WORLD TRADE WT/DS320/R/Add.4 31 March 2008 ORGANIZATION (08-0902) Original: English UNITED STATES – CONTINUED SUSPENSION OF OBLIGATIONS IN THE EC – HORMONES DISPUTE Report of the Panel Addendum This addendum contains Annex D to the Report of the Panel to be found in document WT/DS320/R. The other annexes can be found in the following addenda: – Annex A: Add.1 – Annex B: Add.2 – Annex C: Add.3 – Annex E: Add.5 – Annex F: Add.6 – Annex G: Add.7 WT/DS320/R/Add.4 Page D-1 ANNEX D REPLIES OF THE SCIENTIFIC EXPERTS TO QUESTIONS POSED BY THE PANEL A. GENERAL DEFINITIONS 1. Please provide brief and basic definitions for the six hormones at issue (oestradiol-17β, progesterone, testosterone, trenbolone acetate, zeranol, and melengestrol acetate), indicating the source of the definition where applicable. Dr. Boisseau 1. Oestradiol-17β is the most active of the oestrogens hormone produced mainly by the developing follicle of the ovary in adult mammalian females but also by the adrenals and the testis. This 18-carbon steroid hormone is mainly administered as such or as benzoate ester alone (24 or 45 mg for cattle) or in combination (20 mg) with testosterone propionate (200 mg for heifers), progesterone (200 mg for heifers and steers) and trenbolone (200 mg and 40 mg oestradiol-17β for steers) by a subcutaneous implant to the base of the ear to improve body weight and feed conversion in cattle. The ear is discarded at slaughter. 2. Progesterone is a hormone produced primarily by the corpus luteum in the ovary of adult mammalian females. It is administered to cattle, steers, usually at 200 mg in combination with oestradiol-17β or oestradiol benzoate (usually 20 mg) by a subcutaneous implant to the base of the ear to improve body weight and feed conversion in cattle. The ear is discarded at slaughter. 3. Testosterone is a hormone produced primarily in the testes of adult mammalian males. This 19-carbon steroid has potent androgenic properties. It is administered as testosterone propionate (200 mg) in combination with oestradiol-17β or oestradiol benzoate (20mg) by a subcutaneous implant to the base of the ear to improve body weight and feed conversion in cattle. The ear is discarded at slaughter. 4. Melengestrol acetate is an orally active synthetic progestogen about 30 times as active as progesterone. It is used to improve body weight and feed conversion in female beef cattle. It is fed at daily doses of 0.25-0.50 mg per heifer usually 90-150 days prior to slaughter. 5. Trenbolone acetate is a synthetic steroid with anabolic properties several fold above that of testosterone. It is administered alone (300 mg for heifers) or in combination with oestradiol-17β (20 mg for calves and 40 mg for steers), by a subcutaneous implant to the base of the ear to improve body weight, feed conversion and nitrogen retention in cattle. It is administered to cattle 60-90 days or more before the intended date of slaughter. The ear is discarded at slaughter. 6. Zeranol, is a natural mycooestrogen derived from zearalenone produced by different species of fusarium molds. This non-steroidal anabolic agent is administered to cattle either alone (36 mg) or in combination with trenbolone acetate (140 mg) by subcutaneous implant to the base of the ear to improve body weight and feed conversion in cattle. WT/DS320/R/Add.4 Page D-2 Dr. Boobis1 7. Oestradiol-17β is the most potent mammalian oestrogenic hormone. It is produced in the ovary, placenta, testis, and possibly the adrenal cortex (ChemIDPlus Advanced, National Library of Medicine (http://chem.sis.nlm.nih.gov/chemidplus)) 8. Oestradiol-17β is the most potent form of mammalian oestrogenic steroids. In humans, it is produced primarily by the cyclic ovaries and the placenta. It is also produced by the adipose tissue of men and postmenopausal women (PubChem, National Library of Medicine (http://pubchem.ncbi.nlm.nih.gov/) 9. Progesterone is the principal progestational hormone of the body, secreted by the corpus luteum, adrenal cortex, and placenta. Its chief function is to prepare the uterus for the reception and development of the fertilized ovum. It acts as an antiovulatory agent when administered on days 5-25 of the menstrual cycle (ChemIDPlus Advanced). 10. Progesterone is the major progestational steroid that is secreted primarily by the corpus luteum and the placenta. Progesterone acts on the uterus, the mammary glands and the brain. It is required in embryo implantation, pregnancy maintenance, and the development of mammary tissue for milk production. Progesterone, converted from pregnenolone, also serves as an intermediate in the biosynthesis of gonadal steroid hormones and adrenal corticosteroids (PubChem). 11. Testosterone is a potent androgenic steroid and major product secreted by the Leydig cells of the testis. Its production is stimulated by luteinizing hormone from the pituitary. In turn, testosterone exerts feedback control of the pituitary LH and FSH secretion. Depending on the tissues, testosterone can be further converted to dihydrotestosterone or oestradiol (PubChem). 12. Trenbolone acetate is a synthetic steroid that has been used as an anabolic agent in veterinary practice. (Martindale: The Complete Drug Reference (2006), Pharmaceutical Press, London). 13. Zeranol is a naturally occurring metabolite of the mycotoxin zearlenone which is produced by a number of Fusarium fungal species. The commercial formulation contains specifically the α- isomer. Zeranol is a non-steroidal anabolic agent. (JECFA (1988a). Toxicological Evaluation of Certain Veterinary Drug Residues in Food: WHO Food Additives Series 23, WHO, Geneva, Switzerland). 14. Zeranol is a nonsteroidal oestrogen that has been used for the management of menopausal and menstrual disorders. It has also been used as a growth promoter in veterinary practice (Martindale: The Complete Drug Reference). 15. Melengestrol acetate (MGA) is an orally active 6-methyl progesterone acetate with reported glucocorticoid activity and effect on estrus (PubChem). 16. Melengestrol acetate is a progestogen that is used as an animal feed in beef heifers to improve feed efficiency, increase the rate of body-weight gain, and suppress oestrus (Martindale: The Complete Drug Reference). Dr. Guttenplan 17. Oestradiol-17β: an estrogenic sex hormone, which in the female, functions in the ovarian cycle and maintains uterine health. In males it inhibits the synthesis of testosterone. A member of a 1 A full list of references cited in responses from Dr. Boobis can be found in Attachment 1. WT/DS320/R/Add.4 Page D-3 class of compounds called steroids (which, chemically, have three 6-membered rings and one 5- membered ring). 18. Progesterone: a steroidal anti-estrogen; used as a contraceptive and to correct abnormalities in the menstrual cycle. 19. Testosterone: a steroidal androgenic sex hormone, which in the male leads the production of sperm components. It is also important in promoting the development of secondary sex characteristics. 20. Trenbolone acetate: a synthetic anabolic (growth-stimulating) hormone, often used in cattle. 21. Zeranol: a synthetic nonsteroidal growth promoter often used in cattle. 22. Melengestrol acetate: a synthetic steroidal growth promoter often used in cattle. Also used for estrus synchronization in cattle. (Opinion of SCVPH, 1999 (US Exhibit 4 part 1)) 2. Please provide definitions for the following terms as they relate to the hormones at issue, indicating the source of the definition where applicable: anabolic agents, steroids, steroidal oestrogens, parent compounds/metabolites, catechol metabolites, mitogenicity, mutagenicity, androgenic/oestrogenic activity, genotoxicity, genotoxic potential, carcinogenicity, and tumorigenicity. In your replies, please be sure to identify and describe any relevant differences between the terms. Dr. Boobis Anabolic agent 23. The building up in the body of complex chemical compounds from smaller simpler compounds (e.g., proteins from amino acids), usually with the use of energy. Cf.: catabolism, metabolism. Stedman's Medical Dictionary (2000), Lippincott Williams & Wilkins, Philadelphia, PA 24. Testosterone, or a steroid hormone resembling testosterone, which stimulates the growth or manufacturing of body tissues. Taber's Cyclopedic Medical Dictionary - 20th Ed (2005), F. A. Davis Company, Philadelphia, PA 25. Anabolism: The processes of metabolism that result in the synthesis of cellular components from precursors of low molecular weight. IUPAC(1997). Compendium of Chemical Terminology, 2nd Edition (http://www.iupac.org/publications/books/author/mcnaught.html) Steroids 26. A large family of chemical substances, comprising many hormones, body constituents, and drugs, each containing the tetracyclic cyclopenta[a]phenanthrene skeleton Stedman's Medical Dictionary. Steroidal oestrogens 27. (Steroidal) compounds that produce the behaviour estrus ("the portion or phase of the sexual cycle of female animals characterized by willingness to accept the male"). Hughes, C (1996). Are the WT/DS320/R/Add.4 Page D-4 differences between estradiol and other estrogens merely semantical? (Letter to the Editor). J Clin Endocrinol Metab 81:2405. 28. A more biochemical definition might be: compounds with a steroid structure that possess endocrine effects qualitatively similar to those of oestradiol-17β and that act through oestrogen receptors. Parent compounds/metabolites 29. When related to exogenous compounds, the parent is the compound to which an individual is exposed. The relationship between parent compound and metabolite