topical administration to accurately determine the exact dose administered to each cow. Volume administered will be determined by dividing the weight of the dose administered by the specific activity of the formulation. The test formulation will be administered on the back of the animal, from withers to the tail-head along the spine.

18. Milk Sample Collection Sufficient pre-dose milk samples will be collected from each cow for use as control matrix. Following dosing, milk will be collected in the AM and PM at 10-14 hour intervals. Milk will be collected for 30 days. The date and time of collection and the weight of milk collected will be recorded, then sub sampled for counting and assay. A 10 mL sub-sample of milk from each milking will be stored at <-10 C for the assay. In addition, two 100 mL samples of milk from each milking from each cow will be stored at < -10 C as reserve, until end of analysis (Form 6).

19. Storage, Transfer, and Disposition of Milk Samples All the large volume samples (100 mL) and sub-samples (10 mL) will be placed on dry ice and transported to Pfizer Pharmaceutical Sciences Analytical Laboratory until analysis. The remaining bulk milk will be discarded. A complete chain of custody will be maintained for all milk samples. Storage locations, storage conditions (acceptable temperature ranges), and freezer ID numbers will be recorded in the raw data (Form 11). All the samples collected will be transported to Pfizer Pharmaceutical Sciences Analytical Laboratory with in 72-hours of milk collection.

20. Discarding of milk after sample collection