Project Number: 3060-32420-001-04-I
Project Type: Interagency Reimbursable Agreement
Start Date: Jun 1, 2014
End Date: Sep 30, 2016
Data under this agreement are needed to properly assess the impact of commercial dairy partitioning on veterinary drug residues in pasteurized milk. There are few available data on how drug residues bind to different proteins in milk and milk products. The concentration of potential veterinary drug residues in any finished dairy product depends on the composition of the dairy product, its processing history, and the properties of the veterinary drugs. Two major factors that affect final drug concentration in dairy products are the drug partitioning in the fat and aqueous phases and drug binding to milk proteins. Models of chemical partitioning typically include the octanol-water partitioning coefficient (log Kow). Data on milk products suggests that the log Kow for a drug alone may not provide a quantitative prediction for drug partitioning in these systems. Drug binding to proteins is often specific and complex; very few studies have investigated drug binding to milk proteins and these studies have been limited to only a few veterinary drugs. These proposed experiments will be the first to systematically and comprehensively study the partitioning and protein binding specific to milk and milk products for a wide range of veterinary drugs of interest. This Interagency agreement will experimentally generate data to support the evaluation of the potential impact of partitioning on drug residues, if present, in milk. The data will help evaluate the impact of selective binding of drug residues to the whey and casein fractions of milk, and hydrophobic-based binding to milk fat. Data on the drug residue-fat/protein-binding characteristics will be used to address the following two questions in a risk assessment under development within the FDA: I. If drug residues get into the bulk-tank milk, what is the fate of these residues during processing/manufacturing of various milk products (i.e., in what milk products would these drug residues be found)? II. If present in milk, and commercially processed milk products, which drug residues have the potential for concentration in dairy products? This Interagency agreement will allow the FDA and the USDA-ARS to work collaboratively to develop the experimental data necessary to evaluate these two questions.
Under an Interagency agreement from FDA-CFSAN, scientists at USDA-ARS-NPA will experimentally determine the drug residue partitioning characteristics of a list of pre-determined drugs added to pasteurized milk evaluated through pre-determined milk partitioning procedures. During the performance period of this Interagency agreement, USDA-ARS will: 1. In consultation with FDA, identify and obtain at least 8 veterinary drugs with radio-isotope-labeling for the partitioning study. Depending on the pilot study results, and, if time allows, additional drugs could be added to the list of potential drugs. The list of potential drugs has been provided to USDA-ARS in order of preference, although this prioritized list may change, based on new information obtained on available drugs, and based on the pilot test results. 2. The main focus of the study will be on the extent of binding of drug to fat and protein within dairy milk products. Approach will be to assay radioactivity in milk fractions after (a) skimming of cream, (b) curding, and (c) isolation of whey protein. Triplicates will be used, along with blanks and controls at three environmentally relevant drug concentrations. 3. The work will be divided into three experimental phases. a) Distribution of fortified, radiolabeled, target drugs into the aqueous (skim milk) and cream (fat) phases. This will be accomplished by centrifugation. Radiochemical will be quantitated in both compartments by liquid scintillation counting and combustion analysis, respectively. Aliquots of skim milk and cream extracts will by assessed qualitatively and quantitatively for metabolites/degradates by thin layer chromatography-radioactivity monitoring. b) Partitioning of fortified, radiolabeled, target drugs in skim milk into the curd (insoluble protein) fraction and the whey (water soluble protein) fraction during cheese making. Curding (protein hydrolysis) will be achieved with rennet incubation, followed by physical removal of the curd layer from the whey fraction. Aliquots of whey and curd extracts will by assessed qualitatively and quantitatively for metabolites/degradates by thin layer chromatography-radioactivity monitoring. c) Determine the extent of binding of fortified, radiolabeled, target drugs to whey fraction proteins. This will be accomplished by separation of the whey proteins(>10 kiloDaltons; retentate) from the small molecule/water-soluble fraction (permeate) via ultrafiltration. Aliquots of retentate and/or its extracts as well as permeate will by assessed qualitatively and quantitatively for metabolites/degradates by thin layer chromatography-radioactivity monitoring. 4. USDA-ARS will provide a detailed report of the results and methodology to FDA. 5. Provide the resulting data to FDA in a format mutually agreed upon.