Xenobiotics and Food-Producing Animals. Metabolism and Residues 9780841224728, 9780841213593, 0-8412-2472-2

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Xenobiotics and FoodProducing Animals

In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

ACS

SYMPOSIUM

SERIES

503

Xenobiotics and FoodProducing Animals Metabolism and Residues D. H. Hutson, EDITOR Shell Research Limited

D. R. Hawkins, EDITOR Huntingdon Research Centre

G. D. Paulson, EDITOR U.S. Department of Agriculture

C. B. Struble, EDITOR Hazleton Laboratories Developed from a symposium sponsored by the Division of Agrochemicals of the American Chemical Society and the International Society for the Study of Xenobiotics at the Fourth Chemical Congress of North America (202nd National Meeting of the American Chemical Society), New York, New York, August 25-30, 1991

American Chemical Society, Washington, DC 1992

Library of Congress Cataloging-in-Publication Data Xenobiotics and food-producing animals: metabolism and residues D. H . Hutson . . . [et al.], editor. p.

cm.—(ACS symposium series, ISSN 0097-6156; 503)

"Developed from a symposium sponsored by the Division of Agrochemicals of the American Chemical Society at the Fourth Chemical Congress of Noroth America (202nd National Meeting of the American Chemical Society), New York, New York, August 25-30, 1991." Includes bibliographical references and indexes. ISBN 0-8412-2472-2 1. Veterinary drugs—Congresses. 2. Xenobiotics—MetabolismCongresses. 3. Veterinary drugs—Metabolism—Congresses. 4. Veterinary physiology—Congresses. 5. Veterinary drug residuesCongresses. I. Hutson, D. H . (David Herd), 1935. II. American Chemical Society. Division of Agrochemicals. III. American Chemical Society. Meeting (202nd: 1991: New York, N.Y.) IV. Series. SF917.X46 1992 636.089'57—dc20

92-25718 CIP

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48-1984. Copyright © 1992 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprographic copies of the chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per-copy fee through the Copyright Clearance Center, Inc., 27 Congress Street, Salem, MA 01970, for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval byACSof the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

American Chemical Society Library 1155 16th St., N.W. Washington, D.C. 20036 In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

1992 Advisory Board ACS Symposium Series M . Joan Comstock, Series Editor V . Dean Adams

Bonnie Lawlor

Tennessee Technological University

Institute for Scientific Information

John L. Massingill Mark Arnold

Dow Chemical Company

University of Iowa

Robert McGorrin David Baker

Kraft General Foods

University of Tennessee

Julius J. Menn Alexis T. Bell University of California—Berkeley

Plant Sciences Institute, U.S. Department of Agriculture

Arindam Bose

Vincent Pecoraro

Pfizer Central Research

University of Michigan

Robert F. Brady, Jr.

Marshall Phillips

Naval Research Laboratory

Delmont Laboratories

Margaret A . Cavanaugh

A . Truman Schwartz

National Science Foundation

Macalaster College

Dennis W. Hess

John R. Shapley

Lehigh University

University of Illinois at Urbana-Champaign

Hiroshi Ito IBM Almaden Research Center

Stephen A . Szabo Conoco Inc.

Madeleine M . Joullie University of Pennsylvania

Robert A . Weiss University of Connecticut

Mary A . Kaiser Ε. I. du Pont de Nemours and Company

Peter Willett University of Sheffield (England)

Gretchen S. Kohl Dow-Corning Corporation

In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Foreword IHE A C S SYMPOSIUM SERIES was first published in 1974 to provide a mechanism for publishing symposia quickly in book form. The purpose of this series is to publish comprehensive books developed from symposia, which are usually "snapshots in time" of the current research being done on a topic, plus some review material on the topic. For this reason, it is neces­ sary that the papers be published as quickly as possible. Before a symposium-based book is put under contract, the proposed table of contents is reviewed for appropriateness to the topic and for comprehensiveness of the collection. Some papers are excluded at this point, and others are added to round out the scope of the volume. In addition, a draft of each paper is peer-reviewed prior to final acceptance or rejection. This anonymous review process is supervised by the organizer(s) of the symposium, who become the editor(s) of the book. The authors then revise their papers according the the recommendations of both the reviewers and the editors, prepare camera-ready copy, and submit the final papers to the editors, who check that all necessary revisions have been made. As a rule, only original research papers and original review papers are included in the volumes. Verbatim reproduc­ tions of previously published papers are not accepted. M. Joan Comstock Series Editor

In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Preface GOOD ANIMAL HUSBANDRY IS CRITICAL in the conversion of plantderived feedstuff into animal protein for human consumption. Bioactive chemicals play an important role in maintaining health, rapid growth rate, and efficient feed conversion in animals used for food production. Con­ trol of disease by therapeutic and prophylactic use of veterinary pharma­ ceutical products has gained in importance as the numbers of animals held in close proximity for confined rearing have steadily increased, thus magnifying risk of animal-to-animal disease transmission. However, improvements in the use of pharmaceutical products to control pests in low-intensity animal production are also important. For example, myasis-producing parasitic arthropods, such as the sheep blow fly (Lucila cuprina) and disease-spreading blood-sucking flies, when uncontrolled have devastating effects on the well-being of animals and on the econom­ ics of food production. Thus, an array of bioactive compounds, often derived directly from the pharmaceutical and crop protection industries, are used: antibacterials, anticoccidials, miticides, nematicides, parasiticides (for control of both internal and external parasites), and insecticides. In addition, other speci­ alty chemicals, such as growth-regulating, estrous-synchronizing, and nutrient-repartitioning agents, are in use and are being considered for use in animal production. In the United States, crop protection agents used in food production are regulated by the Environmental Protection Agency, and the use of veterinary products is closely regulated by the Food and Drug Administra­ tion. Clear differences exist between the problems associated with the administration of veterinary products and the exposure of animals to crop protection agents. The use of veterinary products involves their deliber­ ate and controlled application for benefit. Studies conducted in the development phase of a veterinary product address, in addition to the tox­ icology, the fate of the chemical in the test species. Withdrawal periods are set so that residues in edible tissues are at or below an acceptable concentration. In contrast, the exposure of animals to crop protection agents is adventitious and usually via feedstuff. Animal studies on crop protection agents are designed to determine if residue transfer (from feed to animal product) occurs at an acceptably low level. However, there is much in common between studies on veterinary products and crop protection xi In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

agents. Some of the agents (e.g., insecticides) are common. The species exposed to these agents are also common (ruminants, swine, poultry, and fish). Of paramount importance, the terminal residues to which humans are exposed via food consumption present similar toxicological issues. Most of this book concerns the metabolism of veterinary products, as opposed to crop protection agents, because the former grouping covers a wider range of bioactivities. The content will, however, be of interest to those working with both types of agents. Specifically, a substantial amount of information about general methods for studying the fate of xenobiotics in species grown for human consumption is available. Increased attention is being paid to the fate of veterinary drugs in the environment, a major concern for crop protection agents for 30 years. Turning this situation around, environmental scientists concerned with the fate of other (e.g., industrial) chemicals should gain understanding of the capacity of food-producing animals to metabolize and eliminate chem­ icals. This knowledge is important as a basis for judgments about poten­ tial risk in the event of a major chemical contamination. We acknowledge the support of the International Society for the Study of Xenobiotics and the American Chemical Society's Division of Agrochemicals. We thank the authors for their efforts and we are grate­ ful for the help provided by the A C S Books Department. D. H. H U T S O N

Shell Research Limited Kent, England G. D. P A U L S O N

U.S. Department of Agriculture Fargo, ND 58105-5674 D. R. HAWKINS

Huntingdon Research Centre Cambridgeshire, England C. B. S T R U B L E

Hazleton Laboratories Madison, WI 53707 April 6, 1992

xii In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Chapter 1 Uses and Regulation of Veterinary Drugs Introduction Κ.N.Woodward Veterinary Medicines Directorate, Woodham Lane, New Haw, Addlestone, Surrey KT153NB,United Kingdom

There are a variety of veterinary drugs a v a i l a b l e and at the disposal of the veterinarian and the safety of these to the consumer must be assured i n most countries of the world before marketing authorizations can be granted. A large body of pharmacological, toxicological and residues data i s generated and assessed so that the t o x i c o l o g i c a l p r o f i l e of the drug can be established and a maximum residue limit elaborated. This, along with the residues depletion p r o f i l e , allows a withdrawal period to be defined so that the consumer is protected from exceeding the acceptable daily intake for the drug in question. Worker safety is also of paramount formulations importance when assessing a drug and its p r i o r to marketing authorisation. Veterinary medicines take many forms and a few of the more important groups are described in this article. The range of veterinary medicines now available r e f l e c t s both the diseases they are intended to combat and the range of species they are intended to treat. In addition, there are a number of drugs a v a i l a b l e for so-called zootechnical treatment (eg the use of s t e r o i d hormones i n synchronization of oestrus) as opposed to disease treatment or prophylaxis. These aspects w i l l be b r i e f l y discussed i n t h i s a r t i c l e , but a c e r t a i n amount of l i b e r t y has been taken with the t i t l e so that the word "use" i s interpreted i n i t s widest sense to include a description of what occurs before "use" i s allowed! Consequently, a large part of t h i s work w i l l deal with the general requirements of marketing authorization; the regulatory requirements that are applied before a veterinary medicine may be marketed. UB&U

o fVeterinary

Medicines

Veterinary medicines, l i k e t h e i r human counterparts come i n many forms (1). Some would be i n s t a n t l y recognizable as tablets, pills

0097-6156/92/0503-00002$06.00/0 © 1992 American Chemical Society In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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WOODWARD

Uses and Regulation of Veterinary Drugs

3

and i n j e c t a b l e s b u t o t h e r s , because o f c l i n i c a l need, t h e s p e c i e s i n v o l v e d o r t h e needs o f h e r d o r f l o c k s c a l e treatment, differ m a r k e d l y from anything g i v e n t o humans. The major groups o f m e d i c i n e s however a r e r o u g h l y t h e same and t h e s e w i l l be briefly d e s c r i b e d below, w i t h t h e emphasis b e i n g on t h o s e used i n food producing animals. A n t i m i c r o b i a l and a n t i b i o t i c a g e n t s . A l a r g e range of s y n t h e t i c , a r e used f o r the treatment s e m i s y n t h e t i c and n a t u r a l a n t i b i o t i c s of infectious diseases i n food producing animals. In the mid-1950s p e n i c i l l i n was seen as a g e n e r a l tool of s a l v a t i o n i n b o t h v e t e r i n a r y and human m e d i c i n e , b u t now an a r r a y o f B - l a c t a m a n t i b i o t i c s i s a v a i l a b l e f o r a l a r g e range o f i n d i c a t i o n s . The o t h e r major c a t e g o r i e s a r e t h e t e t r a c y c l i n e s , t h e a m i n o g l y c o s i d e s , t h e m a c r o l i d e s and p o l y m i x i n s ( 2 - 5 ) . S u l f o n a m i d e s a r e t h e major c l a s s o f a n t i m i c r o b i a l a g e n t (as opposed t o a n t i b i o t i c s ) and t h e s e c a n be viewed a s d e r i v a t i v e s o f s u l f a n i l a m i d e , the archetypal sulfonamide. A r a n g e o f compounds has been s y n t h e s i s e d for fast, medium o r l o n g a c t i n g abilities (6-7). They a r e o f t e n f o r m u l a t e d w i t h f o l a t e a n t a g o n i s t s s u c h as t r i m e t h o p r i m t o o b t a i n what i s c l a i m e d t o be a s y n e r g i s t i c a c t i o n . used S u l f a m e t h a z i n e ( s u l p h a d i m i d i n e ) i s p e r h a p s t h e most w i d e l y sulfonamide i n v e t e r i n a r y medicine, especially i n p i g production where i t i s u s e d to prevent r e p i r a t o r y disease ( 6 ) . The u s e o f t h i s drug i n p i g production has g i v e n rise t o problems o f r e s i d u e s , p a r t i c u l a r l y i n t h e k i d n e y , i n b o t h Europe and t h e USA. The r e a s o n s for this remain u n c l e a r f o r although failure to observe withdrawal periods plays a part, other c o n t r i b u t i n g factors include i n g e s t i o n o f f a e c e s from t r e a t e d animals and contamination of untreated feed with medicated feed ( 8 ) . Ectoparasiticides. C a t t l e and sheep a r e p a r t i c u l a r l y vulnerable to ectoparasites. I n C e n t r a l and South A m e r i c a , t h e USA and Europe, c a t t l e are attacked by c a t t l e grubs. In the Northern Hemisphere t h e s e "grubs" a r e t h e l a r v a l s t a g e s o f t h e w a r b l e f l y (Hypoderma spp) w h i l e i n South America, l a r v a e o f t r o p i c a l w a r b l e s (Dermatobia h o m i n i s ) a r e t h e c u l p r i t s ( 6 ) . They a r e g e n e r a l l y treated with pour-on formulations which tend t o be viscous p r e p a r a t i o n s o f t e n c o n t a i n i n g organophosphorus compounds. Sheep scab i s a n o t i f i a b l e disease i n both t h e USA and t h e U n i t e d Kingdom, which r e s u l t s i n t h e l o s s o f t h e f l e e c e . Although i t i s n o t g e n e r a l l y r e g a r d e d as a l e t h a l d i s e a s e , i t p o s e s s e v e r e a n i m a l w e l f a r e and economic p r o b l e m s . I t i s almost u n i v e r s a l l y treated by dipping i n aqueous solutions containing organophosphorus insecticides or synthetic pyrethroids (5). Anthelmintics. T h e r e i s i n s u f f i c i e n t scope i n a p a p e r o f this kind to discuss t h e more interesting points of veterinary parasitology. S u f f i c e i t t o say t h a t food producing animals of a l l s p e c i e s a r e l i t e r a l l y plagued by a l a r g e r a n g e o f internal parasites resulting i n distressing diseases and substantial economic l o s s e s ( 6 , 7 ) . The i n t e r e s t e d r e a d e r i s r e f e r r e d t o t h e c h a p t e r s by Roberson i n t h e e x c e l l e n t work e d i t e d by Booth and

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McDonald (31) which w i l l serve as useful introductions. A v a r i e t y of a n t i - p a r a s i t i c drugs have been developed and three examples, the benzimidazoles, levamisole and ivermectin w i l l be mentioned b r i e f l y here. Thiabendazole was the f i r s t benzimidazole to achieve wide use. It i s indicated f o r a range of infestations including those caused by Haemonchus, Trichostrongylus and Strongyloides species i n c a t t l e and sheep. Newer compounds include albendazole, oxfendazole, fenbendazole and mebendazole ( 9 , 1 0 ) . In addition pro-drugs, exemplified by febantel, which undergo c y c l i s a t i o n i n vivo to y i e l d benzimidazoles have now been developed. Most of these drugs have some degree of teratogenic p o t e n t i a l leading to concern about t h e i r residues, but a more p r a c t i c a l concern has arisen over e f f e c t s on the developing fetus i n treated animals. Albendazole, cambendazole and parbendazole are teratogenic i n sheep leading to s p e c i f i c contra-indications i n pregnant animals, while other benzimidazoles are inactive i n t h i s respect. Levamisole i s highly e f f e c t i v e against gastrointestinal nematodes and i s widely used i n c a t t l e , sheep and pigs i n addition to numerous other species. Levamisole i s the 1-isomer of dl-tetramisole. It appears to be the active isomer of the racemic mixture which i s i t s e l f marketed as an antinematodal agent. Concerns have been expressed by Joint FAO/WHO Expert Committee on Food Additives (JECFA) over i t s apparent a b i l i t y to induce agranulocytosis and neutropenia i n humans given the drug f o r therapeutic purposes. This concern led the Committee to set a temporary acceptable d a i l y intake (ADI) of 0 - 0 . 0 0 3 mg/kg body weight pending the r e s u l t s of further research on t h i s phenomenon and i t s relevance to the safety assessment of levamisole residues (11). Ivermectin i s a macrolide compound derived from abamectin, a metabolite produced by Streptomyces a v e r m i t i l i s . More p r e c i s e l y , i t i s a mixture of two compounds, 22,23-dihydroavermectin Β (80%) and 22,23-dihydroavermectin B (20%) (12). I t i s widely and successfully used f o r onchocerciasis treatment i n human medicine and has found widespread use as a nematocidal and cestodocdal agent i n veterinary medicine (12). Although i t i s claimed that i t i n h i b i t s m o t i l i t y of the parasite by acting on ^-aminobutyric acid (GABA) receptors and by blocking chloride ions, the f u l l mechanism i s as yet not f u l l y understood. 1 b

Antifungal agents. Several drugs are a v a i l a b l e as t o p i c a l antifungal agents including thiabendazole, ketoconazole and a number of a l i p h a t i c acids such as undecylenic acid (4,13). Perhaps the two best known systemic drugs are nystatin and g r i s e o f u l v i n . Nystatin i s remarkably low i n t o x i c i t y when given o r a l l y but i s much more toxic a f t e r parenteral administration. As i t i s poorly absorbed a f t e r o r a l administration i t i s useful f o r g a s t r o i n t e s t i n a l t r a c t i n f e c t i o n s . Griseofulvin, a f t e r absorption from the g a s t r o i n t e s t i n a l t r a c t i s deposited i n skin, h a i r and n a i l s and i s useful i n the treatment of dermatomycoses. I t i s , however, teratogenic at high doses, at least i n the cat, and i t s

In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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Uses and Regulation of Veterinary Drugs

use i n p r e g n a n c y i s t h e r e f o r e species.

c o n t r a - i n d i c a t e d i n t h i s and

other

S t e r o i d Hormones. Anabolic hormones l i k e t e s t o s t e r o n e and i t s s y n t h e t i c analogues such a s t r e n b o l o n e have been w i d e l y u s e d i n beef p r o d u c t i o n f o r several years. The n o n - s t e r o i d a l anabolic agent z e r a n o l has a l s o been w i d e l y u s e d f o r t h i s p u r p o s e ( 1 4 ) . Growth p r o m o t i n g u s e s o f a l l s t e r o i d hormones were r e c e n t l y banned by the European Community, but various zootechnical (eg synchronization of estrus) and t h e r a p e u t i c (eg p r e v e n t i o n o f a b o r t i o n ) uses o f t h e endogenous hormones and t h e i r synthetic e s t e r s a r e p e r m i t t e d (15,16). Somatotropins. Somatotropins a r e n a t u r a l l y o c c u r i n g polypeptides found i n a l l s p e c i e s although those intended f o r use i n food may be synthesized production, l a r g e l y to increase milk y i e l d s , recombinant technology (17-19). T h e r e h a s been much using controversy over the use of these materials ranging from t h e q u e s t i o n o f economic need t o f o o d s a f e t y c o n c e r n s (20-22). The European Community's Committee f o rVeterinary Medicinal Products a c o n s i d e r e d t h a t a t l e a s t one o f t h e s e p r o d u c t s d i d n o t p r e s e n t r i s k t o human h e a l t h , and gave a p o s i t i v e o p i n i o n on t h e a g e n t (23). I t i s i m p r a c t i c a l i n an a r t i c l e o f t h i s t y p e t o l i s t a l l t h e arguments f o r and a g a i n s t t h e s e drugs but the c o n t r o v e r s i e s i n v o l v e d seem s e t t o rumble on f o r some t i m e t o come! Currently, the somatotropins a r e not a u t h o r i s e d as milk y i e l d enhancers i n t h e USA o r i n t h e U n i t e d Kingdom. F i s h farming. Although not a " t h e r a p e u t i c use", f i s h farming or a q u a c u l t u r e as i t i s now o f t e n c a l l e d , i s worthy o f m e n t i o n a s i t r e p r e s e n t s a new a r e a o f a n i m a l p r o d u c t i o n and i t p o s e s i t s own problems. One o f t h e major growth a r e a s i n f i s h f a r m i n g i s salmon culture. This i s p a r t i c u l a r l y s u i t e d t o areas of the world where l a r g e i n l a n d expanses o f b o t h s a l t and f r e s h water a r e a v a i l a b l e , i n r e l a t i v e l y s h e l t e r e d environments. These c o n d i t i o n s a l l o w t h e salmon farmer t o grow t h e f i s h i n t h e i r n a t u r a l m a r i n e and f r e s h water e n v i r o n m e n t s thus r e f l e c t i n g t h e i r n a t u r a l h a b i t a t s . Salmon farming i s associated with two major diseases. F u r u n c u l o s i s , a b a c t e r i a l d i s e a s e c a u s e d by Aeromonas s a l m o n i c i d a , attacks and s e a - l i c e , an a r t h r o p o d and an e x t e r n a l p a r a s i t e w h i c h t h e s u r f a c e o f t h e f i s h (24,25). Both d i s e a s e s a r e r a p i d l y fatal and c a u s e s e r i o u s economic l o s s e s . I n t h e U n i t e d Kingdom, t h e o n l y l i c e n s e d medicine used t o c o n t r o l s e a - l i c e i s a formulation c o n t a i n i n g t h e organophosphorus compound d i c h l o r v o s . B e f o r e this c o u l d be a u t h o r i s e d , an enormous amount o f e c o t o x i c o l o g y d a t a had t o be r e v i e w e d a l l o w i n g f o r a r i s k assessment of dangers t o the immediate a q u a t i c environment, i n c l u d i n g the hazards t o other arthropods. Furunculosis i s prevented with the use of v a r i o u s a n t i b i o t i c s i n c l u d i n g a m o x y c i l l i n and o x o l i n i c a c i d ( 2 6 ) . Again, e n v i r o n m e n t a l a s s e s s m e n t s were n e c e s s a r y b e f o r e t h e s e d r u g s could be a u t h o r i z e d . Although i t i s not the t o p i c under d i s c u s s i o n i t i s worth n o t i n g that there i s also considerable here,

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XENOBIOTICS AND FOOD-PRODUCING ANIMALS environmental concern over the "natural" e f f l u e n t s from hatcheries (27). Marketing Authorization It would be impracticable to t r y to describe the various schemes established by regulatory agencies throughout the world to authorize users of veterinary medicines. Instead, the general requirements w i l l be discussed. Usually, three main areas, pharmaceutical quality, e f f i c a c y and drug safety are examined and these must be s a t i s f a c t o r y before marketing authorization i s granted. The corner-stone of these three areas i s product safety. A drug which i s not of the correct q u a l i t y because i t contains toxic contaminants or because i t i s not s t e r i l e i s not safe. S i m i l a r l y , a drug which does not perform i n the way described i n the product l i t e r a t u r e i s also not safe. The term "safety" speaks for i t s e l f : the product i t s e l f must be "safe" f o r the intended to the person using i t , i t must animal, i t must not be hazardous not harm the environment and i t must not leave p o t e n t i a l l y harmful residues i n food intended for human consumption. In the public mind and i n the corporate regulatory mind, i t i s drug safety which i s of paramount importance when food producing animals are being considered. Food safety issues are currently the subject of intense debate i n many areas of the world and many see p e s t i c i d e and veterinary drug residues as posing potential threats to public health. The safety of a veterinary drug to humans depends on a number of factors, some i n t r i n s i c to the animal being treated, some to the properties of the drug i t s e l f , and some to the method of use - or abuse of the veterinary medicine. These can conveniently considered under two main headings: safety to the consumer safety to the operator 1

Safety to the Consumer For any chemical agent to exert a toxic e f f e c t i n humans or i n animals there are two important considerations - the toxic properties of the substance and the dose received. For veterinary drug residues, an assessment of safety involves an investigation of the t o x i c i t y of the drug and a quantitative study of the residues present i n animal tissues. Over the l a s t two decades, a broadly accepted package of t o x i c i t y tests has emerged f o r assessing the t o x i c i t y of chemicals whatever t h e i r intended purpose. The tests are conducted i n laboratory animals, usually rats and mice, and t h e i r objective i s the i d e n t i f i c a t i o n of a dose l e v e l at which a toxic e f f e c t does not occur, the no-effect or no-observed e f f e c t l e v e l (NEL or NOEL) (28,29). From the r e s u l t s of these tests also help to e s t a b l i s h the t o x i c o l o g i c a l p r o f i l e of the chemical can also be established - i s i t a teratogen, a genotoxic carcinogen or an uncoupler of oxidative phosphorylation f o r instance? Different regulatory a u t h o r i t i e s have d i f f e r i n g requirements therefore i t i s impossible

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to compose a precise l i s t of studies required f o r drug approval. Sometimes the requirement to conduct one test depends on the r e s u l t s of another. However, most a u t h o r i t i e s demand more or less the following: a study of acute t o x i c i t y i n rodents a study of short-term t o x i c i t y - 28 or 90 days a battery^ of tests f o r genotoxicity an i n v e s t i g a t i o n of carcinogenic a c t i v i t y an i n v e s t i g a t i o n of teratogenic a c t i v i t y studies of reproductive performance. Current s c i e n t i f i c dogma claims that there i s no safe l e v e l for a genotoxic carcinogen, an agent which causes cancer by a d i r e c t e f f e c t on the genetic material of a c e l l . T h e o r e t i c a l l y a single molecule could give r i s e to a mutation r e s u l t i n g i n a cancer c e l l and then a cancer (30). Whatever the merits of t h i s argument, i t i s widely regarded as unacceptable to be faced with the p o s s i b i l i t y of residues of a genotoxic carcinogen i n food of animal (or any other) o r i g i n . Similar sentiments would apply f o r s i m i l a r reasons, to genotoxic materials with the a b i l i t y to a f f e c t the germ-line c e l l s . Of course, i f a genotoxic carcinogen i s metabolized i n the target animal to non-active residues, then an a l t e r n a t i v e r i s k assessment i s possible and the drug w i l l be viewed as more acceptable. This was i n fact part of the evaluation of the drug carbadox, a growth promoter f o r pigs, by JECFA. I t noted that the drug i t s e l f was both mutagenic and carcinogenic i n laboratory studies but i t s residues were i n a c t i v e and hence acceptable ( 1 1 ) . Assuming that there are no adverse manifestations such as genotoxic carcinogenicity, and assuming again that some t o x i c e f f e c t s have been noted, an NEL should be i d e n t i f i a b l e providing that a suitable range of doses has been chosen f o r the toxicology studies. Once an NEL has been i d e n t i f i e d an acceptable daily intake (ADI) can be calculated using a s u i t a b l e safety factor ( 2 , 3 1 - 3 3 ) . There i s considerable debate over the magnitude of t h i s safety factor but the one usually chosen i s 100. If the drug produces no t o x i c e f f e c t s i n laboratory species but some minor adverse reaction has been noted i n humans, f o r example during use as a human medicine, a smaller factor, usually 10, may be employed. I f the range of tests was l i m i t e d and the r e s u l t s of dubious s i g n i f i c a n c e , or i f the studies were poorly performed, a larger safety factor may be applied and a temporary ADI adopted. The ADI therefore as NEL/100 i s usually quoted i n terms of mg drug/kg body weight/day or mg/kg per day ( 3 2 - 3 6 ) . An ADI can also be calculated f o r a non-genotoxic carcinogen (one which operates v i a an epigenetic mechanism) providing that the mechanism of carcinogenicity i s known. A good example i s provided by the JECFA assessment of the sulfonamide drug sulfamethazine (sulphadimidine). This was shown to be a thyroid carcinogen i n rodents but was accepted to be a non-genotoxic compound. Moreover, i t was concluded that the mechanism of carcinogenicity involved perturbations of the thyroid-pituitary-hypothalamus axis, changes i n thyroid hormone l e v e l s and a r e s u l t i n g hyperplasia of the thyroid. Although the

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f u l l mechanism i s s t i l l p o o r l y u n d e r s t o o d , i t was a c c e p t e d t h a t an on t h e t h y r o i d e f f e c t s and an ADI calculated NEL c o u l d be b a s e d (37). S i m i l a r a p p r o a c h e s have been t a k e n w i t h s t e r o i d hormones where no-hormonal effect levels can be d e t e r m i n e d in suitable e x p e r i m e n t a l models (38,39). H a v i n g d e t e r m i n e d an ADI, i t i s e s s e n t i a l t h a t consumption of o r i g i n by humans w i l l not r e s u l t in this value food of animal T h i s upper l i m i t i s known as t h e maximum residue b e i n g exceeded. l i m i t or MRL (28,31). I t s e l a b o r a t i o n depends on a number of f a c t o r s i n c l u d i n g the l i k e l y degree o f consumption of the food commodity o r commodities i n q u e s t i o n and t h e g e n e r a l q u a n t i t i e s o r d i s p o s i t i o n o f r e s i d u e s i n each t i s s u e . JECFA has p r o p o s e d daily i n t a k e s o f f o o d commodities; 300g muscle, 100g l i v e r , 50g kidney, 50g f a t and 1.51 o f m i l k , which i t u s e s t o e l a b o r a t e t h e MRL and e n s u r e t h a t t h e ADI i s u n l i k e l y t o be exceeded (11,35). There is c u r r e n t l y much d e b a t e over whether these values represent a r e a l i s t i c f o o d i n t a k e f o r t h e commodities i n v o l v e d . For example, do t h e y t a k e i n t o a c c o u n t t h e s o - c a l l e d extreme consumer who might as an instance eat l a r g e d a i l y q u a n t i t i e s of l i v e r ? Do they Or, t o put t h e q u e s t i o n r e p r e s e n t i n t e r n a t i o n a l f o o d consumption? more p l a i n l y , does 300g muscle c o v e r t h e USA consumption o f beef and t h e t h i r d world consumption o f beef? The answer i s q u i t e evidently "no". Nevertheless JECFA, through the Codex A l i m e n t a r i u s system a t t e m p t s t o recommend MRL v a l u e s w h i c h w i l l be u n i v e r s a l l y a p p l i c a b l e and f o r these reasons, some might say l i m i t a t i o n s , what might be seen as average values for food consumption must be a d o p t e d i f a practical s o l u t i o n i s to be found. Having a r r i v e d at an MRL or MRLs for a commodity or commodities, i t i s n e x t e s s e n t i a l to ensure t h a t the t i s s u e s of animals t r e a t e d with v e t e r i n a r y medicines do n o t exceed these i s n o t as s i m p l e as i t might a t first values. In p r a c t i c e t h i s appear. Even f o r a s i n g l e a c t i v e i n g r e d i e n t , t h e r e q u i r e m e n t s of marketing advantages) d i c t a t e that numerous therapy (and f o r m u l a t i o n s a d m i n i s t e r e d by v a r i o u s r o u t e s must be d e v e l o p e d and made a v a i l a b l e f o r t h e v e t e r i n a r i a n and farmer. Consequently, r e s i d u e s d e p l e t i o n i n the l i v i n g a n i m a l w i l l n o t be c o n s t a n t but w i l l vary according to the f o r m u l a t i o n g i v e n and the r o u t e of administration. I t goes a l m o s t w i t h o u t s a y i n g t h a t the species more c o r r e c t l y the metabolism i n that species, will too, or d e t e r m i n e t h e r a t e o f r e s i d u e s d e p l e t i o n f o r any g i v e n f o r m u l a t i o n specific route. This means t h a t residues a d m i n i s t e r e d by a s t u d i e s a r e g e n e r a l l y r e q u i r e d by r e g u l a t o r y a u t h o r i t i e s f o r each species using each formulation and route of administration. Thus, r e s i d u e s d e p l e t i o n below t h e MRL f o r each situation is e n s u r e d (28,29). Drug r e s i d u e s studies usually involve treating drug u s i n g the intended route of the animal i n q u e s t i o n with the a d m i n i s t r a t i o n , the intended formulations, u s u a l l y at the highest recommended dose and the maximum d u r a t i o n of administration. Animals are then s e r i a l l y s l a u g h t e r e d so t h a t r e s i d u e s depletion can be s t u d i e d and t h e time t a k e n t o a c h i e v e l e v e l s below t h e MRL e s t a b l i s h e d (31).

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These studies should, i f conducted properly, show the residues depletion p r o f i l e of the formulation under study and w i l l reveal for example any re-emergence of residues because of entero-hepatic r e c i r c u l a t i o n . The time taken for the residues to be depleted to below the MRL for each of the tissues of interest i s then usually chosen as the withdrawal period or withholding period (or times) for that formulation. Usually the studies must be conducted i n each of the indicated species although simpler and cheaper bi©equivalence studies where pharmacokinetic p r o f i l e s are examined and compared, may be used to evaluate the withdrawal period i n other food-producing species. Withdrawal periods can be a cause f o r dispute between companies and regulatory authorities, often f o r competitive marketing reasons. If two s i m i l a r products are a v a i l a b l e f o r a p a r t i c u l a r therapeutic purpose, the v e t e r i n a r i a n or farmer w i l l usually choose the one with the shorter withdrawal periods so that i f necessary the animal can be sent to slaughter at the e a r l i e s t possible time a f t e r recovery. These considerations are extremely important for milk because i t cannot be kept u n t i l residues have depleted to below the MRL and milking cannot be postponed. Contaminated milk has to be discarded, thus a t t r a c t i n g financial penalties these being a l l the more important the longer the withdrawal period. Drugs with shorter withdrawal periods o f f e r an obvious advantage. Similar considerations can be applied to honey. When bees are treated for disease conditions, the drug accumulates i n the honey (40-42). Here, i t may slowly change to non-biologically active residues (43-45) but i t may p e r s i s t and the honey w i l l need to be discarded u n t i l treatment has f i n i s h e d . Treating f i s h poses d i f f e r e n t technical and therapeutic problems some of which w i l l be mentioned i n a l a t e r part of t h i s Chapter. From a residues point of view, a p a r t i c u l a r problem arises from the general p h y s i o l o g i c a l processes p e c u l i a r to poikilotherms (46). Their metabolic rates are p a r t l y governed by t h e i r body temperature which i s dependent upon the ambient temperature of the water i n which they l i v e ; the cooler the water, the longer residues depletion takes. For t h i s reason, residues depletion studies i n f i s h are usually conducted at several temperatures chosen to represent the range of temperatures to which they w i l l be exposed under farming conditions. Withdrawal periods are then quoted i n degree days these being a function of both time and temperature. S p e c i f i c problems are raised by bound residues. After an animal has been treated with a medicine, i t s residues are present i n plasma and tissues as parent drug and metabolite or metabolites. The residues may be present as free drug and/or metabolites or as covalently bound residues. This then raises the question of the degree of b i o a v a i l a b i l i t y of these bound residues and t h e i r b i o l o g i c a l a c t i v i t y (47,48). In most cases, this question has no simple answers. Of course a drug may be metabolized to carbon dioxide and hence bicarbonate ion or some other simple precursor of normal endogenous biochemicals. If these a r i s e from a r a d i o l a b e l l e d portion of the molecule, measurements of residues simply as incorporated radiolabel will

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lead the investigator to suspect bound residues when i n fact there are only normal bodily constituents containing incorporated isotope. If bound residues are found, t h e i r impact on the ADI must be assessed; can they be ignored or are they of t o x i c o l o g i c a l significance? JECFA has addressed t h i s issue and has recommended a systematic approach to the problem (11,37). JECFA suggests use of a mild extraction procedure to determine those residues which are c l e a r l y b i o a v a i l a b l e . This i s followed by a more vigorous extraction using acids or enzymic techniques to assess whether p o t e n t i a l l y b i o l o g i c a l l y active compounds may be released i n vivo. These studies can be backed-up by relay methodologies (residues transfer studies) whereby tissues from treated animals are fed to moitiés laboratory species and the release of drug-related measured, e.g. i n plasma ( 4 7 , 4 9 , 5 0 ) . The Committee stressed the need to treat each drug on a case-by-case basis (rather than laying down s t r i c t protocols to deal with bound residues as a common issue). The complexity of the problem can e a s i l y be seen by reference to the work of Lu and colleagues i n the United States They investigated bound residues a r i s i n g from use of (51). structurally related to ronidazole, a nitroimidazole drug metronidazole. Among other discoveries, they found that ronidazole covalently bound to proteins i n the pig forming an adduct which they investigated a f t e r acid hydrolysis. Ronidazole i t s e l f i s mutagenic but Lu and h i s co-workers demonstrated that the bound residues were devoid of genotoxic p o t e n t i a l and so did not o f f e r a r i s k to the consumer. A s i m i l a r analysis has been made for residues of furazolidone (52). The current FDA Guidelines outline a series of short-term and i n v i t r o tests for the safety assessment of bound residues A study of together with t h e i r chemical characterisation (53). r e v e r s i b i l i t y of adduct formation i s also included and as with the JECFA recommendation, drugs are investigated i n an i n d i v i d u a l manner. It seems l i k e l y that the JECFA and FDA approaches, at least i n general terms, w i l l become widely adopted i n t h i s p a r t i c u l a r area of hazard and r i s k assessment. Microbiological Risk Assessment Microbiological r i s k i n t h i s context r e f e r s to the possible e f f e c t s of residues of antimicrobial drugs on the gut f l o r a i n humans exerting a s e l e c t i v e pressure favoring e i t h e r the growth of microorganisms with natural resistance to the drug i n question, or the growth of microorganisms with acquired resistance. This i s a the very controversial area of r i s k assessment, for while phenomenon of induction of resistance i s well known (54-58), there i s no evidence for the supposed e f f e c t s i n humans i n vivo as a r e s u l t of ingestion of food containing veterinary drug residues (59-61). While i t i s widely accepted that i t would be virtually impossible to detect a toxic e f f e c t i n humans due to drug residues because of the wide background of disease and the difficulties involved i n a t t r i b u t i n g any e f f e c t to residues, emerging drug resistance should be more evident. Many antimicrobial and

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a n t i b i o t i c s u b s t a n c e s a r e o f low t o x i c i t y and MRLs t h e r e f o r e a r e o f t e n q u i t e generous. However, a p p l y i n g t h e r e s u l t s o f t e s t i n g f o r a n t i m i c r o b i a l r e s i s t a n c e may r e s u l t i n v e r y low MRLs and long withdrawal periods. T h e r e a r e t h r e e b a s i c t y p e s o f s t u d y a v a i l a b l e (11,62,63): s t u d i e s i n human v o l u n t e e r s s t u d i e s i n germ-free (holoxenic) rodents i n v i t r o studies with b a c t e r i a l populations t h e e x a m i n a t i o n o f t h e human fecal The f i r s t o f t h e s e i n v o l v e s f l o r a b e f o r e and a f t e r t r e a t m e n t w i t h a n t i b i o t i c s . Colonisation the g a s t r o i n t e s t i n a l tract, e.g. t h e oral cavity, by of a d v e n t i t i o u s microorganisms i s also investigated. The u s e o f g e r m - f r e e r o d e n t s models t h e human s i t u a t i o n . These a n i m a l s a r e i n o c u l a t e d w i t h human g u t f l o r a and t h e e f f e c t s o f a n t i b i o t i c s and a n t i m i c r o b i a l s c a n t h e n be s t u d i e d . In v i t r o investigations examine t h e e f f e c t s o f v a r y i n g c o n c e n t r a t i o n s o f t h e d r u g o r d r u g s o f i n t e r e s t on c u l t u r e s o f i n d i c a t o r o r g a n i s m s . A l l these studies c a n be u s e d t o d e r i v e n o - e f f e c t l e v e l s f o r t o x i c i t y towards t h e b a c t e r i a employed. More s p e c i f i c a l l y , t h e minimum i n h i b i t o r y c o n c e n t r a t i o n (MIC) v a l u e s c a n be d e t e r m i n e d . An example o f t h i s t y p e o f assessment i s p r o v i d e d by t h e JECFA d e l i b e r a t i o n s on o x y t e t r a c y l i n e a t i t s m e e t i n g i n Rome i n 1990 (11). The Committee n o t e d that the t o x i c o l o g i c a l p o t e n t i a l of oxytetracyline was low b u t studies were a v a i l a b l e on i t s a n t i m i c r o b i a l e f f e c t s i n dogs and human v o l u n t e e r s and a n o - e f f e c t dose o f 2mg p e r day was i d e n t i f i e d from t h e v o l u n t e e r e x p e r i m e n t s . T h i s l e d t o an ADI o f 0-0.003mg/kg body w e i g h t using a safety f a c t o r o f 10. The t o x i c o l o g i c a l s t u d i e s would have g i v e n an ADI o f a r o u n d 0.18 mg/kg body weight u s i n g a s a f e t y f a c t o r o f 100. The i n d u c t i o n o f a n t i b i o t i c r e s i s t a n c e has been r e c o g n i s e d f o r many y e a r s , and t h e r e i s some e v i d e n c e t o s u g g e s t t h a t antibiotic r e s i s t a n c e may d e v e l o p i n pathogens i n a n i m a l s g i v e n antibiotics (58,60). These r e s i s t a n t pathogens may t h e n be t r a n s m i t t e d to humans. There i s however no e v i d e n c e a t t h e moment that a n t i m i c r o b i a l r e s i d u e s i n meat o r o t h e r a n i m a l p r o d u c t s may lead t o e f f e c t s on t h e human g u t f l o r a and more r e s e a r c h i s o b v i o u s l y r e q u i r e d i n t h i s a r e a b e f o r e major r e g u l a t o r y d e c i s i o n s a r e made. I n t h e meantime, many w i l l s e e t h e c a l c u l a t i o n o f ADIs b a s e d upon m i c r o b i o l o g i c a l data a s an i n t e r i m and added safety measure, w h i l s t others w i l l regard i t as dubious s c i e n c e (59). Operator

Safety

A l t h o u g h t h i s a s p e c t i s i r r e l e v a n t t o t h e assessment o f residues and t h e i r i m p l i c a t i o n s f o r consumer s a f e t y , no a c c o u n t o f t h e u s e o f v e t e r i n a r y d r u g s would be complete w i t h o u t some p a s s i n g mention which w i l l r e s t r i c t i t s e l f t o c o n s i d e r a t i o n s o f s a f e t y i n use of medicines rather than safety i n their manufacture. Many f o r m u l a t i o n s used i n v e t e r i n a r y medicine o f f e r very l i t t l e scope f o r s i g n i f i c a n t o c c u p a t i o n a l exposure. T a b l e t s and c a p s u l e s f o r example e n s u r e that occupational exposure i s minimal, i f not non-existent. However, o p e r a t o r e x p o s u r e does o c c u r and means need t o be t a k e n t o r e d u c e t h i s . Organophosphorus compounds a r e

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used in veterinary medicines as ectoparasiticides in i n several countries, food-producing and companion animals; including the UK, these have replaced organochlorine compounds. Companion animal products are exemplified by slow release c o l l a r s used for the control of fleas and other parasites i n cats and dogs. Here, i t i s important to ensure that the product does not allow the rapid release of large quantities of the active ingredient which would otherwise pose a serious r i s k to the owners. Sheep-dips and warblicide formulations contain such ingredients as chlorfenvinphos, diazinon and propetamphos. Apart from the obvious toxic e f f e c t s of anti-cholinesterases (64) over possible long-term effects concern has been expressed following occupational exposure to organophosphorus compounds (65-71). In the United Kingdom, there i s a comprehensive adverse reactions reporting system which covers suspected adverse reactions i n both the animal patient and i n humans using the medicines (72). This has revealed a small series of suspected adverse reactions to sheep dips which some have attributed to the organophosphorus component. These suspected adverse reactions have included wheezing, coughs, i n f l u e n z a - l i k e e f f e c t s and headaches (72). At the present time i t i s unclear i f these are due to the active ingredients, to other excipients such as organic solvent or other, unknown factors. In the United Kingdom and other European Community Member States, a review of veterinary medicines i s currently taking place under European Community l e g i s l a t i o n . This requires that many products currently on the market are assessed for safety, q u a l i t y and e f f i c a c y as i f they were new marketing authorisation applications (73) and as part of t h i s review, the products containing organophosphorus compounds w i l l be rigorously s c r u t i n i z e d to determine whether new precautions or adjustments to the formulations can be made to reduce the frequency of adverse reactions. Another example of occupational problems associated with veterinary medicines i s that of s e l f - i n j e c t i o n of oil-based vaccines (72). This can give r i s e to vascular compression, ischaemia and tissue damage, p a r t i c u l a r l y i f i n j e c t i o n into the tendon sheath occurs (74). In the United Kingdom, t h i s has resulted i n advice being provided to hospital emergency departments so that prompt and appropriate s u r g i c a l treatment can be given (75). Many a n t i b i o t i c formulations are given i n feed and as many a n t i b i o t i c s have a l l e r g e n i c properties, problems can a r i s e i n t h e i r use. Regulatory a u t h o r i t i e s now insist that such formulations are rendered dust-free by admixture with i n e r t oils or by the production of granular formulations so that occupational exposure i s minimized (76). In general, sensible occupational precautions, can be taken so that occupational exposure to veterinary medicines i s reduced or excluded. The use of impervious gloves f o r spreading pour-on formulations, overalls for dealing with sheep-dips and face-masks or r e s p i r a t o r s for handling dusty products can a l l reduce human exposure. I t i s also essential for basic occupational hygiene advice to be given on the product or i n the product l i t e r a t u r e so

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that informed users can take the necessary precautions extreme s i t u a t i o n s , u s e a s u i t a b l e a l t e r n a t i v e ( 7 6 ) .

13 or i n

Summary I t would n o t be e f f e c t i v e i n any way t o r e v i e w a l l t h e d i f f e r e n t t y p e s o f m e d i c i n e s a v a i l a b l e t o t h e v e t e r i n a r y surgeon, n o r i n d e e d dosage forms available and t h e methods of the various administration. The r e a d e r i s r e f e r r e d t o o t h e r works f o r t h a t information. Suffice i t t o say t h a t a variety of active f o r m u l a t i o n s a r e now i n g r e d i e n t s and i n c r e a s i n g l y s o p h i s t i c a t e d s u p p l i e d by p h a r m a c e u t i c a l companies. The onus i s on their t o x i c o l o g i s t s , p h a r m a c o l o g i s t s and r e s i d u e s e x p e r t s t o e n s u r e t h a t t h e i r l a b o u r s g u a r a n t e e s o f a r as i s r e a s o n a b l y p r a c t i c a b l e , that t h e s e p r o d u c t s a r e s a f e f o r t h e consumer and f o r t h o s e otherwise e x p o s e d t o them, i n c l u d i n g t h e v e t e r i n a r i a n and f a r m e r .

Literature cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11 12.

Blodinger, J. In Formulation of Veterinary Dosage Forms; Blodinger, J., Ed.; Marcel Dekker: New York, 1983; pp 135-173. Huber, W.G. In Veterinary Pharmacology and Therapeutics; Booth, N.H.; McDonald, L.E., Eds; Iowa State University Press:Ames, Iowa, 1988; pp 813-821. Huber, W.G. In Veterinary Pharmacology and Therapeutics, Booth, N.H.; McDonald LE. Eds.; Iowa State University Press:Ames, Iowa, 1988; 822-848. Brander, G.C. In Chemicals for Animal Health Control; Taylor and Francis : London, 1986 and references therein. Brander, G.C.; Pugh, D.M.; Bywater, R.J.; In Veterinary Applied Pharmacology and Therapeutics; Balliere Tindall: London, 1982; pp 356-433. Anon. In The Merck Veterinary Manual; 6th Ed.; Merck and Co. Inc.: Rahway, 1986. Bevill, R.F. In Veterinary Pharmacology and Therapeutics; Booth N.H.; McDonald L.E. Eds.; Iowa State University Press:Ames; Iowa, 1988; pp 785-795. McCaughey, W.J.; Elliott C.T.; Crooks S.R.H. Vet. Rec., 1990, 126, 351-354. Roberson, E.L. In Veterinary Pharmaology and Therapeutics; Booth, N.H.; McDonald, L.E. Eds.; Iowa State University Press:Ames; Iowa, 1988 pp 877-881. Roberson, E.L. In Veterinary Pharmacology and Therapeutics; Booth, N.H.; McDonald, L.E. Eds.; Iowa State University Press:Ames: Iowa; 1988 pp 928-949. Joint FAO/WHO Expert-Committee on Food Additives. Evaluation of Certain Veterinary Drug Residues in Food. Technical Report Series 799, WHO:Geneva; 1990. Fink, D.W.; Porras A.G. In Ivermectin and Abamectin; Campbell, W.C.; Ed.; Springer-Verlag, London, 1989; pp 113-130 and references therein.

In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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XENOBIOTICS AND FOOD-PRODUCING ANIMALS 13. 14. 15. 16. 17. 18.

19.

20. 21. 22. 23. 24. 25. 26. 27. 28

29. 30. 31. 32.

33.

34. 35.

Huber, W.G. In Veterinary Pharmacology and Therapeutics; Booth, N.H.; McDonald, L.E. Eds.; Iowa State University Press:Ames, Iowa, 1988; pp 849-860. Lindsay, D.G. Food Chem. Toxicol, 1985, 23, 767-774. Editorial. Vet. Rec., 1986, 118, 522-524. Anon - Council Directive of 7 March 1988; 88/146/EEC. Offic J. Europ Commun., 1988, No L70, 16-18. Vernon R.G.; Flint D.J. In Biotechnology in Growth Regulaton; Heap, R.B.; Prosser, C.G.; Lamming, G.E., Eds.; Butterworths: London, pp 57-71 and references therein. Chilliard, Y. In Use of Somatropin in Livestock Production; Sejrsen, K., Vestergaard, M.; Neimann-Sorensen, Α., Eds; Elsevier Applied Science: London, pp 61-87 and references therein. Burrenich, C.; Vandeputte-van Messom, G.; Roets, E; Fabry, J.; Massart-Leen, A.-M. In Use of Somatotropin in Livestock Production Sejrsen, K.; Vestergaard, M.; Neimann-Sorensen, A. Eds.; Elsevier Applied Science: London, pp 377-280. Hardin P. The Milkweed, February 1990. Epstein S.S. Ecologist, 1989, 19, 191-195. Juskevich J.C.; Guyer C.G. Science, 1990, 249, 875-884 Anon. Animal Pharm 12th April 1991, 1. Roberts, R.J.; Shepherd, C.J. In Handbook of Trout and Salmon Diseases, Fishing News Books: Oxford; 1986, pp. 46-87. Stuart, N. In Practice, March 1988, 47-53. Liao, P.B. Water and Sewage Works, 1970, 117, 291-297. National Office of Animal Health (NOAH). In Compendium of Data Sheets for Veterinary Products 1990-91, Datapharm Publications Limited: London, 1990. Woodward, Κ. N. In Food Contaminants: Sources and Surveillance; Creaser, C.; Purchase, R., Eds.; Royal Society of Chemistry: London, 1991, pp 99-108, and references therein. Woodward, Κ. N. Biologist, 1991, 38, 105-108, and references therein. Somogyi, A. In Carcinogenic Risks. Strategies for Intervention, IARC Scientific Publications, No. 25, IARC: Lyon, 1979; pp 123-127. Booth, Ν. H. In Veterinary Pharmacology and Therapeutics; Booth, N.H.; McDonald, L. E. Eds.; Iowa State University Press: Ames; Iowa, 1988; pp 1149-1205. International Programme on Chemical Safety. In Principles for the Safety Assessment of Food Additives and Contaminants in Food, Environmental Health Criteria 70, WHO: Geneva, 1987, pp.75-86. International Programme on Chemical Safety. In Principles for the Toxicological Assessment of Pesticide Residues in Food, Environmental Health Criteria 104, WHO: Geneva, 1990, pp.76-83. Bigwood, E. J. CRC Crit.Rev. Toxicol., 1973, 2, 41-93. Perez, M. K. J. Toxicol. Environ. Health, 1977, 3, 837-857.

In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

1. WOODWARD 36.

37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

49. 50. 51. 52.

53.

54. 55. 56. 57.

Uses and Regulation of Veterinary Drugs

15

Vettorazzi, G.; Radaelli - Benvenuti, B. In International Regulatory Aspects for Pesticide Chemicals, Volume II, Tables and Bibliography; CRC Press: Boca Raton, Florida, 1982, pp 1-9. Joint FAO/WHO Committee on Food Additives. Evaluation of Certain Veterinary Drug Residues in Food, Technical Report Series 788, WHO: Geneva, 1989. Joint FAO/WHO Committee on Food Additives. Evaluation of Certain Veterinary Drug Residues in Food, Technical Report Series 763, WHO: Geneva, 1988. Foxcroft, G. R.; Hess, D. L. In Drug Residues in Animals, Rico, A. G. Ed; Academic Press: London, 1986 pp 147-174. Barry, C. L . ; Mac Eachern, G. M. J. Assoc. Off. Anal. Chem, 1983, 66, 4-7. Grandi, Α.; Piastrelli, G. Rass. Chim., 1977, 29, 113-120. Gilliam, M.; Argauer, R. J. Environ. Entomol., 1981, 10, 479-482 Gilliam, M.; Taber, S. J. Invert. Pathol., 1978, 31, 128-130. Gilliam, M.; Taber, S.; Argauer, R. F. Am. Bee J., 1979, 118, 722-723. Gilliam, M.; Taber, S.; Argauer, R.J. J.Apic. Res., 1979, 18, 208-211 EuroResidues. In Conference on Veterinary Drugs in Food. Workshop Reports. Noordwijkerhout, Netherlands, May 1990 Burgat-Sacaze, V.; Rico Α.; Panisset, J-C. In Drug Residues in Animals, Rico, A.G., Ed.; Academic Press: London, 1986; pp 2-31. MacDonald, Α.; Laurencot, H. In Residues of Veterinary Drugs in Food; Haagsma, N.; Ruiter, Α.; Czedik-Eysenberg, P.B., Eds.; Proceedings of the EuoResidue Conference, Noordwijkerhout, The Netherlands, May 1990, pp 259-266. Gallo-Torres, Η. E. J. Toxicol. Environ. Health, 1977, 2, 827-845. Jaglan, P.S.; Glen, M.W.; Neff, A.W. J. Toxicol. Environ. Health, 1977, 2, 815-826. Lu, A.Y.H.; Miwa, G.T.; Wislocki, P.G. Rev. Biochem. Toxicol., 1988, 9, 1-27. Vroomen, L.H.M.; Berghmans, M.C.J.; vanBladeren, P.J.; Groken, J. P.; Wissink, A.C.J.; Kuiper, H.A. In Veterinary Pharmacology, Toxicology and Therapy in Food Producing Animals, Simon, F.; Lees, P.; Semjen, G., Eds; Unipharma: Budapest, 1990, 259-266. Weber, N.E.; Brynes, S.D. In Residues of Veterinary Drugs in Food; Haagsma, N.; Ruita, Α.; Czedik-Eysenberg, P.B., Eds., Proceedings of the EuroResidue Conference, Noordwijkerhout, The Netherlands, May 1990,. pp 404-410. Walton, J.R. Vet. Rec., 1988, 122, 249-251 Anon The Public Health Aspects of the Use of Antibiotics in Food and Feedstuffs, Technical Report Series 260, WHO: Geneva; 1963. Greenwood, D. Pig Vet. J., 1990, 24, 38-46. Washington, J.A. Rev. Infect. D i s . , 1979, 1, 781-785.

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XENOBIOTICS AND FOOD-PRODUCING ANIMALS 58.

Jackson, G. Vet. Rec., 1981, 108, 325-328 and references therein. 59. Somogyi, A. J . Vet. Med., 1989, Suppl. 42, 10-17. 60. Linton, A. H. Vet. Rec., 1981, 108, 328-331. 61. Institute of Medicine. Human Health Risks with the Subtherapeutic Use of Penicillin or Tetracyclines in Animal Feed, National Academy Press: Washington D.C. 1989. 62. Raynaud, J . P.; Mackinnon, J . ; Kemp, G.; Beukers, B.; Metzger, K. J. Vet. Med., 1989 Suppl. 42, 40-55. 63. Tancrede, C.; Barakat R. J. Vet. Med., 1989, Suppl. 42, 35-39. Ecobichon, D. J. Pesticides and Neurological Diseases. 64. Ecobichon, D. J; Joy, R.M. Eds.; CRC Press: Roca Baton, Florida, pp 151-203. 65. Bartle, H. New Scientist, 18 May 1991, 30-35. 66. Burns, J . Farmers Weekly, 3 June 1991. 67. Swanston, D.; Shaw, I. Sheep Farmer, October 1990, 31-33. 68. Brown, S.K.; Ames, R.G.; Mengle, D.C. Arch. Environ. Health, 1989, 44, 34-39. 69. Rodnitzsky, R.L.; Levin, H.S.; Mick, D.L. Arch. Environ. Health, 1975, 30, 98-103. 70. Savage, E.P.; Keef, T . J . ; Mounce, L.M.; Heaton, R.K.; Lewis, J.Α.; Burcar, P.J. Arch. Environ. Health, 1988, 43, 38-44. 71. Duffy, F.H.; Burchfield, J . L . ; Bartels, P.H.; Gaon, M; Sim, V.M. Toxicol. Apppl. Pharmacol., 1979, 47, 161-176. 72. Woodward, K.N.; Gray, A.K. Human Exp. Toxicol., 1990, 9, 326 73. Woodward, K.N; Cameron, R.S.; Peirce, M.A. Vet. Rec., 1991, 128, 23-24. 74. Mrvos, R.; Dean, B.S.; Krenzelok, E.P. Clin. Toxicol., 1987, 25, 297-304. 75. Anon, Brit. Med. J . 1987, 294, 652 76. Woodward, K.N. Vet. Human Toxicol., 1991, 33, 168-172 Received February 28, 1992

In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Chapter 2

Use of Xenobiotics in Food-Producing Animals in the United States Regulatory Aspects Ν.

E.

Weber

Division of Chemistry, Center for Veterinary Medicine, Food and Drug Administration, Rockville,MD20855 A FEDERAL REGISTER document published December 31, 1987 permits the use of carcinogenic animal drugs in food animals as allowed by the DES proviso to the Delaney Clause found in the Food, Drug and Cosmetic Act. A set of guidelines was developed to support the regulation. This chapter will explain the significant toxicology and chemistry elements of those guidelines including metabolic and kinetic aspects that are employed in the regulation of carcinogenic as well as non-carcinogenic animal drugs and feed additives. This report is designed to give the reader an overview of the regulation of veterinary drugs in the United States. Particular emphasis will be given to the human food safety aspects after the other parts of the regulatory process are outlined so that a broadened perspective of the regulatory scheme may be seen. A profile of regulation of veterinary drugs includes not only the awareness of the need to demonstrate the efficacy of the drug and human food safety aspects but also the aspects of target animal safety, environmental safety and manufacturing controls. Since the animal drug amendments of 1968, veterinary drugs have had their own section of the Code of Federal Regulations (CFR), section 512. Under this section the sponsor of a new animal drug is required to demonstrate not only the efficacy of the drug, but also the safety to the target animal. These studies must be scientifically valid and well controlled to support the approval and are codified in 21 CFR 514. Before continuing on to the human food safety and environmental portions, mention should be made that there is a large amount of information required concerning the manufacture and controls for production of the drug prior to its approval. These requirements are very similar if not identical to those for human drugs. The manufacturer must comply with good manufacturing practices (GMPs) and have acceptable stability tests for the product both prior to as well as after the approval. Environmental Issues Under the National Environmental Policy Act (ΝΕΡΑ) of 1969, the Center for Veterinary Medicine must consider the potential environmental impact of the actions (decisions) that it takes. The types of action that most commonlyrequirean environmental evaluation are

This chapter not subject to U.S. copyright Published 1992 American Chemical Society In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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XENOBIOTICS AND FOOD-PRODUCING ANIMALS

authorizations to investigate uses of new animal drugs and approvals of new animal drugs for marketing. The environmental evaluation of the impact of these actions (and most others) can take one of three forms: (i) a categorical exclusion from preparing an environmental assessment, (ii) an environmental assessment (EA) and (iii) an environmental impact statement (EIS). The procedures and reports needed to address the environmental issues are described in Title 21 Part 25 of the Code of Federal Regulations (CFR). There is a significant amount of documentation needed to support each of the forms identified above. That documentation usually consists of describing the specific action, describing any controls used to limit the release of the animal drug into the environment and, as necessary, providing information concerning the potential fate and effects of the animal drug in the environment. Often information from the drug metabolism studies used to determine food residue chemistry as well as results of toxicology studies used to determine human food safety can be used as part of the fate and effects information needed to evaluate the potential environmental impact of a new animal drug. The latter also includes safety to the persons who handle and administer as well as produce the drug. The Environmental Sciences Staff at the Center for Veterinary Medicine, FDA, reviews the information submitted. Human Food Safety The remainder of this article will outline the major parts of the human food safety portion of the new animal drug approval process with emphasis on the important interface between drug toxicology and residue chemistry. However, before the discussion of the guideline material a brief discussion of some important historical aspects and definitions needs to be given. Residue. To begin, the definition of a residue has been around for a long time. One of the definitions of a residue comesfromthe 1958 food additive amendments of the Federal Food, Drug, and Cosmetic Act (FFD&CA), As Amended (/), Sec. 409. This section addresses residues from the standpoint of methods as well as safety as follows: (b) (2)(D) a description of practicable methods for determining the quantity of such additive in or on food and any substance formed in or on food, because of its use; (c) (5)(A) the probable consumption of the additive and of any substance formed in or on food because of the use of the additive; Time has not signiflcandy changed the definition of a residue. A recent definition that was given in a FDA 1985 proposed regulation (2) is: "Residue" means any compound present in edibletissuesof the target animal that results from the use of the sponsored compound, including the sponsored compound, its metabolites, and any other substances formed in or on food because of the sponsored compound's use. The phrase "drug residues" is used synonymously with residue. The concepts apply whether the residue comes from an animal drug or feed additive. Another way of visualizing drug residues is seen in the following outline.

In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

2.

WEBER

Use of Xenobiotics in Food-Producing Animals in the U.S.

19

DRUG RESIDUES 1. Parent compound 2. Metabolites- addition,cleavage, oxidation, reduction of functional groups 3. Conjugates- small molecules (glucuronides, etc.) macromolecules (bound residues) Origin of No Residue. The 1958 amendments also had another important food safety concept, the anticancer proviso or the Delaney Amendment as it is often referred to. This provision is incorporated in Section 409 (c)(3)(A) and states "That no additive shall be deemed safe if it is found to induce cancer when ingested by man or animal or if it is found, after tests which are appropriate for the evaluation of the safety of food additives, to induce cancer in man or animal." In 1962, section 409 of the FFD&CA was amended, in which the same section, (c)(3)(A), quoted above now was given additional language that exempted feed additives through the so called DES PROVISO by stating after the above wording "that this proviso shall not apply with respect to the use of a substance as an ingredient of feed for animals which are raised for food production, if the Secretary finds (i) that, under the proposed conditions of use ...such additive will not adversely affect the animals... and (ii) that no residue of the additive will be found (by methods of examination prescribed or approved by the Secretary ...) in any edible portion of such animal after slaughter or in any food yielded or derived from the living animal." When the FFD&C Act was amended in 1968 to include a new section (Section 512) on New Animal Drugs, the wording of the DES Proviso was included in that section, Sec. 512 (d)(1)(H). The SOM Procedure. The no residue wording of the DES Proviso which states "...no residue by a method prescribed or approved by the Secretary..." became the foundation by which FDA regulates not only carcinogenic but also non-carcinogenic animal drugs. Beginning in 1973 and continuing for a period of 14 years, the FDA attempted to finalize a regulation to implement the concept by which carcinogenic animal drugs could be approved. The final regulation was published on December 31,1987 (J). The title of the rule is "Sponsored Compounds in Food- Producing Animals; Criteria and Procedures for Evaluating the Safety of Carcinogenic Residues; Final Rule." However, it has become known as the Sensitivity of the Method (SOM) document because it is based on the concept that the Secretary prescribes or approves the methods for carcinogens as permitted by the DES Proviso and through a process determines the level (sensitivity/concentration) required for no residue. A scientific principle applied is that once a drug is given to an animal, residues will not deplete to absolute zero. The focal point of the SOM rule became the procedure by which no residue is determined The regulation uses a simple approach which basically involves extrapolating cancer data from laboratory animal models (usually mice or rats) from the observed natural or background incidence to a predicted increased incidence of no more than 1 tumor in 1 million test animals as a result of ingesting the sponsored compound. These calculations involve the number of animals with tumors compared to the total number of animals exposed at a dose in their diet over a lifetime. Various mathematical models are discussed for calculating the 1 in 1 million dose. However, the FDA preferred the multistage model (4) at thetimethe final rule was written. The 1 in 1 million dose becomes the permitted concentration that is used to calculate the no residue level required for the method of analysis for residues in food for human consumption. Although this value is involved in the calculation, an additional calculation is needed to take into consideration total residues in the food animal before a specific value for a specific analyte can become the no residue value by which the drug is regulated. In the following paragraphs, the procedure by which this is done will be outlined. In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

20

XENOBIOTICS AND FOOD-PRODUCING ANIMALS

Carcinogenic Residues and General Food Safety - A Unified Concept The SOM concept became fully integrated into a general food safety concept that the FDA had developed throughout most of the period prior to the publication of the final rule. FDA uses a similar chemistry approach for determining the tolerance for residues for any compound including "no residue" for a carcinogen. The unified concept applies to carcinogens and non-carcinogens. Before the carcinogenicity of a compound can be evaluated, all compounds have to be treated similarly to assess their carcinogenic potential. An initial decision tree approach that all compounds must undergo was outlined in the SOM document Threshold Assessment is the name given to this process. The process initially involves a structure-activity assessment to determine whether the sponsored compound is a suspect carcinogen. The compound must also be tested in a battery of mutagenicity tests and must tested in subchronic 90 day studies-usually in the rat and dog. The carcinogenic potential of the compound may be suggested by these tests. If any of the tests signal a potential for carcinogenicity, then chronic lifetime studies are required. When a carcinogenic potential is not seen, the level of residue in edibletissuesfurther determines whether a sponsored compound has to undergo chronic studies. Threshold assessment is outlined below to show the interaction of its elements. Categories D and C refer to suspect and non-suspect carcinogens respectively based on structure-activity relationships. Assignment of category A or Β resultsfromthe outcome of biological tests which include the mutagenesis battery described below and 90 day subchronic studies in laboratory animals. General Food Safety (GFS) is the category into which a compound is placed when it is determined not to be a carcinogen. The calculation of a safe concentration for residues for GFS is discussed later. DRUG Structure Activity

(Suspect) Biology

C (Non-suspect)

+

—I CHRONIC STUDIES

Xs Use Level

LOW ,250ppb

LiGH

,>10ppb

CHRONIC STUDIES

liGH

,

Η

ο r m m ζ

42

XENOBIOTICS AND FOOD-PRODUCING ANIMALS

2.2 rag f l u n i x i n NMG/kg body weight/day for three consecutive days vas homogenized i n 60% p e r c h l o r i c a c i d , incubated at 25°C overnight to digest the t i s s u e , and then extracted with chloroform or methylene c h l o r i d e under a c i d i c , n e u t r a l and basic conditions. The combined organic extracts were evaporated to dryness under reduced pressure, redissolved i n methanol and analyzed by HPLC. Recovery of sample r a d i o a c t i v i t y i n the combined organic extracts ranged from 69Z to 83X f o r the 12-hour l i v e r samples and SX to 22% f o r the 24-hour samples. F l u n i x i n was the major residue i n the organic extract from l i v e r of c a t t l e at 12 and 24 hours post f i n a l dose, representing greater than 952 of the extractable r a d i o a c t i v i t y . In an attempt to enhance recovery, two a l t e r n a t e e x t r a c t i o n methods, including aqueous hydrochloric acid e x t r a c t i o n and methanol-hydrochloric acid:potassium hydroxide e x t r a c t i o n were employed. In each case, recoveries were comparable to the i n i t i a l r e s u l t s , and f l u n i x i n s t i l l represented the major residue detected i n the 12- and 24-hour l i v e r samples. Following i n i t i a l p r o f i l i n g of the 12- and 24-hour post f i n a l dose l i v e r t i s s u e , the l i v e r s from two of three animals at each s a c r i f i c e i n t e r v a l (12, 24, 72 and 120 hours post f i n a l dose) as w e l l as kidney tissue from each animal at each s a c r i f i c e i n t e r v a l were extracted and the metabolite p r o f i l e s determined. Each l i v e r and kidney sample was homogenized i n hexane to remove l i p i d , centrifuged, the tissue p e l l e t hydrolyzed i n hydrochloric a c i d , the hydrolyzed homogenate extracted with e t h y l acetate at pH 4-4.5, and the e t h y l acetate extract concentrated and analyzed by HPLC. With few exceptions, f l u n i x i n accounted f o r at least 50X of extractable tissue r a d i o a c t i v i t y and was the major residue in the l i v e r s and kidneys of male and female feeder c a t t l e . The 4'-hydroxy metabolite of f l u n i x i n was also present and represented a major residue i n female l i v e r samples at 12 and 24 hours post f i n a l dose and i n selected male and female kidney samples at 72 and 120 hours. Minor amounts of the other hydroxylated metabolites, including 5-hydroxyflunixin and 2'-methylhydroxyflunixin, were also detected. These r e s u l t s are in agreement with the p r o f i l i n g data from the i n i t i a l t o t a l residue study and indicate that the primary routes of metabolism of f l u n i x i n are v i a oxidation of the pyridine and phenyl r i n g systems as w e l l as the methyl substituent on the phenyl moiety. The data also indicate that f l u n i x i n i s the most appropriate marker residue i n the target tissue ( l i v e r ) . Despite use of a l t e r n a t e methods of tissue e x t r a c t i o n , the percent of t o t a l radiolabeled residue extractable from the l i v e r or kidney decreased with increasing length of time a f t e r dosing. In l i v e r , the percent recovery of t o t a l r a d i o a c t i v i t y i n the organic extract ranged from 70-942 at 12 hours, 61-72X at 24 hours, and had decreased to between 21 and 48% at 72 and 120 hours post f i n a l dose. These r e s u l t s suggest that bound residues may be formed during the metabolism of f l u n i x i n i n cattle.

In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

4.

CLEMENT ET AL.

Residue Depletion and Metabolism of Flunixin in Cattle

Surveillance and Confirmatory Assay Development To e s t a b l i s h a q u a n t i t a t i v e r e l a t i o n s h i p between the depletion of t o t a l residue of t o x i c o l o g i c a l concern and the marker residue i n the target t i s s u e , a r e l i a b l e a n a l y t i c a l method for the marker residue must be developed. This method w i l l undergo interlaboratory v a l i d a t i o n t r i a l s and must, therefore, be rugged and of s u f f i c i e n t s e n s i t i v i t y to detect the marker residue at concentrations at which the t o t a l residue i s at or below the safe concentration. The method u s u a l l y consists of two components: a s u r v e i l l a n c e (determinative) assay to quantify the marker residue, and a confirmatory assay to v e r i f y the i d e n t i t y of the compound. Each assay must be of acceptable s p e c i f i c i t y ( i n c l u d i n g freedom from interference by p o t e n t i a l l y coadministered medications), s e n s i t i v i t y (accurate detection of the marker compound at a concentration equal to one-half the concentration at a time when t o t a l residue i s at the safe concentration), accuracy and p r e c i s i o n . Each method should u t i l i z e commercially a v a i l a b l e reagents and standard a n a l y t i c a l laboratory instrumentation, and be capable of being performed by experienced analysts w i t h i n a 48-hour period. For a detailed discussion of assay requirements, the reader i s referred to guideline V of "General P r i n c i p l e s for Evaluating the Safety of Compounds Used i n Food-Producing Animals' (1). 1

Assay Development f o r F l u n i x i n (Marker Residue) i n L i v e r (Target Tissue). A s u r v e i l l a n c e assay was developed which detects and quantitates f l u n i x i n i n bovine l i v e r by ion-pair reverse phase high performance l i q u i d chromatography. In the assay, l i v e r samples are homogenized i n T r i s Buffer (0.1 M, pH 10.7), extracted with dichloromethane/isopropyl a l c o h o l (9:1 v/v) to extract l i p i d s and p r e c i p i t a t e proteins, and then, following c e n t r i f u g a t i o n , the aqueous layer i s a c i d i f i e d to pH 90% of the dose was excreted across the gills. Because its properties are such that it can be either excreted across the gills, or conjugated with sulfate to a readily excreted metabolite, phenol was not retained by the lobster. Other Decapod Crustacea. The fate of the pesticide fenitrothion has been studied in blue crabs, Callinectes sapidus, after water-borne exposure to 5.2 ppb for 48 hours (80). The hepatopancreas was the major organ of uptake (0.3 μg fenitrothion equivalents/g after 48 hours), and concentrations in muscle were very low (0.026 μg/g), although muscle concentrations were almost 4 times higher than hemolymph concentrations (80). The major metabolites of fenitrothion found in hepatopancreas were fenitrooxon, desmethylfenitrooxon and 3-methyl-4-nitrophenol, and these metabolites were also recovered from the tank water (80). The biotransformation of water-borne methyl parathion has been studied in two prawn species, the Malaysian prawn, Macrobrachium rosenbergii, and the ridgeback prawn, Sicyonia ingentis, and a crayfish, Procambarus clarkii. (81). Animals were exposed to C-methyl parathion, 0.01 mg/litre for 15 to 24 hours then placed in clean, flow-through water. The tank effluent containing excreted metabolites was passed over XAD-4 resin and the metabolites were subsequently eluted from the resin (72). In all three species, the major primary metabolite 14

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XENOBIOTICS

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Remainder Shell

0 •

20 Η

ANIMALS

•

Muscle

•

Hepatopancreas

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"D

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Ό CO

Time, weeks after oral dose 14

Figure 9a. Tissue distribution of C-erythromycin as percent of dose after oral administration (50 mg/kg) to lobsters (Homarus americanus). (n = 6).

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Figure 9b. Concentration of total radioactivity in tissue after oral administration (50 mg/kg) of C-erythromycin to lobsters (Homarus americanus) (η = 6). 14

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was 4-nitrophenol, which was excreted predominantly as sulfate and glucoside conjugates (81). These studies did not report tissue concentrations of the pesticide. The tissue distribution and metabolism of the herbicide triclopyr was studied in crayfish, Procambarus clarkii, after static exposure of animals to 1 or 2.5 ppm for 11 days (25). After exposure at the higher concentration, tail muscle contained 0.3 μg triclopyr equivalents/g, and the elimination half life was 10 days. The concentration in muscle was more than 10-fold lower than the hemolymph concentration (25). The hepatopancreas attained higher concentra­ tions than muscle but lower concentrations than hemolymph at both exposure concentrations. Triclopyr was metabolized in the hepatopancreas to the taurine conjugate and to other polar metabolites (25). The fates of the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) 2, 4, 5-trichlorophenoxyacetic acid (2,4,5-T) and the DDT metabolite, bis-(4chlorophenyl) acetic acid (DDA) were studied in the spiny lobster, Panulirus argus (24). All acids were injected intrapericardially at 10 mg/kg. Both 2,4-D or 2,4,5-T were extensively excreted, unchanged, in urine in the first 24 hours after the dose. About 10% of the dose of 2,4,D and 2,4,5T was taken up by hepatopancreas where the taurine conjugate was formed and subsequently excreted either in urine or feces. Part of the DDA dose was excreted in urine, as unchanged DDA and as DDA-taurine, but DDA was more extensively taken up by shell and hepatopancreas and more slowly excreted than 2,4D or 2,4,5-T. For all three carboxylic acids, muscle concentrations were lower than hemolymph concentrations at 24 hours after the dose, and were less than 5μg/g. Summary. The available data indicates that compounds that are readily watersoluble, or can be biotransformed into water-soluble conjugates, are more rapidly excreted from Crustacea than are lipid soluble drugs that must be metabolized by phase 1 monooxygenases in order to introduce polar functionalities into the molecule. Very lipophilic drugs and pesticides can be expected to attain much higher concentrations in the hepatopancreas than in other tissues, and to be slowly excreted in feces after metabolism to more polar metabolites. For some drugs, hemolymph concentrations are the same order of magnitude as muscle concentrations, but this relationship is different for each drug. Acknowledgments The authors would like to acknowledge financial support NIEHS: ES04184; FDA: FD-U-000158 and FD 01466-01. Literature Cited 1.

Creavan, P.J.; Parke, D.V.; William, R.T. Biochem. J. 1965, 96, 879-885.

2.

Dewaide, J.H.; Henderson, P.T., Biochem. Pharmacol. 1968, 17, 19011907.

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128 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

XENOBIOTICS AND FOOD-PRODUCING ANIMALS Buhler, D.R.; Rasmusson, M.E. Comp. Biochem.Physiol.1968, 25, 223239. Pohl, R.J.; Bend, J.R.; Guarino, A.M. Drug Metab. Dispos. 1974, 2, 545555. Ahokas, J.T.; Pelkonen, O.; Karki, N.T. Cancer Res. 1977, 37, 37373743. Bend, J.R.; James, M.O. In Biochemical and Biophysical Perspectives in Marine Biology; Malins, D.C.; Sargent J.R. eds.; Academic Press: New York, 1978, Vol. 4, pp. 125-188. Elcombe, C.R.; Lech, J.J. Toxicol. Appl. Pharmacol. 1979, 49, 437-450. Stegeman, J.J.; Woodin, B.R. Fed. Proc 1980, 39, 1752. Statham, C.N.; Szyjka, S.P.; Menhahan, L.A.; Lech, J.J. Biochem. Pharmacol. 1977, 26, 1395-1400. Miranda,C.L.;Wang, J.L.; Henderson, M.C.; Buhler, D.R. Biochem. et Biophys. Acta. 1990, 1037, 155-160. Vodicnik, M.J.; Elcombe, C.R.; Lech, J.J. Toxicol. Appl. Pharmacol. 1981, 59, 364-374. Addison, R.F.; Zinck, M.E., Willis, D.E. Comp. Biochem. Physiol. 1977, 57C, 39-43. Kleinow, K.M.; Haasch, M.L.; Williams, D.E.; Lech, J.J. Comp. Biochem. Physiol. 1990, 96C, 259-270. Franklin, R.B.; Elcombe, C.R.; Vodicnik, M.J.; Lech, J.J. In Microsomes, Drug Oxidations and Chemical Carcinogenesis; Coon, M.J.; Conney, A.H.; Estabrook, R.W. eds., Academic Press: New York, NY, 1980, pp. 833-836. Lech, J.J.; Bend, J.R.; Environ. Health Perspect. 1980, 34, 115-131. Khan, M.A.; Lech, J.J.; Menn, J. American Chemical Society Symp. Ser. No. 99. Am. Chem. Soc., Washington, D.C. 1979. James, M.O., Xenobiotica, 1989, 19, 1063-1076. Khan, M.A.Q.; Coello, W.; Khan, Α.Α.; Pinto, H. Life Sciences 1972, 11, 405-415. Singer, S.C.; March, P.E., Jr.; Gonsoulin, F.; Lee, R.F. Comp. Biochem. Physiol. 1980, 65C, 129-134. O'Hara, S.C.M.; Neal, A.C.; Corner, E.D.S., Pulsford, A.L. J. Marine Biol. Assoc. UK 1985, 65, 113-131. James, M.O.: Bowen, E.R., Dansette, P.M.; J.R. Chem. Biol. Interact. 1979, 25, 321-344. James, M.O.: In Xenobiotic Metabolism and Disposition. Kato, R.; Estabrook, R.W.; Cayen, M.N. eds. Taylor and Francis, U.K., USA, 1989. pp. 283-290. Schell, J.D.; James, M.O. J. Biochem. Toxicol. 1989, 4, 133-138. James, M.O.: Drug Metab. Disp. 1982, 10, 516-552.

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Barron M.G.; Hansen, S.C.; Ball, T. Drug Metab. Disp. 1991,19,163167. James, M.O.; Barron, M.G. Vet. Human Toxicol. 1988, 30 (Supplement 1), 36-40. Kleinow, K.M.; Lech, J.J.; Vet. Human Toxicol. 1988, 30, Suppl. 1, 2630. Endo, T.; Onozawa, M. 1987, Nippon Suisan Gak. 53, 551-555. McKim, J.M.; Goeden, H.M. Comp. Biochem. Physiol. 1982, 72C, 65-74. Heming, T.A. Am. J. Vet. Res. 1989, 50, 93-97. Droy, B.F.; Goodrich, M.S.; Lech, J.J.; Kleinow, K.M. Xenobiotica. 1990, 20, 147-157. Squibb, K.S.; Michel, C.M.F.; Zelikoff, J.T.; O'Connor, J.M. Vet. Human Toxicol. 1988, 30, Suppl. 1, 31-35. Aida, Κ.; Hirose, Κ.; Yokote, M. Bull. Jap. Soc. Sci. Fish 1973, 39, 1,107- 1,115. Heidiger, R.C.; Crawford, S.D. J. Fish Res. Bd. Can. 1977, 34, 633-638. Kleinow, K.M.; Droy, B.F.; Buhler, D.R.; Williams, D.E. Toxicol. Appl. Pharm., 1990,104,367-374. Miller, M.R.; Hinton, D.E.; Blair, J.J.; Stegeman, J.J. Mar. Environ. Res. 1988, 24, 37-39. Statham, C.N.; Melancon, M.J.; Lech, J.J. Science. 1976, 193, 680-681. Klaassen,C.D.;Plaa, G.L. J. Appl. Physiol. 1967, 22, 1,151-1,155. Gingerich, W.H.; Weber, L.J.; Larson, R.E. Comp. Biochem. Physiol. 1977, 58C, 113-120. Kleinow, K.M.; In Aquatic Toxicology and Risk Assessment; Mayes, M.A.; Barron M.G., Eds. ASTM STP 1124; American Society for Testing and Materials: Philadelphia, PA, 1991, Vol. 14; pp 131-138. McKim, J.M., Heath, E.M. Toxicol. Applied Pharmacol., 1983, 68, 177187. Droy, B.F.; Tate, T.; Lech, J.J.; Kleinow, K.M. Comp. Biochem Physiol. 1989, 94C, 303-307. Beyenbach, K.W.; Kirschner, L.B. Am J. Physiol. 1975, 225, 389-393. James, M.O.; Pritchard, J.B. Drug Metabol. Disp. 1987, 15, 665-670. Plakas, S.M.; James, M.O. Drug Metabol. Disp. 1990, 18, 552-556. Pritchard, J.B.; James, M.O. J. Pharmacol. Exptl. Therap. 1979, 208, 280-286. Bungay, P.M.; Dedrich, R.L.; Guarino, A.M. J. Pharmacokinet. Biopharm. 1976, 4, 377-388. Plakas, S.M.; Dickey, E.W.; Barron, M.G.; Guarino, A.M. Can J. Fish Aq. Sci. 1990, 47, 766-771. Fabacher, D.L.; Comp. Biochem. Physiol., 1982, 73C, 277-283. Fabacher, D.L.; Comp. Biochem. Physiol., 1982, 73C, 285-288. Hunn, J.B.; Willford, W.A. Comp. Biochem. Physiol. 1970, 33, 805. Matsubara, T.; Koike, M.; Touchi, Α.; Tochino, Y.; Sugeno, K. Anal. Biochem. 1976, 75, 596-603.

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Kleinow, K.M.; McElroy, A.E.; Bull Mt. DesertIsl.Biol.Lab. 1990, 29, 135-136. 54. Jarboe, H.H.; Kleinow, K.M. Pharmacologist 1990, 32, 149. 55. Sake, R.; Liestol, K. Acta. Vet. Scand. 1983, 24, 418-430. 56. Ueno, R.; Okumura, M.; Horiguchi, Y.; Kubota, S.S.; Nippon Suisan Gak. 1988, 54, 485-489. 57. Jacobsen, M.D.; J. Fish Diseases, 1989, 12, 29-36. 58. Kasuga, Y.; Sugitani, Α.; Yamada, F.; Arai, M.; Morikawa, S.; J. Food Hyg. Soc. Jap. 1984, 25, 512-516. 59. Endo, T.; Onozawa, M. Nippon Suisan Gak. 1987, 53, 557-562. 60. Endo, T.; Onozawa, M. Nippon Suisan Gak.1987, 53, 551-555. 61. Ishida, N.; Nippon Suisan Gak. 1990, 56, 55-59. 62. Stenger, V.G.; Maren, T.H. Comp. Gen. Pharmacol., 1974, 5, 23-35. 63. Hunn, J.B.; Schoettger, R.A.; Willford, W.A. J. Fish. Res. Bd. Canada. 1968, 25, 25-31. 64. Phillips, B.F.; Cobb, J.S.; George, R.W. In Physiology and Behavior; Cobb, J.S.; Phillips, B.F. Eds.; The Biology and Management of Lobsters; Academic Press; New York, NY, 1980, Vol.1, 2-82. 65. Mykles, D.L. J. Exp. Biol. 1980, 84, 89-101. 66. Conklin, D.E. In Physiology and Behavior; Cobb, J.S.; Phillips, B.F. Eds.; The Biology and Management of Lobsters; Academic Press: New York, NY, 1980 Vol.1, 277-300. 67. James, M.O.; Shiverick, K.T.: Arch Biochem. Biophys. 1984, 233, 1-9. 68. James, M.O.; Schell, J.D.; Barron, M.G.; and Li, C-L. J. Drug Metab. Disp. 1991, 19, 536-542. 69. Barron, M.G.; Gedutis, C.; James, M.O. Xenobiotica, 1988, 18, 269-276. 70. Holliday, C.W. Comp. Biochem. Physiol. 1977, 58A, 119-120. 71. Synder, M.J.; Chang, E.S. Gen. Comp. Endocrinol. 1991, 82, 118-131. 72. Foster, G.D.; Crosby, D.G. Environ.Toxicol.Chem. 1986,15,1059-1071. 73. James, M.O.; Schell, J.D.; Magee, V. Bull. Mt. Desert Isl. Biol. Lab., 1989, 28, 119-121. 74. James, M.O. In Pesticide Chemistry, Advances in International Research, Development and Legislation; H. Frehse, Ed.; VCH Weinheim, Germany, and New York, USA, 1991, pp 277-286. 75. Holwerda, D.A.; Vonk, J.K. Comp. Biochem. Physiol. 1973, 45B, 51-58. 76. James, M.O. Arch. Biochem. Biophys. 1990, 282, 8-17. 77. Friedman, C.S. Vet. Human. Toxicol. 1991, 33 (Supplement 1), 30-33. 78. James, M.O.; Herbert, A.H. Pharmaceutical Res. 1988, 5 S196. 79. James, M.O. The Toxicologist 1990, 10, 176. 80. Johnston, J.J.; Corbett, M.D. Toxicol. Appl Pharmacol. 1986, 85, 181188. 81. Foster, G.D.; Crosby, D.G. Xenobiotica 1987, 17, 1393-1404. 82. McLaughlin, P.A. In Internal Anatomy; Mantel, L. Ed; The Biology of Crustacea; Academic Press: New York, 1983, Vol.5. Received March 19, 1992

In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Chapter 9 Pirlimycin in the Dairy Cow Metabolism and Residue Studies R. E. Hornish, T. S. Arnold, L. Baczynskyj, S. T. Chester, T. D. Cox, T. F. Flook, R. L. Janose, D. A. Kloosterman, J. M. Nappier, D. R. Reeves, Yein, and M. J. Zaya F. S. Animal Health Drug Metabolism, Upjohn Laboratories, The Upjohn Company, Kalamazoo, MI 49001 Pirlimycin hydrochloride (I), a lincosaminide antibiotic, is a new therapeutic agent under development for the treatment of mastitis in the dairy cow. Absorption, distribution, metabolism, excretion and residue decline studies of I have been conducted in the dairy cow following intramammary infusion of an aqueous gel formulation of I into all four quarters of the udder via the teat canals. Total milk residues accounted for only 50% of the dose and the milk residue concentration- time course was bi-phasic. Nearly half of the dose was thus absorbed for systemic circulation. Drug residue concentrations in blood were best fit to a two­ -compartment pharmacokinetic model. Pirlimycin accounted for ≥95% of the drug residue in milk and was excreted predominantly as parent compound in the urine and feces. Pirlimycin sulfoxide was the major residue found in the liver, the target tissue for residue analysis. GI tract microflora converted part of the fecal drug residue to 3-(5'-ribonucleotide) The comparative adducts of pirlimycin and pirlimycin sulfoxide. metabolism of I in the rat following oral administration was nearly identical to that in the cow following intramammary infusion.

Pirlimycin (I), Figure 1, is a semi-synthetic member of the lincosaminide antibiotics derived from lincomycin (II) and clindamycin (III). Its activity against most grampositive organisms is comparable to clindamycin and several times as active against Staph.aureus (1,2). The proposed use of pirlimycin as a therapeutic agent for the treatment of bovine mastitis was initially investigated by Yancey in an in vitro lactating mouse model developed for estimating mastitis activity (3). Pirlimycin hydrochloride is now under development as a dairy cow mastitis therapeutic agent. The development of any drug or chemical entity targeted for food-producing animals must undergo a six-step safety evaluation process encompassing the study of the adsorption, distribution, metabolism and excretion ( A D M E studies) to address drug residue concerns in the consumable products as outlined in the landmark papers by

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Perez (4) and Weber (5). This report describes part of the A D M E studies carried-out for the safety evaluation of pirlimycin in the dairy cow for the treatment of mastitis. All four udder quarters of twelve dairy cows in mid-lactation were treated at 4 times the therapeutic dose of 50 mg/ quarter by intramammary infusion of an aqueous gel containing 200 mg of pirlimycin free base equivalents, including the labeled C-pirlimycin hydrochloride. The pharmacokinetic parameters for total pirlimycin residue in blood and milk were determined. Three cows were sacrificed at each of four post-treatment intervals (4, 6, 14, and 28 days) to establish tissue residue depletion kinetics. Metabolite profiles of the residues in milk, liver, urine and feces were obtained and the unknown radiolabeled components identified. A comparative metabolism study in the rat was also conducted to address its relevancy as a toxicological test species. The residue concentration decline kinetics were also determined for pirlimycin in milk from cows treated at a dose rate of 50 mg per quarter in all 4 quarters to establish a milk discard interval for the proposed use. 14

Materials and Methods 14

14

C-pirIimycin. C-Pirlimycin hydrochloride was synthesized by the sequence shown in Figure 2. Final purification was accomplished by recrystallization to a radiochemical purity >98% as measured by H P L C radioactivity monitoring techniques (HPLC/RAM). The specific activity of the purified material was 11.7 mCi/mmole. Cow animal husbandry for radiolabeled studies. Twelve Holstein cows in mid-2nd or mid-3rd lactation were housed individually in stainless-steel metabolism stalls and maintained therein through 4 or 6 days post-last-treatment then allowed freedom of movement in an enclosed corral until sacrificed. The cage floor was fitted with a plastic-coated rubber mat to reduce the stress of standing for long periods of time. The cages were equipped with manual-fill feed bins and automatic-fill water troughs as well as a drainage system to collect urine and a rear access door to approach the animal for milking and feces collection. 14

Dose preparation and administration. A mixture of C-pirlimycin hydrochloride and non-labeled pirlimycin hydrochloride (99% chemical purity) was prepared to adjust the specific activity to ^ 10,000 dpm^g. The dose formulation was prepared to contain total pirlimycin free base equivalents at 20 mg per mL in an aqueous gel containing 2% by weight carboxymethylcellulose. Plastets, polyethylene dosing devices used for udder infusions, were filled with 10.1 mL of formulation. The contents of one Plastet was administered into each quarter the udder through the teat canal immediately after milk-out. All four quarters were treated to simulate the rare maximal use situation. A second dose was administered similarly 24 hours following the first dose. Collection of samples and total residue analysis. Blood (10-15 mL) was collected by jugular venipuncture into heparinized syringes at 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 24, 30, 36, 48, 60, 72 and 96 hours after the 1st dose. Sub-samples of 200-300 mg were immediately weighed in triplicate for radiolabel quantitation by combustion analysis. The remainder of the sample was centrifuged for the separation of plasma, which was

In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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:

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iso-Pirlimycin HCI

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Figure 2. Synthetic sequence for C-pirlimycin hydrochloride.

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then sub-sampled and stored at -20°C for subsequent analysis. The cows were milked by commercial single cow milking machines at regular 11-13 hour intervals to provide a composite milk sample (4 quarters combined) per cow per time interval. Milk was collected for analysis through 6 days post-last-treatment or until the animal was sacrificed (4 day animals). Urine was collected at 24- hour intervals through the drainage system of the metabolism cage into 5 gallon plastic containers. Each collection was weighed, homogenized and sub-sampled for analysis. Total feces was collected at 24-hour intervals into 5 gallon plastic containers. The net fecal weight was measured and an equal weight of water added to prepare a homogenate slurry for sub-sampling and analysis. Each animal was sacrificed by captive bolt after the appropriate withdrawal interval (4, 6, 14 and 28 days after the 2nd dose) and processed as in an abattoir. The entire liver, kidneys and udder were excised and 1-2 kg samples of muscle from both the flank and the udder diaphragm and 1-2 kg samples of fat from the abdominal area were collected. Each organ and tissue was minced and processed three times through a commercial meat grinder to prepare respective homogenate samples. Sub-samples (200-300 mg) were prepared in triplicate for total residue analysis. Total radioactivity concentrations, expressed as pirlimycin free base equivalents, were determined by direct liquid scintillation counting (liquids) or combustion analysis (solids) following standard techniques. Metabolite/residue analysis. Milk, urine and plasma samples were first analyzed by a microbiological cylinder/plate procedure against M.luteus which has a limit of detection of 0.02 ppm. A sub-sample of the milk was prepared for this assay by a centrifugation step followed by a pH adjustment to 8.5. In addition, an HPLC/RAM analysis was conducted after treating another sub-sample with FTSH (10% formic acid, 30% trifluoroacetic acid, 2% sodium chloride, 2N hydrochloric acid) followed by centrifugation to precipitate the proteins. The supernatant was basified and concentrated by C-18 solid phase extraction (SPE) techniques. The HPLC conditions were: Column - 20 cm χ 4.8 mm C-8; Mobile-phase -linear gradient at 5%/minute from 90:10 0.1M pH 7 phosphate buffenmethanol to 20:80; Detectors - UV operated at 214 nm and a radioactivity flow detector operated in the C DPM mode. 14

Tissues and feces were processed as follows: The extraction of >90% of the total C residue was accomplished for all samples by homogenizing 1 part sample with 2 parts FTSH, centrifugation, followed by a second extraction of the solids with 20% FTSH. The acid extracts were combined, basified to pH 8.5 ± 0.5 with cone, ammonium hydroxide and processed through C-18 solid phase extraction columns. Pirlimycin and the metabolites were eluted from the columns with methanol and 1% HC1 in methanol, respectively. These samples were evaporated to dryness and takenup in buffer for microbiological or HPLC/RAM analysis. 14

FAB/MS and NMR. FAB/MS spectra were obtained on a VG magnetic sector instrument. The samples were placed on the FAB target probe containing 2-hydroxyethyldisulfide as the matrix solvent. The target was bombarded with xenon at 8-9 KV and the data recorded with a UPACS II data system. Matrix ions were subtracted from the samples ions. All NMR experiments were performed at 25° on a Bruker

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AM-500 spectrometer operating at 500.13 MHz for proton magnetic resonance. Samples were prepared in d -DMSO and 1-D and 2-D spectra obtained. Several experiments were run for the 2-D spectra: COSY, Relay COSY, HOHAHA, and NOESY which were zero-filled in the F l dimension only. D 0 exchange spectra were also obtained. 6

2

Reference standards. Pirlimycin was obtained as an Upjohn Control Reference Standard of purity >99% as the hydrochloride hydrate. Pirlimycin sulfoxide was prepared from the treatment of pirlimycin with hydrogen peroxide followed by recrystallization. Samples of pirlimycin adenylate and pirlimycin sulfoxide adenylate were prepared by the procedures described by Argoudelis et al (6). Comparative metabolism in the rat. Adult male (6) and female (6) Sprague-Dawley rats were housed individually in polycarbonate metabolism cages and were orally administered by gavage an aqueous solution of C-pirlimycin HCI. Five daily doses of 29 mg/kg/day were administered at 24-hour intervals to each rat. Urine and feces were collected at 24-hour intervals just before dose administration. The animals were sacrificed at 2 to 3 hours post-last-dose and liver, kidneys, and samples of flank muscle and abdominal fat carefully excised and placed into tared bottles. Homogenates of 2:1 water.tissue were prepared for combustion/LSC analysis. Metabolite profiles were obtained for liver, urine and feces as described above. 14

Milk decline study at 50 mg/quarter (IX). Twenty-six lactating cows (Holstein) identified to be mastitic in one or more quarters were treated with two intramammary infusions of 50 mg/quarter of pirlimycin hydrochloride into all four quarters at a 24hour interval. All cows were milked at 11-13 hour intervals following standard dairy farm practices. Samples from the individual cows (composite of all 4 quarters) were taken for analysis out to 96 hours post-last-dose. The samples were analyzed by the M.luteus cylinder/plate microbiological assay. Results And Discussion i4

14

Radiolabeled C-pirlimycin was readily synthesized from C sodium cyanide and 4ethyl-pyridine N-oxide as shown in Figure 2. The final reduction step produced a 2:1 mixture of desired product and a biologically inactive stereoisomer. Recrystallization preferentially produced pirlimycin HCL in >98% radiochemical purity and >99% chemical purity with an overall radiochemical yield of 25%. 14

ADME studies. Twelve cows were administered two doses of C-pirlimycin at a dose rate of 200 mg/quarter into all 4 quarters at a 24-hour interval. This dose rate was selected as the highest potential dose rate before the final efficacious dose of 50 mg/quarter had been firmly established. This treatment rate thus resulted in a 4-fold overdose. Blood, milk, urine and feces were collected at various times following the first dose. Combustion analysis of whole blood produced the time course of total residue, as illustrated in Figure 3 for three of the cows. There was a slow absorption of pirlimycin across the udder membrane/blood barrier with maximum concentrations occurring in the 6- to 12-hour posttreatment period. The terminal depletion of the

In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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total residue following the second dose appeared to correspond to a two-compartment pharmacokinetic model and suggests a very slow overall elimination. Various pharmacokinetic parameters were derived following noncompartmental analysis (7) and are summarized in Table I. Subsequent analysis of plasma, which contained total residue at a concentration approximately equal to whole blood, showed that the plasma residue consisted almost exclusively of unchanged pirlimycin. Thus, the parameters in Table I are useful indicators of the overall pharmacokinetic behavior of pirlimycin in the dairy cow following intramammary administration.

Table I. Whole Blood Pharmacokinetics of Pirlimycin in the Dairy Cow by the Intramammary Route Parameter

Mean Value, η = 12

AUCo_ t abs

2.27 to 7.11 μg/hr/mL 2.89 ± .46 hours 12 hours 6-12 hours 0.083 ± .03 μ g / m L 0.131 ± .047 μ g / m L 0.0224 ± .009 hour 37.6 ± 17.4 hours

120

1/2

trnax-2

1

t

1/2

el

Typical depletion of total residue in milk, expressed as a concentration-time course, is illustrated in Figure 4 for three of the cows. Milk can be treated as an elimination pathway and estimates of pharmacokinetic parameters can be made by the Sigma minus technique (7). However, the true focus of milk residue concentration determinations as a function of time is the decline of these residues to levels below the "safe concentration." This will be addressed later when the 50 mg/quarter dose rate study is discussed. The important observation made clear by Figure 4 is the biphasic shape of the concentration-time course following the second dose. We interpret this to reflect an initial rapid udder emptying of unabsorbed pirlimycin during the first 2 to 3 milkings post-treatment since each milking is not 100% efficient in milk removal. The slow terminal depletion phase represents systemic elimination of absorbed drug as it is transported back across the udder membrane/blood barrier.

Tissue residues.

The concentrations of total residues resulting from the 200 mg/quarter/ dose study in the various tissues at various withdrawal times are presented in Table II. Muscle and fat contained little or no detectable residue beyond day 6. Liver is clearly the target tissue for residue analysis and showed a first order depletion (r = .995) with a t of 5.7 days. Kidney depleted at a faster rate, with a t of 3.3 days. Pirlimycin was not sequestered in the udder as demonstrated by the relatively low concentration of total residue detected in udder. 1/2

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XENOBIOTICS AND FOOD-PRODUCING ANIMALS

0.3-1

0.003 \

0

ι

1

1

1

1

\

1

1

12

24

30

48

00

72

84

96

Time, hours 14

Figure 3. Time-course of C-pirlimycin total residue in whole blood in 3 cows treated twice at a 24-hour interval by the intramammary infusion of Cpirlimycin hydrochloride into all 4 quarters at 200 mg/quarter. 14

0.014

1

1

1

1

1

1

0

24

48

72

96

120

144

f 168

Time, hours 14

Figure 4. Time-course of C-pirlimycin total residue in milk in 3 cows treated twice at a 24-hour interval by the intramammary infusion of C-pirlimycin hydrochloride into all 4 quarters at 200 mg/quarter. 14

In Xenobiotics and Food-Producing Animals; Hutson, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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Table Π. Total

1 4

Pirlimycin

139

in the Dairy Cow

C Pirlimycin Residues in Tissues - 4X I M M Dose Mean Concentration (n=3) in Parts Per Million 28 Day 14 Day 4 Day 6 Day

Tissue

Liver Kidney Muscle Fat Udder

9.18 1.96 0.10 0.22 0.97

± 1.37 ± 0.71 ± 0.04 ± 0.22 ± 0.62

7.13 0.78 0.05 0.03 0.13

± 1.28 ±0.17 ± 0.01 ± 0.01 (n=l)

0.50 ± .37 0.01 ± .01 (0) (0) (0)

3.57 ± .39 0.26 ± .05 0.02 ±