UNITED STATES – CONTINUED SUSPENSION OF OBLIGATIONS IN THE EC – HORMONES DISPUTE
Document: Report of the Panel Addendum
Date: 31 March 2008
Document codes: WT/DS320/R/Add.4; WT/DS320/R.
Complainant: EU
Respondent: U.S.
Third parties: Australia, Brazil, Canada, China, Taiwan, India, Mexico, New Zealand, Norway
Dispute stage: Appellate Body
Agreements cited: Art. I, II  GATT 1994Art. 3.7, 22, 23.1, 23.2, 21.5  Dispute Settlement Understanding (DSU)

UNITED STATES – CONTINUED SUSPENSION OF
OBLIGATIONS IN THE EC – HORMONES DISPUTE

Report of the Panel

Addendum

 This addendum contains Annex D to the Re port of the Panel to be found in document
 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

ANNEX D

REPLIES OF THE SCIENTIFIC EXPERTS
TO QUESTIONS POSED BY THE PANEL

A. G
ENERAL DEFINITIONS
1. Please provide brief and basic definitions for the six hormones at issue (oestradiol-17 β,
progesterone, testosterone, trenbolone acetat e, zeranol, and melengestrol acetate),
indicating the source of the definition where applicable.

Dr. Boisseau

1. Oestradiol-17β is the most active of the oestroge ns 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) a nd trenbolone (200 mg and 40 mg oestradiol-17 β 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-17 β or oestradiol benzoate (20mg) by a subcutaneous
implant to the base of the ear to improve body we ight 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 fo r heifers) or in combination with oestradiol-17 β
(20 mg for calves and 40 mg for steers), by a subcut aneous implant to the base of the ear to improve
body weight, feed conversion and nitr ogen 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 ze aralenone 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.

Dr. Boobis
1

7. Oestradiol-17β is the most potent mammalian oestroge nic 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 placen ta. 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 func tion 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 main tenance, 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 luteinizin g 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 comme rcial 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.

class of compounds called steroids (which, chemic ally, 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, whic h 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 te rms as they relate to the hormones at issue,
indicating the source of the definition where applicable: anabolic agents, steroids,
steroidal oestrogens, parent compounds/metabo lites, catechol metabolites, mitogenicity,
mutagenicity, androgenic/oestrogenic activi ty, 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 te stosterone, which stimulates the growth or
manufacturing of body tissues. Taber's Cyclopedic Medical Dicti onary - 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, 2
nd
Edition (http://www.iupac.org/publications/books/author/mcnaught.html)
Steroids

26. A large family of chemical substances, co mprising 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 behavi our estrus ("the portion or phase of the sexual
cycle of female animals characterized by willingness to accept the male"). Hughes, C (1996). Are the

differences between estradiol and other estrogens merel y 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-17 β 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 betwee n parent compound and metabolite is that the parent serves as a
substrate for biotransformation (enz ymatic conversion) to yield a produc t that is chemically distinct
from the parent, a metabolite (A. Boobis). With respect to metabolites of veterinary drugs, it is
possible that the residue in meat comprises, at least in part, one or more metabolites of the drug used
to treat the animals. Ingestion of such metabolites can lead to their metabolism in human subjects.
Hence, there will be a parent/metabolite relationship even for such compounds.
30. Metabolite
: Any intermediate or product resulti ng from metabolism. National Library of
Medicine (1993). Glossary for Chemists of Terms Used in Toxicology
(http://www.sis.nlm.nih.gov/enviro/glossarymain.html; Pure Appl Chem, 1993, 65, 2003-2122)
31. Metabolism: in a narrower sense, of drugs, one mechanism of clearance, is the irreversible
biochemical transformation of a compound to anothe r chemical (metabolite). The metabolite is
usually more polar (water-soluble) and, therefore, more readily excreted, than the parent compound;
thus, metabolism facilitates drug excretion. Absorption Systems (2006). Glossary Terms
(http://www.absorption.com/Site/Glossary/Default.aspx)
Catechol metabolites

32. Any intermediate or product resulting from metabolism (enzymatic transformation)
containing the core structure benzene-1,2-diol IUPAC (1993). A Guide to IUPAC Nomenclature of
Organic Compounds, Blackwell Science, Oxford, UK
Mitogenicity

33. The property of an agent whereby it induces mito sis and cell proliferation. Mitosis is the
process by which a cell nucleus divides into two daughter nuclei, each having the same genetic
complement as the parent cell: nuclear di vision is usually followed by cell division (NLM Glossary
for Chemists of Terms Used in Toxicology).
Mutagenicity

34. Ability of a physical, chemical, or biological ag ent to induce heritable changes (mutations) in
the genotype in a cell as a consequence of altera tions or loss of genes or chromosomes (or parts
thereof).
35. Mutation: Any relatively stable heritable change in genetic material that may be a chemical
transformation of an individual gene (gene or point mutation), altering its function, or a
rearrangement, gain or loss of part of a chromosome, that may be microscopically visible
(chromosomal mutation); mutation can be either germinal and inherited by subsequent generations, or
somatic and passed through cell lineage by cell division. NLM Glossary for Chemists of Terms Used
in Toxicology

Androgenic activity

36. Having the property to interact with androgen receptors in target tissues to bring about the
effects similar to those of testosterone. Depending on the target tissues, androgenic effects can be on
sexual differentiation; male reproductive orga ns, spermatogenesis; secondary male sex
characteristics; libido; development of muscle mass, strength, and power.
37. Capacity to promote the development and maintenance of male sex characteristics. National
Library of Medicine, Genetics Home Reference (http://ghr.nlm.nih.gov/ghr/glossary/Glossary)
Oestrogenic activity

38. Biological activity similar to that of an oestrogen.
39. Oestrogens cause the thickening of the lining of the uterus and vagina in the early phase of the
ovulatory, or menstrual, cycle; in lower animals cy clical oestrogen secretion also induces oestrus, or
"heat". The oestrogens are also responsible fo r female secondary sex characteristics such as, in
humans, pubic hair and breasts, and they affect ot her tissues including the genital organs, skin, hair,
blood vessels, bone, and pelvic muscles. The Columbia Electronic Encyclopedia (2003), Sixth
Edition, Columbia University Press, New York City, NY
40. Oestrogenic activity can arise through several possible mechanisms, by mimicking natural
oestrogens and interacting with oestrogen receptors, by affecting oestrogen-sensitive pathways by
some other mechanism and by altering the levels of endogenous oestrogens, be changing the rate of
synthesis or degradation. Lintelmann J, Katayama A, Kurihara N, Shore L, and Wenzel A (2003).
Endocrine disruptors in the environment (IUPAC Technical Report) Pure Appl Chem, 75, 631–681.
Miyamoto J and Burger J (Editors) (2003). Special Topic Issue on the Implications of Endocrine
Active Substances for Humans and Wildlife. Pure Appl Chem, 75: 1617-2615.
Genotoxicity

41. Ability to cause damage to genetic material . Such damage may be mutagenic and/or
carcinogenic. NLM Glossary for Chemists of Terms Used in Toxicology
42. Mutagenicity is a form of genotoxicity. However, not all genotoxicity is necessarily
mutagenicity. Examples include adduction to DNA and damage to DNA that does not lead to
heritable change. Whilst adduction can lead to mutation, the presence of adducts per se is a measure
of genotoxicity and not of mutagenicity.
Genotoxic potential

43. Of a compound, it possesses characteristics such that it might be capable of causing
genotoxicity (usually in vivo), based on considerations such as the results of tests in vitro. It remains
to be determined whether genotoxicity is indeed expressed in vivo, i.e. that the potential is realized (A.
Boobis interpretation of usage by JECFA and elsewhere).
Carcinogenicity

44. Process of induction of malignant neoplasms by chemical, physical or biological agents.
45. Malignant neoplasm: a population of cells show ing both uncontrolled growth and a tendency
to invade and destroy other tissues; a malignancy is life-threatening.

46. Neoplasm: new and abnormal formation of tissue as a tumour or growth by cell proliferation
that is faster than normal and continues after the initial stimulus that initiated the proliferation has
ceased. NLM Glossary for Chemists of Terms Used in Toxicology
Tumourigenicity

47. Process of inducing tumours, i.e. any abnormal swelling or growth of tissue, whether benign
or malignant. NLM Glossary for Chemists of Terms Used in Toxicology
48. Hence, whilst a carcinogen produces tumours (whi ch are malignant), tumourigenic agents do
no necessarily produce malignant neoplasia.
Dr. Guttenplan

49. Anabolic agents: agents promoting build-up - in animals, usually muscle mass, in
biochemicals, building larger molecules from smaller ones.
50. Steroids: Metabolites of cholesterol, containing three 6-membered rings and one 5-membered
ring.
51. Steroidal oestrogens: Estrogens that contain the steroidal ring system.
52. Parent compounds/metabolites : in a chemical conversion, the initial chemical is called the
parent compound and the product, the metabolite.
53. Catechol metabolites: Catechols are compounds containing a benzene ring with two hydroxyl
groups on the benzene ring. When they are conve rted to a different compound, a catechol metabolite
results.
54. Mitogenicity: Relating to or causing cell division.
55. Mutagenicity: Relating to or causing a change in D NA composition. May also relate to a
change in protein structure
56. Androgenic activity: acting like a male sex hormone.
57. Oestrogenic: acting like a female sex hormone.
58. Genotoxicity: Relating to or causing damage to DNA.
59. Genotoxic potential: The possible ability of an agent to cause damage to DNA.
60. Carcinogenicity: Relating to or causing a process leading to cancer.
61. Tumorigenicity: Relating to or causing the formation of tumors. This term refers to tumor
formation, whereas carcinogenicity may also refe r to the process by which tumors are induced.
(Codex Microbiological RA).
B. RISK ASSESSMENT TECHNIQUES
3. Please identify any international guidance documents relevant to the conduct of a risk
assessment with respect to veterinary drug residues. Since when have they been
available? Please also indicate if there is any relevant ongoing work at Codex.

Dr. Boisseau

62. To my knowledge, there is no international gui dance document relevant to the conduct of a
risk assessment with respect to veterinary drug residues. Currently, there is no Codex guidance
document relevant to the conduct of a risk assessment with respect to veterinary drug residues. The
situation is similar in the European Union. Th e CVMP has assessed all the pharmacologically active
substances used in veterinary medecine without any written guideline about risk assessment.
63. I have proposed some 15 years ago to CCRVDF (Codex Committee on Residues of
Veterinary Drugs in Food) to de velop and adopt a guidance about risk management including a risk
assessment policy. In its last session held in Ma y 2006 in Cancun, Mexico, CCRVDF has decided to
propose to the Codex Committee on General principles (CCGP) and to the Codex Commission a draft
project concerning a rationale about the risk anal ysis to be implemented by CCRVDF. This draft
project includes two parts: (1) a procedure with the interactions between CCRVDF, responsible for
risk management, and JECFA (Joint Expert Co mmittee on Food Additives) responsible for risk
assessment, with, in annex, the format to be used by member states for establishing a risk profile; (2)
the principles of a risk assessment policy.
Dr. Boobis

International guidance documents

64. The following guidance documents relevant to the conduct of a risk assessment with respect
to veterinary drug residues are available:
 WHO (2001): Residues of veterinary drugs in food (current version Jan 2001).

 WHO procedural guidelines for the Joint FAO/WHO Expert Committee on Food Additives

 WHO (1996): Residues of veterinary drugs in food (current version August 1996) Guidelines
for the preparation of toxicological work ing papers for the Joint FAO/WHO Expert
Committee on Food Additives

 Residues of veterinary drugs in food (Sept 2002)

 FAO (2002a) procedural guidelines for the Joint FAO/WHO Expert Committee on Food
Additives

 Procedures for Recommending Maximum Residue Limits – Residues of Veterinary Drugs in
Food (1987-1999) (FAO, 2000a)

 Envionmental Health Criteria (EHC) 70: Principles For The Safety Assessment Of Food
Additives And Contaminants In Food (IPCS, 1987)

 Environmental Health Criteria (EHC) 104: Pr inciples For The Toxicological Assessment of
Pesticide Residues In Food (IPCS, 1990)

65. Also available are relevant sections from Ge neral Consideration Items in JECFA reports,
which document guidance developed by JECFA over the years and are provided as an ongoing update
to its risk assessment procedures (relevant volumes of WHO Technical Report Series).

66. Codex publishes a Procedural Manual that contains generic guidance on risk analysis and risk
assessment policies. This is updated regularly, the latest version (15th) having been published
in 2005: Codex Alimentarius Commission (CAC) (2005). Procedural Manual, Fifteenth edition,
WHO and FAO, Rome, Italy (ftp://ftp.fao.org/codex/Publications/ProcManuals/Manual_15e.pdf).
67. Codex is currently developing a risk assess ment policy for recommending maximum residue
limits for veterinary drugs in food. To my knowledge this is still in the dr afting stage (see JECFA,
2006a).
 CCRVDF (2005). Risk Management Methodologies, Including Risk Assessment Policies in
the Codex Committees on Residues Of Veterinary Drugs in Foods
(ftp://ftp.fao.org/codex/Ccrvdf16/rv16_10e.pdf).

 JECFA (2006a). Summary and Conclusions of Si xty-sixth meeting (Residues of veterinary
drugs), Rome, 22-28 February 2006
 (http://www.who.int/ipcs/food/jecfa/summaries/summary66.pdf)

4. The European Communities states that there is "no Codex standard specifically on the
risk assessment of effects of residues of veterinary drugs" but a general one on
microbiological assessment. Is this correct? Which guidelines or principles have been
used by JECFA in the conduct of its risk as sessments with respect to the hormones at
issue? [see para. 192 of EC Rebuttal Submission (US case)].

Dr. Boisseau

68. The European Communities is right when it says that "there is no Codex standard specifically
on the risk assessment of effect of residues of veteri nary drugs". In the conduct of its risk assessment
with respect to the hormones at i ssue, as for all the other pharmacol ogically active substances used in
veterinary medecine, JECFA has followed the genera l rationale used by all the countries which have
assessed the safety of veterinary drug residues. Th is rationale has been internationally harmonised
through scientific conferences and it is possible to say that there was an international non written
agreement on this rationale. Nevertheless, the International Programme on Chemical Safety (IPCS)
has sponsored in the 1980s the preparation and the publication of the Environmental Health Criteria
(EHC) monograph No 70 entitled " Principles for the safety assessment of food additives and
contaminants in foods". Then, JECFA has, in its meetings, regularly developped and consolidated the
principles of this monograph EHC No 70 but it has ne ver published the outcome of this work in any
official document or monograph on risk assessment of veterinary drug residues, the only exception
being for microbiologicals.
Dr. Boobis

Codex standards for risk assessment

69. It is not clear what is meant by the EC asserti on that there is "no Codex standard specifically
on the effects of residues of veterinary drugs", but a general one on microbiological assessment. It is
certainly true that there is no detailed guidance manual from Codex on the assessment of the effects of
residues of veterinary drugs. However, there are guiding principles in place, that have been in
existence since before 1999. These relate to the pr ocedures for risk assessment, the implications and
meaning of an ADI (Acceptable Daily Intake) and procedures for setting MRLs. As indicated above,
JECFA was guided by a number of relevant documents in its risk assessment procedures. JECFA
developed an approach to the risk assessment of residues of antimicrobials, which was novel and not
covered in detail in such guidance. Specific guidance was therefore developed by JECFA and
adopted by Codex. In contrast, the approach es used in the assessment of the hormones followed

established risk assessment principles for toxicolo gically (as opposed to microbiologically) active
compounds.
Dr. Guttenplan

70. It is correct that there is "no Codex standard specifically on the risk assessment of effects of
residues of veterinary drugs".
71. A monograph "TOXICOLOGICAL EVALUATI ON OF CERTAIN VETERINARY DRUG
RESIDUES IN FOOD" WHO Food Additives Series: 43, Prepared by the Fifty-second meeting of
the Joint FAO/WHO Expert Committee on Food A dditives (JECFA) describes the data used to
determine the ADI for estradiol, progesterone and testosterone. The principles of risk assessment
(described below) were used in determining ADI's for estradiol, progesterone, and testosterone.
5. Please briefly describe the three components of a risk analysis exercise (risk assessment,
risk management and risk communication) and explain how they differ.

Dr. Boisseau

72. The following brief description of the three components of a risk assessment exercise is
given with respect to veterinary drugs residues likely to be present in food of animal origin.
73. Risk assessment is a procedure run by persons having the relevant scientific and technical
expertise. It is intended to determine the likel yhood and the gravity of any unexpected unwanted
effect for the consumer which may result from the i ngestion of veterinary drugs residues likely to be
present in food of animal origin. Only scientific da ta, relevant with regard to assessing this risk, have
to be taken into consideration in this procedure. In the Codex procedure, JECFA is responsible for
conducting the risk assessment for veterinary drug residues.
74. Risk management is a procedure run by persons having political or administrative
responsabilities. It is intended to protect consum ers from any problem of public health associated
with veterinary drugs residues likely to be present in food of animal origin. Other criteria than
scientific ones, such as economical, sociological, cultura l etc., can be taken into consideration in this
procedure. Usually, this procedure leads to regulatory and/or administrative decisions.
 The risk management procedure usually implies four steps:

 (1) Risk evaluation

 – identification of a food safety problem
 – establishment of a risk profile
 – ranking of the hazard for risk assess ment and risk management priorities
 – establishment of a risk assessment policy
 – commissioning of risk assessment
 – consideration of the results of the risk assessment

 (2) Assessment of the different possible risk management options

 – identification of the different possible risk management options
 – selection of the preferred risk management option
 – final risk management decision

 (3) Implementation of the risk management decision

 (4) Monitoring and review

 – assessment of the effectiveness of the measures taken
 – review of risk management and /or risk assessment as necessary

75. The risk management procedure has been considered by a joint FAO/WHO expert
consultation in 1997.
76. In the Codex procedure, Codex is responsib le for conducting the risk management for
veterinary drug residues.
77. Even if communication between persons respons ible for the risk assessment and the risk
management is desirable and useful, scientific pers ons running the risk assessment procedure must be
in the position to perform their work without an y influence from the persons having political or
administrative responsabilities. In order to guara ntee their independence, these scientific persons,
very often, carry out their work within independent agencies, at national or regional level. JECFA is
an expert committee independent from the Codex. It carries out, among others, the risk assessment
for veterinary drug residues on the request of Code x. Codex, including the CCRVDF and the Codex
Commission, is, together with the member states, involved in the risk management. Risk assessors
have to publish the conclusions of the risk assess ment they have performed. JECFA does through
monographs on toxicology and residues published r espectively by WHO and FAO. Risk assessors
may, in their conclusions, address some recommanda tions to the persons/bodies responsible for the
risk management but they have not the power to take any regulatory or administrative decision.
78. Risk communication
 is an interactive process of excha nge of informations and opinions on
the potential risks associated with veterinary drug residues likely to be present in food of animal
origin, among
 (1) Risk assessors

 (2) Risk managers

 (3) Other interested parties such as

 – consumers
 – veterinarians
 – technicians in animal husbandry
 – animal owners
 – animal health industry
 – food processing industry

 Risk communication should, among others,

 – promote awareness and unders tanding of the specific issues under
consideration during the risk analysis

 – promote consistency and transparen cy in formulating risk management
options/recommendations

 – provide a sound basis for understanding the risk management decisions
proposed

 – improve the overall effectiveness and efficiency of the risk analysis

79. Scientific persons in charge of the risk assessment procedure ar e responsible of the
communication on the issues associated with the ri sk assessment and the persons having political or
administrative responsabilities are responsible of th e communication on the issues associated with the
risk management.
80. The risk communication has been considered by a joint FAO/WHO expert consultation.
Dr. Boobis

Components of risk analysis

81. The three components of a risk analysis exercise can be described as follows:
Risk Assessment

82. A process intended to calculate or estimate the risk to a given target organism, system, or
(sub)population, including the identification of attendant uncertainties, following exposure to a
particular agent, taking into account the inherent ch aracteristics of the agent of concern as well as the
characteristics of the specific target system.
 From IPCS (2004). Risk Assessment Terminology, WHO, Geneva
(http://www.iseaweb.org/ipcsterminologyparts1and2.pdf)

Risk Management

83. The process, distinct from risk assessment, of weighing policy alternatives, in consultation
with all interested parties, considering risk assess ment and other factors relevant for the health
protection of consumers and for the promotion of fair trade practices, and, if needed, selecting
appropriate prevention and control options.
84. Risk management comprises three elements: risk evaluation; emission and exposure control;
and risk monitoring.
 From Codex Alimentarius Commission(2005). 15th Procedural Manual

Risk Communication

85. The interactive exchange of information and op inions throughout the risk analysis process
concerning risk, risk-related factors and risk perceptions, among risk assessors, risk managers,
consumers, industry, the academic community and ot her interested parties, including the explanation
of risk assessment findings and the basis of risk management decisions.
 From Codex Alimentarius Commission (2005). 15th Procedural Manual

86. Risk assessment
 is a scientific process in which the data are evaluated and on this basis,
together with weight of evidence and expert judgmen t a conclusion is reached as to the nature of the
hazards, the potential risk to exposed individuals and the extent to which exposure (measured or
estimated) approaches those levels considered to be without appreciable risk. The output of the risk
assessment is a health based guidance value, the allowable daily intake (ADI), in the case of a
veterinary drug residue in food. An important aspect of risk assessment is to identify and describe the
uncertainties associated with the ev aluation. The MRL is an exposure level that it compatible with

both health protection and good veterinary pr actice. The ADI does not determine the MRL
recommended by JECFA for consideration by Codex. However, in the risk characterization stage of
risk assessment, comparison of ex posure based on GVP (good veterinary practice) resulting in levels
at the MRL, with the ADI establish es whether the exposure is adequately protective. If not, the risk
assessment may be refined, or the conclusion may be that it is not possible to establish an MRL such
that exposure would be consistent with public health protection.
87. Risk management
 is not a scientific process but a procedure whereby policies are established
with respect to the use and acceptability of, in this case, a veterinary drug, compatible with protection
of the public, good veterinary practice and efficacy and ensuring fair trade. Hence, the output of a
risk assessment is one input to risk management d ecision-making. However, it is not the only one, as
indicated above. Issues such as the veterinary need for the product and the security of the food supply
may also be considerations. Normally, the risk manager accepts the output of the risk assessor, as this
is the conclusion of the scientific e xperts in the field. There should be a clear separation between risk
assessment and risk management. That is not to say that there should be no communication, but that
the conclusions of the risk assessors should be their own, uninfluenced by any policy needs of the risk
manager. Similarly, the risk manager should accept the conclusions of the risk assessor, unless there
is a transparent reason to challenge them. If the ri sk manager chooses a course of action that is more,
or less, precautionary than that justified on the basi s of the risk assessment, the reasons for this should
be clear and distinct from the risk assessment.
Dr. Cogliano

88. Risk assessment
 is the use of scientific data to desc ribe the adverse effects of exposure to
hazardous agents. Risk communication is the art of explaining these risks to different audiences.
Risk management is the process of considering a risk along with other factors (for example, legal
mandates, technical feasibility, cost, equity, and social norms) and making a decision about whether
and how to mitigate the risk. The three are separate activities carried out for separate purposes.
Dr. Guttenplan

89. Risk Assessment is defined as the scientifically based process consisting of the following
steps: (i) hazard identification, (ii) hazard charact erization, (iii) exposure assessment, and (iv) risk
characterization. It basically attempts to evaluate risk.
90. Risk Management is defined as the process of weighing policy alternatives in the light of the
results of risk assessment and, if required, selec ting and implementing appropriate control options,
including regulatory measures. It basically refers to dealing with the hazard, generally to reduce risk.
91. Risk Communication is defined as the interactive exchange of information and opinions
concerning risk and risk management among risk assessors, risk managers, consumers and other
interested parties. It is basically concerned with making known the risks to interested and/or affected
parties. (Codex Microbiological RA).
6. Please briefly describe the four steps of a risk assessment (hazard identification, hazard
characterization, exposure assessment and risk characterization) as identified by Codex,
indicating any relevant sources.

Dr. Boisseau

92. The brief description of the four steps of a risk assessment procedure is given with respect to
veterinary drugs residues likely to be present in food of animal origin. These four steps are:

(1) hazard identification, (2) hazard character ization, (3) exposure assessment, (4) risk
characterization
93. The goal of the hazard identification is (1) to identify all the residues of the veterinary drugs
under review likely to pose problems of health to consumers. The residues of concern for this
substance imply both the parent compound and a ll the pharmacologically active metabolites derived
from this parent compound; (2) to determine the c oncentrations of all these residues in the different
edible tissues and products derived from animals trea ted by this veterinary drug; (3) to determine the
evolution over the time of the concentrations of all these residues in the different edible tissues and
products after animals have been treated by this vete rinary drug; (4) to identify the marker residue to
be used for the monitoring of residues in order to be sure that the animal derived food intended to the
human consumption does not contain concentrations of residues exceeding th e MRLs established for
this veterinary drug.
94. The goal of the hazard characterization is to assess qualitatively and quantitatively all the
adverse effects associated with the residues of ve terinary drugs which may negatively impact the
health of consumers or the environment. An importa nt component of this step is to ascertain wether
or not it is possible to establish a dose-effect re lationship and a threshold which is the quantity of
residues under which no adverse effect towards th e health of consumers can be expected. The
outcome of this step is, when possible, to est ablish a NOAEL (No Observed Adverse Effect Level)
from the scientific data base available and to deri ve an ADI (Acceptable Daily Intake) from this
NOAEL using an appropriate safety factor, the value of which depends on the toxicological profile of
the residues. The NOAEL is the highest quantity of the veterinary drug at issue which is not
associated with any adverse effect in toxicity tests carried out in animals or in studies carried out in
humans.The ADI represents the maximum amount of residues of concern for the veterinary drug
under review which can be daily ingested by consum ers over a life time without any risk for their
health. NOAELs and ADIs are expressed in mg or μg/kg/day.
95. The goal of the exposure assessment is to assess quantitatively the exposure of consumers to
the residues of the veterinary drugs under review through the consumption of food of animal origin.
This exposure os determine through a standard food basket determined by JECFA which encompasses
mainly 500g musle, 100g liver, 50g kidney, 50g fat, 1,5l milk and 100g eggs.
96. In general terms, the goal of the risk characterization is to assess qualitatively and
quantitatively the likelihood and the gravity of a gi ven hazard for a human population exposed to this
hazard. This assessment is based on the conclusions of the three former steps of hazard identification,
hazard characterisation and exposure assessment. In the specific case of veterinary drug residues, this
step does not exist as the goal of the risk anal ysis for these compounds is not to assess qualitatively
and quantitatively the likelihood and the gravity of th e adverse effects for the health of consumers
associated with the veterinary drug residues they are exposed to through the animal derived food but
to protect consumers' health from any adverse effect associated with these residues. In order to do so,
MRLs are established , when possible. MRLs repres ent the highest concentrations of the residues of
concern which can be accepted for the different ed ible tissues and products derived from the animals
treated by the veterinary drug under review so that the quantity of residues daily ingested by
consumers does not exceed the established ADI. This establishment of MRLs, aimed at providing an
efficient protection of consumer health, is, theref ore, a component of the risk management. Thus,
when JECFA proposes MRLs to CCRVDF, it is also involved in a risk management component of the
risk analysis procedure but, as these MRLs have to be adopted by Codex (CCRVDF and
Commission), Codex is really, together with the member states, the body responsible for the risk
management.

Dr. Boobis

Four steps of risk assessment

97. The four stages of risk assessment are as follows (IPCS (2004). Risk Assessment
Terminology):
Hazard Identification

98. The identification of the type and nature of adverse effects that an agent has an
inherent capacity to cause in an organism, system, or (sub)population. This has been described as the
intrinsic toxicological properties of the compound.
Hazard Characterisation

99. The qualitative and, wherever po ssible, quantitative description of the inherent property of an
agent or situation having the potential to cause adverse effects. This should, where possible, include a
dose–response assessment and its attendant uncertainties. It also entails determining whether or not
there is a threshold for the toxicological effect, i.e. a dose below which no effect occurs.
Exposure Assessment

100. The qualitative and/or quantitative evaluation of the likely intake of biological, chemical, and
physical agents via food as well as exposures from other sources if relevant ( Codex Alimentarius
Commission (2005).
Risk Characterisation

101. The qualitative and, wherever possible, quan titative determination, including attendant
uncertainties, of the probability of occurrence of know n and potential adverse effects of an agent in a
given organism, system, or (sub)population, under defined exposure conditions.
Dr. Guttenplan

102. Hazard identification is defined as the identification of biological, chemical, and physical
agents capable of causing adverse health effects and which may be present in a particular food or
group of foods. It is concerned with recognizi ng potential harmful agents. Hazard characterization is
defined as the qualitative and/or quantitative evaluati on of the nature of the adverse health effects
associated with the hazard. Exposure assessment is defined as the qualitative and/or quantitative
evaluation of the likely intake of biological, chemical, and physical agents via food as well as
exposures from other sources if relevant. It basically attempts to estimate the quantity of the agent to
which individuals or populations ar e exposed. Risk characterization
 is defined as the process of
determining the qualitative and/or quantitative estimation, including attendant uncertainties, of the
probability of occurrence and severity of known or potential adverse health effects in a given
population based on hazard identification, hazard characterization and exposure assessment. For
instance the risk of lung cancer in smokers is 1 in 10. (Codex Microbiological RA).
7. Please comment on the EC statement made in para. 140 of the EC Replies to Panel
Questions that "which ever approach of a risk assessment is followed, they are all based
on a deterministic approach to risk characterization [and that they] have serious
limitations in non-linear situations, such as in the current case regarding hormones".
Are these situations, in your view, addressed by the risk assessment guidance currently
available from the Codex Alimentarius Commis sion? Have they been addressed in the

1988 and 1999 JECFA risk assessments of thes e hormones? [see Cana da's comments in

Dr. Boisseau

103. The EC statement in para. 140 of the EC Repli es to Panel questions indicating that "which
ever approach of a risk assessment is followed, they are all based on a deterministic approach to risk
characterization (the level of exposure amounts propor tionally to the level of risk for a given hazard)
(and therefore they) have serious limitations in non linear situations, such as in the current case
regarding hormones" refers to th e genotoxic effect of oestradiol-17 β and expresses the view that, for
this hormone, a threshold should not be set. This situation is addressed by the risk assessment
guidance currently under discussion within the CCRVDF. In 1987 and 1999, at the time of the
assessment of oestradiol-17 β, there was no risk assessment guidance available on this issue.
Nevertheless, JECFA was perfectly aware about this kind of non lin ear situations. Thus, in 1987 and
1989, although the relevant data bases were not complete, JECFA considered that, for compounds
such as Chloramphenicol, associat ed with aplasic anemia, and genotoxic nitroimidazole compounds
such as Dimetridazole and Ronidazole, it was not po ssible to establish an effect/dose relation, and
decided to base its conclusions on a qualitative ri sk assessment and did not recommend any ADI for
these compounds.
104. In its 32nd session held in 1987, JECFA did not address this kind of non linear situation for
oestradiol-17β because it concluded that the tumorigeni c effect associated with this compound was
related to its hormonal activity and that it was therefore possible to consider a threshold in this case.
105. If, in 1999, the 52nd JECFA recognized that oestradiol-17 β "has a genotoxic potential", it
concluded nevertheless that "the carcinogenicity of oestradiol-17 β was probably a result of its
interaction with hormonal receptors". Therefore, it did not take into consideration a non linear
situation in its risk assessment and decided to confirm its conclusions made in 1987 and to establish
an ADI of 0-0.05 μg/kg of body weight.
Dr. Boobis

Deterministic risk assessment

106. This question presupposes a specific outcome of the risk assessment, that there is no threshold
for the toxicological effects of the hormones. The JECFA risk assessment concluded that the dose-
response relationship for all of the endpoints was non-linear and that there was a threshold dose below
which there was no appreciable risk over a lifetime of exposure. Hence, a deterministic approach, via
the establishment of ADIs, was appropriate accordi ng to the procedures followed by the Committee.
Should the Committee have concluded that the dose -response relationship was linear and that there
was no dose below which there was no appreciable ri sk, there would have been two options. These
would have been to have declined to establish an ADI on the basis that no exposure would be
acceptable. The second would have been to establis h a margin of exposure below which exposures
would have been judged to pose a minimal (though no n-zero) risk. Such an approach has recently
been formalised (IPCS (2005). Draft ECH, Principles fo r Modelling Dose-Response for the Risk
Assessment of Chemicals, WHO, Geneva, Switzerland ) and was utilized by JECFA for its evaluation
of certain contaminants in 2005 (JECFA (2006b). Evaluation of C ertain Food Contaminants, WHO
Technical Report Series 930, WHO, Geneva). In practice, it is likely that as veterinary drug residues
in food are avoidable by not using the drug, the Committee would have declined to establish an ADI.
8. Please describe the procedure followed by JECFA in the identification of ADIs and the
development of recommendations on MRLs. Please identify and describe any steps that

are taken in the risk assessment process to build a margin of safety into the final
recommendation.

Dr. Boisseau

107. The procedure followed by JECFA for estab lishing ADIs and recommending MRLs includes
three steps.
108. Establishment of a no observed adverse effect level (NOAEL) . This NOAEL is established
after JECFA has considered the data obtained from a ll the available in vivo toxicity studies run in
laboratory animals and from all the epidemiological studies and observations carried out in humans.
JECFA considers also all the available in vitro tests, such as batteries of mutagenicity tests, which are
likely to make easier the understanding of the mechanis m of action of the toxicological effects of the
veterinary drug under review. For each of these stud ies, except for in vitro tests, JECFA establishes a
NOAEL which is the highest dose of the veterinary dr ug under review which is not associated with an
observed adverse effect in humans or in animals. When the review of all these studies is completed,
JECFA adopts, among the different NOAELs establis hed for these studies, the final NOAEL which,
once combined with an appropriate safety factor, will lead to the most conservative, the lowest, ADI.
109. Establishment of an ADI (Acceptable Daily Intake) in humans. ADI is the highest quantity of
residues of the veterinary drug under review wh ich can be daily ingested over a life time by
consumers through animal derived food which will not pose a problem of health. JECFA derives such
an ADI from the established NOAEL by using a safety factor. The value of this safety factor depends
on the nature of the toxic effect associated w ith the NOAEL finally adopted by JECFA. If the
NOAEL is derived from an in vivo toxicological study run in a laboratory animal, the value of such a
safety factor is usually 100 as it associates two 10 safety factors. The first 10 safety factor is for the
extrapolation from this laboratory animal to huma n as it is assumed, for caution reason, that humans
may be 10 times more sensitive to this toxic effect than the laboratory animal involved in the study.
The second 10 safety factor is for taking into consideration the diversity of humans, resulting from the
sex, age, race, which can lead to a different sensitivity with regard to this toxic effect. If the toxic
effect associated with the NOAEL finally adopted by JECFA is considered as being serious, as long
as it is nevertheless still possible to consider that this toxic effect is compatible with a linear situation
and the establishment of a threshold, the value allocat ed to the safety factor can be higher, up to 1000.
On the contrary, if the adverse ef fect, associated with the NOAEL fi nally adopted by JECFA, is only
derived from observations made in humans, the value of this safety factor, for exemple in the case of a
reversible physiological effect, can be 10. In conclusion, the value of an ADI is usually 100 times less
than the value of the corresponding NOAEL but may be also much lower.
110. Proposal of MRLs (Maximum Residue Limits). As an ADI is the final end point of the risk
assessment procedure, there is a need for an operati onal tool which offers a practical way to be sure
that this ADI will not be exceeded. That is the reason for which MRLs, already defined in my reply
to the question No 6, are established so that it is possible for analytical laboratories to check that
animal derived food do not contain residues of the veterinary drug under review in such amounts that
the established ADI would be exceeded. In order to establish these MRLs for all the different edible
tissues and products derived from the animals trea ted by the veterinary drug under review, JECFA
uses a very conservative estimation of the human consumption of these tissues and products which
represent an important additional safety factor. This food basket has been al ready described in my
reply to the question No 6. Thus, MRLs are estab lished in such a way that the quantities of residues
potentially daily ingested resulting from this theoriti cal consumption of animal derived food do not
exceed the value of the corresponding ADI. In additi on, when it is not possible, for a veterinary drug
under review, to identify and quantify all the residues associated with the toxi c effect of concern,
JECFA uses an additional safety factor in considering that all the residues derived from this veterinary
drug have the same potential toxicity. On the othe r hand, all the residues which have not been proven

as being non bioavailable after oral ingestion are consid ered among the residues of concern. It is of
special importance for the three natural hormones which are poorly bioavailable through oral route.
111. In conclusion, in order to build a margin of safety into the final recommendations, JECFA
includes at different steps of its risk assessment the following different safety factors:
 (1) establishment of ADI: humans are 10 times more sensitive than the animals involved
in the most sensitive toxicity test; some humans may be 10 times more sensitive than
others with regard to this toxic effect; th e value of the safety factor can be increased
in case of some serious adverse effects,

 (2) exposure assessment: the human consumpti on of animal derived food is definitively
overestimated,

 (3) MRLs establishment: all residues, which are not clearly demonstrated that they are
not associated with the toxic effect on which the ADI is based, are considered as
being as toxic as the metabolite responsible for this toxic effect. All residues, which
are not clearly demonstrated as being not bioavailable via oral route, are also included
in the daily intake of residues of concern.

Dr. Boobis

JECFA procedure for establishing ADIs and MRLs

112. The procedure adopted by JECFA to establish ADIs is as outlined in the guidance on risk
assessment principles ( Codex Alimentarius Commission (2005) ; see also my reply to question 3
above). Specifically, the hazard identification involved a systematic examination of the studies in
experimental animals, together with studies in humans, where available and in vitro studies as
appropriate. The extent of these varied with th e hormone, being much greater with the natural
hormones than with the synthetic ones. Human studies comprised epidemiological investigations,
clinical trials and experimental studies. This evaluation enabled the range of effects of the
compounds to be identified. In the hazard charact erization stage, the mode of action and the dose-
response curve for the toxicological endpoints were determined, to the extent possible. Understanding
the mode of action helped inform the interpretation of the dose response relationship. Hence, once the
Committee had concluded on the weight of evidence that the carcinogenic effects observed were most
likely due to an endocrine mode of action, the identification of a threshold in the dose-response
relationship was consistent with this. The do se at which no effect could be observed for each
endpoint was determined (NOAEL) by inspection of the data, and failing that the dose producing the
lowest observable adverse effect was identified (LOAEL). These data were used as the starting points
(points of departure)for the derivation of the ADIs. To allow for human interindividual variability
due either to differences in sensitivity (dynamics) or kinetics, a 10-fold factor was applied. When
extrapolating from studies in experimental animals an additional 10-fold factor was applied to allow
for possible inter-species differen ces in dynamics and kinetics. If a LOAEL was used an additional
factor of up to 10 was used, depending on dose sp acing, the shape of the dose-response curve above
the LOAEL, and the magnitude of the response. Finally, where there was an identifiable sub-group
who might reasonably be expected to be more sens itive than the group in whom data were obtained,
for example children relative to ad ults, an extra factor was applied. Exposure assessment was based
on determining residues in edible tissues after controlled trials in cattle. Using radiolabel, unless there
was evidence to the contrary, all radioactive material was assumed to be parent and biologically active
(e.g. for MGA (see JECFA (2000a). Residues of Some Veterinary Drugs in Foods And Animals,
FAO Food and Nutrition Paper 41/13, FAO, Rome) . This is a cautious assumption, as often some or
even most of the radiolabel is in the form of biologically less active or inactive metabolites (see
JECFA (2004). Residues of Some Veterinary Drugs in Foods And Animals, FAO Food and Nutrition

Paper 41/16, FAO, Rome). Standard food consumption figures were used for different segments of
the population, which again were re lative conservative. Using these data the predicted exposure of
high consumers was obtained, i.e. theoretical maximum daily intakes (TMDIs). In risk
characterization, comparison of the estimated exposure (TMDI) with the ADI showed whether
lifetime exposure at the levels predicted would be e xpected to be associated with any appreciable risk
of adverse effects. This was undertaken for different age groups within the population. For all of the
hormones under consideration, the estimated daily intake was well below the ADI, and hence use
according to GVP would be without appreciable risk. Steps where a margin of safety is built in to the
procedure are indicated above. However, to emphasize a few of these: risk assessment is based on the
most sensitive endpoint, it assumes high level consumption over a lifetime, it often assumes that all of
the residue is as active as the parent, default safety factors are used which are generally conservative.
9. Please confirm or comment on the following Canadian statement: "it is recognized that
JECFA only allocates an ADI for a food addi tive or veterinary drug under review when
JECFA considers that its scientific data base is complete and that there are no
outstanding scientific issues". [see para. 68 of Canada Rebuttal Submission]

Dr. Boisseau

113. The Canadian statement stipulating that "it is recognized that JECFA only allocates an ADI
for a food additive or a veterinary drug under review when JECFA considers that its scientific data
base is complete and that there is no outstanding scientifific issue" is correct.
Dr. Boobis

Influence of completeness of scientific database on establishing an ADI

114. I would qualify the statement that "it is r ecognized that JECFA only allocates an ADI for a
food additive or veterinary drug u nder review when JECFA considers that its scientific data base is
complete and that there are no outstanding scientific i ssues" as follows: This is certainly normally the
case, but there are exceptions. The critical issue is whether a sufficiently cautious default can be
adopted in the absence of certain information. For example, there may not be a NOAEL in a study,
but it might be judged acceptable to use the LAOEL with an additional safety factor of up to 10.
Similarly, the nature of the residue might not be fully defined in which case it would be assumed that
it was all as active as the most active moiety, often th e parent compound. As often some of the reside
will be less active or inactive metabolites, this assu mption is generally conservative. Hence, JECFA
would require a complete data base unless it could a dopt default assumptions that would if anything
lead to a more conservative risk assessment than would be the case otherwise.
10. In paras. 129 and 168 of its Replies to th e Panel Questions, the European Communities
states that "JECFA's traditional mandate does not allow it to examine all risk
management options but restricts it to either propose MRLs or not". Does Codex have
risk management options other than (1) the establishment of an MRL, (2) establishment
that an MRL is not necessary or (3) no recommendation?

Dr. Boisseau

115. As already written in my replies to the questions No 5 and 6, JECFA is only responsible for
conducting the risk assessment and C odex is responsible for conducting the risk management even if
JECFA is also partly involved in the risk management in proposing MRLs to Codex. To my
knowledge, Codex has no other risk management opti ons concerning veterinary drug residues than
(1) the establishment of a MRL, (2) establishing that a MRL is not necessary, (3) no recommandation.

116. Nevertheless, as already said in my reply to the question No 7, when JECFA decided that it
was not possible to propose any ADI for Chlo ramphenicol and nitroimidazole compounds, it
suggested to Codex that efforts should be made to replace or prohibit the use of these veterinary
drugs.
11. What should, in your view, be the components of a qualitative risk assessment,
compared with a quantitative risk a ssessment? [see para. 82 of Canada Rebuttal
Submission]

Dr. Boisseau

117. A qualitative risk assessment should be based on the following components: (1) hazard
identification, (2) hazard characterization and (3 ) qualitative exposure assessment. A qualitative risk
assessment can be applied to a veterinary drug for which it has been demonstrated that (1) according
to the hazard identification step, it leads to residues in animal derived food, (2) according to the
hazard characterisation step, some of these residues ar e responsible of an adverse effect (a) which,
such as genotoxicity, can not be associ ated with a relation effect/dose, (b) which can be expressed in
humans, (c) for which it is not possible to establis h a threshold under which an amount of residues,
even very limited, cannot generate this adverse effect in humans, (3) according to the exposure
assessment step, consumers are likely to ingest these residues through animal derived food.
118. As already said in my reply to the question No 7, JECFA based its conclusions on such a
qualitative risk assessment for chloramphenicol, dimetridazole and ronidazole and did not recommend
any ADI for these compounds.
Dr. Boobis

Qualitative versus quantitative risk assessment

119. The risk assessment paradigm is such that it is not appropriate to conduct a qualitative risk
assessment a priori. This is because such an assessment requires knowledge of hazard and mode of
action, either determined experimentally or assumed. Hence, a qualita tive assessment might be
undertaken after conducting at least part of a conve ntional risk assessment, when it was apparent, or
assumed, that there was no exposure that did not po se some risk and thus establishing a safe dose (an
ADI) would not be possible.
120. Hence, a qualitative risk assessment should compri se all four steps of the conventional risk
assessment paradigm, but with certain differences. There would still be need of hazard identification
and some form of hazard characterization. During hazard characterization, if possible, the mode of
action should be determined through mechanistic considerations. The potential relevance of this to
human risk should be considered. Where mode of action cannot be established, human relevance is
assumed in the absence of evidence to the contrary. Certain modes of action are considered to possess
no threshold based on the intrinsic hazard (most notably DNA-reactive genotoxicity). For compounds
exhibiting such properties it is assumed that ther e is no threshold for the response. In such
circumstances, current practice in many regions, including WHO and the EU, would be that it would
be inappropriate to derive a health based guidance value (ADI), as any exposure would be considered
to pose a risk. The need for detailed dose-response analysis would be questionable. However, in a
risk assessment, as opposed to risk management, there is still need fo r scientific rigour. Hence, the
conclusion that exposure is irrelevant because of the nature of the effect is a risk management
decision. In risk assessment, even if establishment of an ADI is considered inappropriate, it would be
of value to risk managers to provide a margin of exposure estimate, to determine how great the risk is
likely to be. This would require exposure assessment . This would be of help in considering the
relative risk compared with background exposure, particularly for compounds occurring

endogenously. Finally, risk characterization w ould be necessary to consider the relevance of
experimental observations to human s. There may be kinetic or dynamic factors indicating that
although theoretically there was no exposure with zero risk, in practice the risk would be minimal and
therefore acceptable (e.g. PPR Opinion on daminozid e, which contributed to EC decision to approve
annex 1 listing of the compound .(PPR (2004). Opinion of the PPR Panel related to the evaluation of
daminozide in the context of Council Directive 91/414/EEC (May 2004)
(http://www.efsa.eu.int/science/ppr/ppr_opinions/453_en.html; Official Journal L 241 , 17/09/2005 P.
0051 – 0056))
Dr. Cogliano

121. The components of a qualitative risk assessment are (1) a critical review of the pertinent
scientific information on an agent and (2) an evalua tion of the weight of th e evidence that the agent
can alter the risk of one or more adverse effects.
122. Paragraph 82 of Canada's Rebuttal Submissi on seems confused about the role of dose-
response analyses in a qualitative assessment. A qualitative risk assessment can consider the presence
or absence of dose-response relationships in evaluating epidemiological and experimental
information. For example, the IARC Monographs do this in their evaluations of whether an agent can
alter the incidence of cancer in humans. This is a completely different matter from estimating the
dose of an agent that may provoke a specific level of adverse effect. This latter activity is part of
quantitative risk assessment and it can be delineated as a separate ac tivity from the qualitative risk
assessment.
12. How is scientific uncertainty addressed in ri sk assessments in general? With respect to
the assessment of risks from the consumpt ion of meat treated with the growth
promotion hormones at issue, how has sci entific uncertainty been considered by
JECFA/Codex? How does it differ from the way it has been considered by the
European Communities in its assessment of risks from the consumption of meat treated
with the growth promotion hormones at issue?

Dr. Boisseau

123. In assessing the risk for human health associat ed with the exposure to veterinary drug
residues, JECFA adresses the scien tific uncertainty by using the safety factors listed above in my
reply to question 8 describing, among others, how JECFA builds a margin of safety into its final
recommendations.
124. For the hormonal growth promoters, JECFA ha s considered that, given the quality and the
quantity of the available data, it was possible to carry out a complete quantitative risk assessment. For
establishing ADIs and MRLs for the three synthetic hormones, melengestrol, trenbolone and zeranol,
JECFA has implemented the usual procedure regard ing the safety factors. For the three natural
hormones, oestradiol-17β, progesterone and testosterone, JECFA has decided that the margin of safety
deriving from the values of the established ADIs a nd from a maximum estimated intake of residue
was such that it was not necessary to set up MRLs.
125. For oestradiol-17β, the European Communities did not consider any scientific uncertainty as
it decided that it was not possible, for reasons of principle, to establish an ADI for a genotoxic
compound. For the five other hormones at issue, the European Communities did not really consider
any scientific uncertainty as it decided that the ava ilable data were too limited to allow a complete
quantitative risk assessment to be carried out.

Dr. Boobis

Addressing scientific uncertainty in risk assessment

126. Scientific uncertainty is dealt with in a variet y of ways in risk assessment. A description of
some of the issues can be found in the draft repor t of the UK VUT (Variability and Uncertainty in
Toxicology) working group of the COT (April, 2006) at
http://www.food.gov.uk/multimedia/pdfs/vutdraftreport.pdf.
127. One way of dealing with uncertainty is to defau lt to the worst case in the absence of evidence
to the contrary. Hence, the most sensitive relevant endpoint in the most sensitive species is used as
the basis of the risk assessment. In extrapolating to humans a default factor of 10 is used to allow for
species differences, which assumes that humans are mo re sensitive than the experimental species. A
further factor of 10 is included for interindivi dual differences. These differences may be due to
gender, genetics, life stage or other factors. Howeve r, to some extent such differences have already
been taken into account in the choice of endpoint , as this will usually represent the most sensitive
lifestage, gender and to some extent genetics by using data from the most sensitive species. Where
there are additional uncertainties, such as no NOE AL or the absence of a non-critical study, an
additional safety factor will be included, and this is almost always conservative, as when the data gaps
have been completed, the appropriate safety factor is almost always less than that used to account for
these data gaps. The residue may be assumed to be all as active as the most active moiety, which is
almost always a conservative assumption. Dietar y intake is based on conservative data for food
consumption. It is also assumed that all meat th at could contain veterinary drug residue will contain
the residue and that this will be present at the high end of the range (MRL or other appropriate level).
In respect of the ADI, the assumption is that intake will be at this high level for a lifetime, when in
reality there will be occasions when little or no meat is consumed or that which is consumed contains
less or even no residue. In their risk assessment of the hormones, JECFA applied all of these
approaches to dealing with the uncertainty.
128. In dealing with scientific uncertainty much depends on the expert judgment of the risk
assessor. Issues such as biologi cal coherence, whether effects are considered compound related,
relevance to humans, the reliability of model systems at predicting effects in vivo all impact on the
interpretation of the data. Within the EU, it is clear that there are also differences in the interpretation
of data, as illustrated by the differing conclusi ons of the Committee on Veterinary and Medicinal
Products - CVMP (1999) and the Scientific Comm ittee on Veterinary Measures relating to Public
Health - SCVPH (1999). In part, the EC assessme nt of the hormones did not go as far as including
some of the considerations for uncertainty used by JECFA because of the co nclusion that there was
insufficient information to determine whether ther e was a threshold for the carcinogenic effects.
However, for some of the compounds this was based on the results of a small number of non-standard
tests of genotoxicity, with equivocal of very weak responses. It is not clear whether the EC applied a
weight of evidence approach to evaluating the genotoxicity of all of the compounds, taking into
account the totality of the available data, as was the case by JECFA.
 CVMP (1999). Report of the CVMP on the Safety Evaluation of Steroidal Sex Hormones in
particular for 17β-Oestradiol, Progesterone, Altrenogest, Flugestone acetate and Norgestomet in the
Light of New Data/Information made available by the European Commission. EMEA/CVMP/885/99
(http://www.emea.eu.int/pdfs/vet/srwp/088599en.pdf)

 SCVPH (1999). Opinion of the Scientific Committee on Veterinary Measures Relating to
Public Health: Assessment of potential risks to hum an health from hormone residues in bovine meat
and meat products (http://ec.europa.eu/food/fs/sc/scv/out21_en.pdf)

C. A
SSESSMENT OF OESTRADIOL-17Β
13. To what extent, in your view, does the EC risk assessment identify the potential for
adverse effects on human health, including th e carcinogenic or genotoxic potential, of
the residues of oestradiol-17 β found in meat derived from cattle to which this hormone
had been administered for growth pr omotion purposes in accordance with good
veterinary practice? To what extent does th e EC risk assessment evaluate the potential
occurrence of these adverse effects?

Dr. Boisseau

A. CARCINOGENIC POTENTIAL OF THE RESIDUES OF OESTRADIOL-17β

129. There is a general international agreem ent to recognize that oestradiol-17 β is associated with
a carcinogenic potential resulting from its interaction with hormonal receptors.
130. For exemple, in its fifty second session held in 1999, JECFA noted that "in long term studies
in carcinogenicity in animals, reviewed at its thirty second meeting, oral and parenteral administration
of oestradiol-17β increased the incidence of tumors only in hormone dependent tissues including the
kidney of male Syrian hamsters " and concluded that "the carcinogenicity of oestradiol-17 β is most
probably a result of its interacti on with hormonal receptors". Considering also epidemiological
studies on women who took oestrogens, either alone or in combination with progestogens and
androgens, JECFA concluded that " the available da ta suggest that the increase of cancers of the
breast and the endometrium observed in women re ceiving post menopausal oestrogen replacement
therapy is due to the hormonal effect of oestrogens . Therefore, JECFA has considered appropriate to
establish a NOAEL on the basis of the changes in several hormone dependent parameters in post
menopausal women and to derive from this NOAEL an ADI using two safety factors of 10, one to
account for normal variation among individuals and a second one to protect the sensitive human
populations.
131. In its 1999 report, CVMP concluded also that "hormonal carcinogens in humans and
experimental animals are characterized by (1) tu morigenic action typically in various endocrine-
responsive organs and/or tissues and (2) the need for a prolonged exposure to high concentrations
before tumorigenic effects become apparent".
132. In its 1999 report, SCVPH concluded also that "w hether it is clear that exogenous oestrogens,
present in oral contraceptives or used in hormonal re placement therapy in women, are responsible for
an increase risk of endometrial cancer and, to lesser extent, some increased risk of breast cancer, there
is no direct evidence on the consequences of the contribution of exogenous oestradiol-17 β originating
from the consumption of treated meat".
B. GENOTOXIC POTENTIAL OF THE RESIDUES OF OESTRADIOL-17 β

133. The amounts of substance needed to be used in toxicological studies in general are, by far,
higher than the levels of residues likely to be present in food derived from animals treated by
veterinary drugs. If these studies would have been carried out with the very little amounts of
substances such as those corresponding to the residue le vels in food, they would have always led to
negative results. That is the reason for which these studies are, practically, always carried out with
the parent substances and not with the residues a nd it is assumed that the residues derived from the
parent substances have the same toxicological potential as these parent substances. As far as they are
concerned, genotoxicity tests are mainly carried out in order to understand the mecanism of the
carcinogenic effects, if any, of the substance under review and even the in vivo tests, because it is
obvious for the in vitro studies, are not scheduled to determine a dose-response relationship and to

establish a threshold. Therefore, when genotoxicity tests give positive results, it is only possible to
conclude that the parent substance itself has been shown genotoxic in the conditions of these tests and
that its residues, given their very low levels in animal derived food, may have also a genotoxic
potential.
134. There is currently some general agreement on the fact that oestradiol-17 β is associated with a
genotoxic effect.
135. Thus, although it recognized that oestradiol-17 β does not lead to positive results in all the
classical tests which have been used to demonstrat e its genotoxicity and its mutagenicity (oestradiol-
17β did not cause gene mutations in vitro and gives, in some other assays, sporadic but unconfirmed
positive results), JECFA, in its fifty second session held in 1999, concluded "that oestradiol-17 β has a
genotoxic potential".
136. In its 1999 report, the Committee for Veterinary Medicinal Products (CVMP) of the European
Medicine Agency (EMEA) released the following conclusions: "oestradiols and/or their synthetic
analogues are devoid of the ability to induce gene mu tations or chromosomes aberrations in vitro.
With regard to the studies of Rajah and Pento (1995) and Thibodeau et al. (1998), those are
considered inconclusive and, therefore, additional experiments are needed before making any
statement that oestradiol-17 β induces MTX resistance and/or HPRT-deficient gene mutations.
Tsutsui and Barret and Tsutsui et al. hypothesi sed that oestradiols ar e capable of inducing
aneuploidy, followed by malignant transformation a nd the studies of Abul-Hajj et al., Paquette, and
Anderson et al. may suggest that oestradiol-17 β and/or its metabolites induce DNA damage or
genomic instability. However, the demonstration remains to be made that the observed indicator
effects are representative of mutagenesis at the gene or chromosome level and also occur in somatic
cells in vivo. This is not likely in the view of the following: earlier studies had mostly indicated that
hormones do not induce micronuclei or other chromosomes aberration types in vivo. With the
exception of the study reported by Dhilon and Dhillon, the recent data confirm the earlier findings and
clearly indicate that hormones and/or their synthe tic analogues are not associated with genotoxicity
properties in the bone marrow micronucleus assay in vivo.
137. The sub-group of the UK Veterinary Product Committee (VPC) concluded in its 1999 report
that "there is currently no positive results from in ternationally acccepted test systems which indicate
that the hormones considered in the report are genotoxic".
138. In its 2002 opinion, SVCPH reported a series of new assays in which oestradiol-17β and/or its
metabolites induce positive results but it has to be noted that all these assays ha ve been carried out in
vitro studies with cell cultures and no one in an in vivo study.
139. If there is currently some general agre ement on the fact that oestradiol-17 β is associated with
a genotoxic effect, there is nevertheless no agreement on the fact that this genotoxic potential could be
expressed in vivo in order to give to oestradiol-17 β the capacity to act as a complete carcinogen,
responsible of both initiation and promotion of tumours.
140. CVMP, quoting JECFA(1999) and IARC(1999) c oncluded that the potential genotoxic
properties of the compounds(hormones and in particular oestradiol-17 β) would not be expressed in
vivo and/or not play a role in the tumorigenic activit y. Therefore, it does mean that, even it has been
considered that oestradiol-17 β has a genotoxic potential, the tumorigenic activity of this hormone is
not associated with its genotoxic potential but with its hormonal activity.
141. If SCVPH, in its 1999 report, expresses its concern in concluding that "Finally, in
consideration of the recent data on the formation of genotoxic metabolites of oestradiol suggesting
oestradiol-17β acts as complete carcinogen by exerting tumour initiating and promoting effects … no

quantitative estimate of the risk related to residu es in meat could be presented", it provides no data
indicating that oestradiol-17β is associated with the increase of tu mours in tissues or organs which are
not hormone dependent.
142. In conclusion, the EC risk assessment did not support that residues of oestradiol-17 β, despite
the genotoxic potential of this hormone, can initiate and promote tumours in humans.
C. OTHER ADVERSE EFFECTS ON HUMAN HEALTH

143. In its 1999 Opinion, SCVPH has also identif ied that hormonally active substances could be
associated with other adverse effects concerning, for example, the intrauterine and perinatal
development, the growth and puberty in humans an d the immune system. Nevertheless, these data
have not been used by the Eu ropean Communities to conduct any quantitative risk assessment likely
to lead, for these effects associated with the hor monal properties of growth promoters, to the
establishment of thresholds and ADIs different from those proposed by JECFA.
Dr. Boobis

EC risk assessment of hormone residues in meat

144. The EC has not identified the potential for adve rse effects on human health of residues of
oestradiol found in meat from treated cattle. This is because the analysis undertaken was focused
primarily on hazard identification. There was little in the way of hazard characterization, and no
independent exposure assessment was undertaken. Data from the JECFA evaluation were used,
together with speculative assumptions about misuse or abuse of the product. No adequate assessment
of exposure following use according to GVP was undert aken. Hence, it was not possible to complete
the risk characterization phase of the assessment. The EC's evaluation essentially stopped once it was
concluded that the effects of the hormone were such that there were no thresholds (genotoxic
carcinogenicity and hormonal effects). There was no attempt to estimate the potential occurrence of
adverse effects in humans following exposure to le vels of the hormones found in meat from treated
animals.
Dr. Guttenplan

145. I believe the EC has done a thorough job in identifying the potential for adverse effects on
human health of oestradiol-17 β found in meat derived from cattle to which this hormone had been
administered. They have identified a number of potential adverse effects of oestradiol-17β in humans.
They have established metabolic pathways relevant to these effects, and have examined mechanisms
of these effects. In addition they have performed thorough studies of residue levels in cattle, and the
environment. The evidence evaluati ng the occurrence of adverse effects is weak. Animal models are
very limited and the target organs do not coincide we ll with the target organs in humans. There are
basically no epidemiological studies comparing ma tched populations consuming meat from untreated
and hormone-treated cattle. Thus, little can be infe rred about the potential occurrence of the adverse
effects, the potential for adverse effects seems reasonable. (JECFA Meeting 52-WHO-FAS 43,
SCVPH Opinions 1999, 2002).
14. In your view, does the risk assessment undertaken by the European Communities on
oestradiol-17β follow the Codex Guidelines on risk assessment, including the four steps
of hazard identification, hazard charac terization, exposure assessment and risk
characterization with respect to oestradiol-17β?

Dr. Boisseau

146. The European Communities does not indicate anywhere in its submission that it does not
intend to follow the Codex guidelines on risk assessment including the four steps of hazard
identification, hazard characterisation, exposure assessment and risk characterisation. On the
contrary,the following indicates that the Europ ean Communities considers the same approach for
assessing the risk associated with the residues of growth promoters. It only claims, on the basis of the
opinion released by the Scientific Committee on Veterinary Measures relating to Public Health
(SCVPH) in 1999, as the two following opinions of this SCVPH, released successively in 2000 and
2002, did not amend this conclusion adopted in 1999, that it is not possible to carry out a quantitative
risk assessment with regard to the six hormones in general and to oestradiol-17β in particular. For the
European Communities, such a quantitative risk assessment cannot be carried out because "In
consideration of the recent concerns relating to the lack of understanding of critical developmental
periods in human life as well as the uncertainties in the estimates of endogenous hormone production
rates and metabolic clearance capacity, in particul ar in prepubertal children, no threshold and
therefore no ADI can be established for any of the six hormones".
147. After re-appraisal of 17 studies launched early in 1998 and recent literature, SCVPH, in its
opinion released in 2002, adopted conclusions wh ich do not challenge th e Codex guidelines on risk
assessment. SCVPH concluded, among others, that (1) "the consequence of the consumption of
lipoidal esters of oestradiol-17 β needs to be considered in a risk assessment", (2) "experiments with
heifers, one of the major target animal groups fo r the use of hormones, indicated a dose dependent
increase in residue levels of all hormones, partic ularly at the implantation site", "Epidemiological
studies with opposite-sexed twins suggest that the exposure of the female co-twin in utero to
hormones results in an increased birth weight and, consequently, an increase adult breast cancer risk"
(These two statements call for refining the exposure assessment to hormone residues).
Dr. Boobis

Adherence of EC assessment to Codex risk assessment guidelines

148. As indicated above, the EC risk assessment of o estradiol does not follow the four steps of the
Codex risk assessment paradigm. Even if it were concluded that oestradiol is a genotoxic carcinogen,
the four steps should have been followed, for the reasons explained in answer to question 11 above,
and as described further in the next section.
Dr. Guttenplan

149. The EC has been thorough in following Code x guidelines on hazard identification and very
thorough in exposure assessment. The hazard characte rization is more limited since there is only one
animal model that is well characterized and this is in the hamster kidney. As kidney is not a known
target of estradiol in humans the extrapolation to humans is uncertain. The risk characterization is
very qualitative at best. There is also a mouse uter us model, but this has not been characterized with
respect to dose-response and mechanism. More limited data is available in certain other animal
systems and these are older studies with no reports of replication. There are no epidemiological
studies comparing cancer incidence or prevalence in populations consuming hormone-treated or
untreated meat, and, as indicated above, the hazard characterization is limited. Thus, taken together,
the risk assessment has a mixed rating in following the Codex guidelines.
[The references for the two questions above are: pa ra. 77 of EC Replies to Panel Questions and
the Opinions in Exhibits US-1, 4, and 17; paras. 194-207 of EC Rebuttal Submission (US case),
paras. 115-127 of EC Rebuttal Submission (Canad a case), paras. 85-91, 134-153 of EC Replies
to Panel Questions; paras. 35-40 US Rebuttal Subm ission, paras. 72-73 of US Replies to Panel

Questions, paras. 140-160 of US First Submission; paras. 70-111 of Canada Rebuttal
Submission and paras. 88-106 of Canada First Submission]

D.
CONSUMPTION OF MEAT CONTAINING HORMONES
(a) Carcinogenicity
15. Does the identification of oestradiol-17 β as a human carcinogen indicate that there are
potential adverse effects on human health when it is consumed in meat from cattle
treated with hormones for growth promotion purposes? Does your answer depend on
whether good veterinary practices are followe d? [see paras. 206-207 of EC Rebuttal
Submission (US case), para. 121 of EC Rebuttal Submission (Canada case), para. 97-98
of EC Replies to Panel Questions, paras. 76- 77, 150 and 155-156 of US First Submission,
paras. 35-40 and 46 of US Rebuttal Submission]

Dr. Boisseau

150. Considering my reply to question 13, it is leg itimate to conclude that (1) the carcinogenic
potential of oestradiol-17β results from its hormonal activity, (2) it is possible to establish a NOAEL
and, by using an appropriate safety factor, to de rive from this NOAEL an ADI which represents the
highest quantity of oestradiol-17 β causing in humans no hormonal effect and therefore no
carcinogenic effect. On these grounds, it is possible to conclude, in agreement with JECFA, that
oestradiol-17β, even it has been recognized as being able to generate tumours, is not likely to produce
adverse effects on human health when it is consum ed in meat from cattle treated with hormones for
growth promotion purposes.
151. My reply depends on the efficient implementation of good veterinary practices. It has to be
clearly understood that if these good veterinary prac tices are not implemented or if the conditions of
use of the veterinary drugs in animal husbandry ar e different from those which have been taken into
consideration by JECFA in its risk assessments, all the work carried out since years by both JECFA
and Codex to establish MRLs to guarantee the hygien ic quality of animal derived food and to protect
human health with regard to veterinary drug residues is meaningless.
Dr. Boobis

Relevance of carcinogenicity of oestradiol-17β

152. The entire basis of risk assessment is based on th e fact that there is a relationship between
dose and effect. This is true even for compounds for which there is no threshold in their dose-
response curve. Hence, the greater the dose the greater the risk. Th e corollary is that the lower the
dose, the lower the risk. A key consideration in the risk assessment is whether there is a threshold in
the dose-response. If not, whilst risk declines w ith dose, it does not reach zero until there is no
exposure (zero dose). However, in the case of oestradi ol, the issue is complicated by the fact that the
compound is produced naturally in the body. Hence, an additional factor in the risk assessment of this
compound is whether the levels from consumption of meat from treated animals impacts on the
circulating levels of the hormone. If not, then there should be no change in risk.
153. JECFA concluded that whilst oestradiol is a human carcinogen, its mode of action is such that
there would be no appreciable risk of cancer at exposures up to the ADI. The risk of cancer at
exposures above the ADI would depend on the dura tion of exposure, which would need to be
relatively prolonged (in the order of years rather than months) and on the magnitude of the exposure.
It is likely that at exposures slightly above the ADI , the risk would be minimal. However, it is not
possible to estimate with any accuracy at which le vel of exposure risk would become significant.

This would also vary with the individual. E xposure from meat of cattle treated according to GVP
would be substantially below the ADI and hence the threshold for any carcinogenic effects. If GVP is
not followed, then whether there is a carcinogeni c risk would depend on whether the ADI is exceeded
and by what margin. However, even if the ADI is ex ceeded, this would have to be on a regular basis.
As indicated above, the occasional exposure above the ADI, such as might occur if GVP is not
followed, would not be associated with any increase in risk of cancer.
Dr. Cogliano

154. The identification of oestradiol-17 β as a human carcinogen indicates that there are potential
adverse effects on human health when oestrodiol-17 β is consumed in meat from cattle treated with
hormones for growth promotion purposes. This answer does not depend on whether good veterinary
practices are followed. It depends on the presence of the hormone in the meat that people consume.
Dr. Guttenplan

155. If potential is taken to mean possible, then an adverse effect cannot be ruled out, but it is
unlikely if good veterinary practices are followed. If good veterinary practi ces are not followed, the
potential for adverse effects may be significant. (JECFA Meeting 52-WHO-FAS 43, SCVPH
Opinions 1999, 2002).
16. Does the scientific evidence relied upon in the SCVPH Opinions support the conclusion
that carcinogenic effects of the hormones at issue are related to a mechanism other than
hormonal activity? [see para. 148 of the EC Replies to Panel Questions and paras. 35-40
and 46 of US Rebuttal Submission]

Dr. Boisseau

A. OESTRADIOL-17 β

156. Considering my reply to the question 13, the scientific evidence relied upon in the SCVPH
Opinions does not support the conclusion that carcinogenic effects of oestradiol-17 β are related to a
mechanism other than hormonal activity.
B. PROGESTERONE

157. In its thirty second session, JECFA concluded th at "Although equivocal results have been
reported for the induction of single-strand DNA brea ks and DNA adducts have been seen in vivo and
in vitro in some studies, progesterone was not mu tagenic … progesterone has no genotoxic potential".
It concluded also that "these effects on tumour production occurred only with doses of progesterone
causing obvious hormonal effects … the effects of progesterone on tumour production was directly
related to its hormonal activity".
158. In its 1999 report, SCVPH concluded, about th e carcinogenicity of progesterone, that "At
present, the data are insufficient to make any quantitative estimate of the risk arising from the
exposure to residues in meat" Therefore, the scientific evidence relied upon in the SCVPH Opinions
does not support the conclusion that the carcinogenic effects of progesterone are related to a
mechanism other than hormonal activity.
C. TESTOSTERONE

159. In its thirty second session, JECFA concluded that the increase of the incidence of prostactic
and uterine tumours observed in rodents treated with high doses of testosterone resulted from the

hormonal activity of testosterone". In its fift y second session held in 1999, JECFA concluded that "In
mammalian cells, no chromosomal aberrations, mu tations or DNA adducts were found following
treatment with testosterone … testosterone has no genotoxic potential".
160. In its 1999 report, SCVPH concluded, about the carcinogenicity of testosterone, that, given
the limited data on genotoxicity and on carcinoge nicity in humans, no conclusive quantitative
estimate of the risk arising from the excess intake with meat from treated animals can be made.
Therefore, the scientific evidence relied upon in th e SCVPH Opinions does not support the
conclusion that the carcinogenic effects of testos terone are related to a mechanism other than
hormonal activity.
D. MELENGESTROL

161. In its fifty fourth session, JECFA concluded from the review of a range of assays in vitro and
in vivo that melengestrol acetate is not genotoxic. It also agreed upon the fact that "no firm
conclusion could be drawn about the carcinogenic potential of melengestrol acetate in ICR mice …
the increased incidence of malignant tumors in the highest-dose group of prepuberal C3Han/f mice
was assumed to be due not to a direct carcinogenic e ffect of melengestrol acetate but to the promoting
effect of increased prolactin concentrations".
162. In its 1999 report, SCVPH concluded, about the carcinogenicity of melengestrol, that "in view
of the lack of data on mutagenicity/carcinogenicity and on DNA interactions and in consideration of
carcinogenicity studies conducted only in one animal species, these data are inadequate to assess the
carcinogenic potential of melengestrol. Therefore, the scientific evidence relied upon in the SCVPH
Opinions does not support the conclusion that the car cinogenic effects of melengestrol are related to a
mechanism other than hormonal activity.
E. TRENBOLONE

163. In its thirty second session held in 1987, JE CFA concluded from carcinogenic studies in
animals that "the liver hyperplasia and tumours in mice … and the slight increase in the incidence of
islet-cell of the pancreas of rats arose as a conse quence of the hormonal activity of trenbolone". In its
thirty fourth session held in 1989, JECFA, having reviewed a comprehensive battery of short term
tests, concluded that " it was unlikely that tre nbolone acetate was genotoxic" and decided to confirm
its previous conclusion to base the evaluation of trenbolone acetate and its metabolites on their no-
hormonal-effect.
164. In its 1999 report, SCVPH concluded, about th e carcinogenicity of trenbolone, that "in
consideration of the lack of in vitro short term assays on mutagenicity and genotoxicity of other
trenbolone metabolites other than α-trenbolone and in consideration of the equivocal results of the
transformation assays and the in vivo studies, the ava ilable information is insuficient to complete a
quantitative risk assessment". Therefore, the scientific evidence relie d upon in the SCVPH Opinions
does not support the conclusion that the carcinogenic effects of trenbolone are related to a mechanism
other than hormonal activity.
F. ZERANOL

165. In its thirty second session held in 1987, JECF A concluded that zeranol and its metabolites,
zearalanone and taleranol, were not mutagenic in a number of tests in bacterial and mammalian
systems even if it has noted that zeranol gives a positive result in the Rec-assay and taleranol gives a
positive result in the test with Ch inese hamster ovary cells in the absence of activation but a negative
result with activation. After having reviewed the carcinogenicity studies in animals, JECFA
concluded that " the tumorigenic effect of zeranol was associated with its oestrogenic properties".

166. In its 1999 report, SCVPH concluded, about the carcinogenicity of zeranol, that "in
consideration of the lack of data on mutagenicity /genotoxicity and the clear evidence for an induction
of liver adenomas and carcinomas in one animal species, no assessment of the possible
carcinogenicity of zeranol can be made". Therefore, the scientific evidence relied upon in the
SCVPH Opinions does not support the conclusion that the carcinogenic effects of zeranol are related
to a mechanism other than hormonal activity.
167. In conclusion, considering my reply to ques tion 13 above, the scientific evidence relied upon
in the SCVPH Opinions does not support the conc lusion that carcinogenic effects of oestradiol-17 β
are related to a mechanism other than hormonal activity. On the other hand, considering the
conclusions of JECFA and the fact that SCVPH bases always its reservations on the lack of data more
than on data establishing the genotoxicity and the ca pacity of the five other hormones (progesterone,
testosterone, melengestrol, trenbolone and zeranol) to act as complete carcinogens, it can be said that
the scientific evidence relied upon in the SCVPH Op inions does not support the conclusion that the
carcinogenic effects of these five hormones are related to a mechanism other than hormonal activity.
Dr. Boobis

Mechanism of carcinogenicity of oestradiol-17β

168. There is no doubt that some of the hormones in dispute are genotoxic and mutagenic in some
assays in vitro . However, the conduct and interpretati on of these assays requires expert judgment.
Some endpoints are prone to artefactual positives, fo r example due to cytotoxicity and even a true
positive may be a reflection of the non-physio logical conditions of the in vitro system (Greenwood et
al, 2004; Kirkland et al, 2005) . Hence, the guidelines on genotoxic ity testing require confirmation of
an in vitro positive using an appropriate in vivo assay (CVMP, 2004) . An additional factor is the
testing of metabolites or putative metabolites. In vitro it is possible to test these unopposed by
detoxication or excretion processes. However, in vi vo, it is often the situation that some metabolites
are not formed in sufficiently high concentrati ons for a sufficient period of time to cause any
genotoxicity. A key example is the formation of reactive oxygen species. Whilst this is an
established mechanism for mutagenicity in vitro, there is very little evidence for such an effect in vivo
(Bianco et al, 2003; Brusick, 2005 ). Further, there is a threshol d for this mechanism, due to the
efficiency of endogenous antioxidant systems (Aria et al, 2006; Russo et al, 2004) . This is because
endogenous production of reactive oxygen species during intermediary metabolism is substantial, and
hence efficient protective systems have evol ved to maintain the integrity of the cells (Russo et al,
2004). For these reasons, in vitro studies implica ting metabolites in the mode of action of a
carcinogen should be supported by mechanistic studi es in vivo. In particular, evidence for the
formation and genotoxic effects of such metabolit es should be sought in vivo. Whilst there is
adequate evidence that some of the hormones are genotoxic in some in vitro assays, there are data
supporting mechanisms other than direct reactivity with DNA. The possibility of redox cycling of
some metabolites, with the generation of reactive oxygen species that can give rise to 8-hydroxylation
of guanine has been discussed above (see also Yagi et al, 2001 ). Redox cycling may give rise to
adducts by other mechanisms, such as formation of aldehydes (Lin et al, 2003) . There are clear
thresholds for these interactions (see above). Th e evidence is against any direct interaction of
oestradiol or its metabolites with DNA (Chen et al, 2005; Hurh et al, 2004; Huez et al, 2004) .
Oestradiol can cause genotoxicty by effects other than direct or indirect interaction with DNA. These
include induction of micronuclei (Fischer et al, 2001) and promotion of DNA instability (Stopper et
al, 2003), both of which exhibit thresholds.
169. The carcinogenic effects of oestradiol appear to be a consequence of its endocrine activity.
Some of the evidence for this is the target tissu es, which are hormonally r esponsive, the concordance
of carcinogenic effect with oestrogenic potency, the absence of reliable evidence for genotoxicity,
including DNA binding, in target tissues (see above). It is notable that specific antagonism of the

oestrogen receptor in women with drugs such as ta moxifen, markedly reduces the risk of oestrogen-
related cancers, such as of the breast in those w ith high risk factors due to endocrine status (Fisher et
al, 2005). This suggests that the carcinogenic effects of o estradiol are mediated, to the extent that can
be estimated from such studies, by activation of the oestrogen receptor. The importance of the
oestrogen receptor (ER α) in the carcinogenic effects of oestradi ol is reinforced by the results of
experimental studies in genetically engineered mice (Tilli et al, 2003).
170. As indicated above, the studies in which positive results were obtained for the genotoxicity of
oestradiol and upon which the conclusions of the EC regarding mechanism were based, should have
been evaluated on a weight of evidence basis. Several of the studies suffered from significant
limitations and there were a number of well conduc ted studies on a variety of endpoints that should
have been included in such an evaluation.
 Arai T, Kelly VP, Minowa O, Noda T and Nishim ura S (2006). The study using wild-type and
Ogg1 knockout mice exposed to potassium bromat e shows no tumor induction despite an extensive
accumulation of 8-hydroxyguanine in kidney DNA. Toxicology 221:179-186

 Bianco NR, Perry G, Smith MA, Templeton DJ and Montano MM. Functional implications of
antiestrogen induction of quinone reductase: inhi bition of estrogen-induced deoxyribonucleic acid
damage (2003). Mol Endocrinol 17:1344-1355

 Brusick D (2005). Analysis of genotoxicity and the carcinogenic mode of action for ortho-
phenylphenol. Environ Mol Mutagen, 45:460-481

 Chen ZH, Na HK, Hurh YJ and Surh YJ (2005). 4-Hydroxyestradiol induces oxidative stress
and apoptosis in human mammary epithelial cells: possible protection by NF-kappaB and
ERK/MAPK. Toxicol Appl Pharmacol, 208:46-56

 CVMP (2004). Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human
Food: Genotoxicity Testing, European Medicines Agency, London

 Fisher B, Costantino JP, Wickerham DL, Cecch ini RS, Cronin WM, Robidoux A, Bevers TB,
Kavanah MT, Atkins JN, Margolese RG, Runowicz CD, James JM, Ford LG and Wolmark N (2005).
Tamoxifen for the prevention of b reast cancer: current status of the National Surgical Adjuvant
Breast and Bowel Project P-1 study. J Natl Cancer Inst, 97:1652-1662.

 Fischer WH, Keiwan A, Schmitt E and Stopper H (2001). Increased formation of micronuclei
after hormonal stimulation of cell proliferation in human breast cancer cells.
Mutagenesis, 16:209-212

 Greenwood SK, Hill RB, Sun JT, Armstrong MJ, Johnson TE, Gara JP, Galloway SM (2004).
Population doubling: a simple and more accurate estimation of cell growth suppression in the in vitro
assay for chromosomal ab errations that reduces irrelevant positive results. Environ Mol Mutagen
43:36-44. Erratum in: Environ Mol Mutagen, 2004, 44:90

 Hurh YJ, Chen ZH, Na HK, Han SY and Sur h YJ (2004). 2-Hydroxyestradiol induces
oxidative DNA damage and apoptosis in human mamma ry epithelial cells. J Toxicol Environ Health
A, 67:1939-153

 Huetz P, Kamarulzaman EE, Wahab HA and Mavri J (2004). Chemical reactivity as a tool to
study carcinogenicity: reaction between estradiol and estrone 3,4-quinones ultimate carcinogens and
guanine. J Chem Inf Comput Sci, 44
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 Kirkland D, Aardema M, Henderson L, Muller L. Evaluation of the ability of a battery of
three in vitro genotoxicity tests to discriminat e rodent carcinogens and non-carcinogens I.
Sensitivity, specificity and relative predictivity (2005). Mutat Res 584:1-256. Erratum in: Mutat Res,
2005, 588:70

 Lin PH, Nakamura J, Yamaguchi S, Asakura S and Swenberg JA (2003).

 Aldehydic DNA lesions induced by catechol estrogens in calf thymus DNA. Carcinogenesis
24:1133-1141

 Russo MT, De Luca G, Degan P, Parlanti E, Dogliotti E, Barnes DE, Lindahl T, Yang H,
Miller JH and Bignami M (2004). Accumulation of th e oxidative base lesion 8-hydroxyguanine in
DNA of tumor-prone mice defective in both the Myh and Ogg1 DNA glycosylases. Cancer Res
64:4411-4414

 Stopper H, Schmitt E, Gregor C, Mueller SO and Fischer WH (2003). Increased cell
proliferation is associated with genomic instab ility: elevated micronuclei frequencies in estradiol-
treated human ovarian cancer cells. Mutagenesis, 18:243-247

 Tilli MT, Frech MS, Steed ME, Hruska KS, Johnson MD, Flaws JA and Furth PA (2003).
Introduction of estrogen receptor-alpha into the tT A/TAg conditional mouse model precipitates the
development of estrogen-responsive mammary adenocarcinoma. Am J Pathol, 163:1713-1719

 Yagi E, Barrett JC and Tsutsui T (2001). The ability of four catechol estrogens of 17beta-
estradiol and estrone to induce DNA adducts in Syrian hamster embryo fibroblasts.
Carcinogenesis, 22:1505-1510

Dr. Guttenplan

171. The SCVPH Opinions (SCVPH Opinions 1999, 2002) do indicate that a mechanism other
than hormonal activity is possible, "In acknowle dging the recent findings on the metabolism based
genotoxicity of 17-ß oestradiol (see chapter 2.5 of th e report) it has to be stated that the assumption
that the carcinogenic potential is exclusively rela ted to the hormonal activity is no longer valid."
However, the US and Canada cite other reports indicating that genotoxic effects of estrogens are
unlikely. It should also be noted that more recent reports support a role for a genotoxic mechanism by
which hormones contribute to cancer (SCVPH Opinion, 2002).
17. Could you comment on Canada's statemen t that "the studies commissioned by the
European Communities also failed to find ev idence of "catechol metabolites" – that is
the oestradiol metabolites identified as the source of the genotoxic potential – in meat
from treated animals"? What would be the implication of an absence or presence of
catechol metabolites? [see para. 102 of Canada Rebuttal submission, EC Exhibit 51A]

Dr. Boisseau

172. The Canada statement that " the studies comm issioned by the Eur opean Communities also
failed to find evidence of " catechol metabolites "in meat from treated animals" seems right if the
study reported in the Exhibit EC-51A is considered. It is written in this report, page 15, that, in an in
vivo metabolic study, "(1) the presence of methoxy-oestrogens that derive by catechol-O-
methyltransferase activity from catechol oestrogens was demonstrated neither in liver nor in kidney…
(2) Residues … are scarcely detectable 12 days after injection of oestradiol-17 β, that could be
explained by a fast turn over of metabolites covalently bound to macromolecules, if really present,
which should be different from catechol oestrogens a dducts in proteins ... (3) However, gluthathione

or glucuronide conjugates of catechol oestrogens coul d be present at very low concentrations in liver
or kidney extracts and could correspond to sub-minor peaks we have isolated without being able to
identify them due to the too low amounts we have pu rifed … Nevertheless, in urine of one steer, we
have identified a glucuronic acid derivative of a methoxy-estrone as a minor metabolite, which
demonstrates that catechol oestrogen biosynthesis activity is present although very weak…. (4) no
trace of catechol oestrogen adducts could be detected at the same time in this fraction " page 16, that,
in in vitro studies, "No metabolites coming from the catechol oestrogen biosynthesis could be
isolated" page 18, that "metabolic studies performed in vivo …and in vitro …failed to demonstrate a
significant aromatic hydroxylation activity that would lead to catechol oestrogen derived metabolites.
173. In conclusion, (1) it can be said that this study could not find evidence of metabolites coming
from the catechol oestrogen biosynthesis. Nevert heless, it cannot be excuded that such catechol
oestrogen biosynthesis may exist although being very weak, (2) if the amount of catechol metabolites
would have been demonstrated as being significant, which is not the case, the genotoxic potential of
these metabolites would have to be taken into c onsideration in assessing the genotoxicity potential of
oestradiol-17β.
Dr. Boobis

Relevance of catechol metabolites

174. The analytical data certainly show that leve ls of catechol metabolites in meat from treated
animals were below the limits of detection of th e method. This is consistent with the rapid
detoxication and elimination of these metabolites in vivo. The implications for the risk assessment of
oestradiol would depend on the underlying assumptions for the carcinogenic effects of the compound.
For the catechols to be significant it would be necessary for these to be responsible for the
carcinogenic effects of oestradiol, it would be necessar y for there to be no threshold for their effects
and if there were it would be necessary for intake to exceed this threshold. Oestradiol is itself
carcinogenic at high doses in human subjects. Henc e, there is no need for exposure to preformed
catechols for a carcinogenic effect If these are ne cessary for the carcinogenicity, sufficient can be
formed in vivo. However, as indicated above, th ere is no good evidence implicating catechols in the
carcinogenic effects of the hormones. Further, al so as discussed above, any genotoxicity of these
compounds due to redox cycling would be militate d against by endogenous anti-oxidant systems.
Hence, whilst the absence of detect able catechols in meat from treated animals is reassuring, even if
they were detected at low levels, it would not impact on the risk assessment.
Dr. Cogliano

175. The presence of catechol metabolites would support th e potential for adverse effects to occur.
The absence of catechol metabolites could imply e ither (1) that detectable levels of catechol
metabolites were not formed from the parent compound or (2) that some level of catechol metabolites
was formed that the test methods were not sufficiently sensitive to detect it.
Dr. Guttenplan

176. It is true that only very small amounts of cat echol metabolites were detected in meat from
treated animals. However, significant levels of est radiol and estrone were detected. These can be
metabolized in humans to catechols (Rogan EG. Badawi AF. Devanesan PD. Meza JL. Edney JA.
West WW. Higginbotham SM. Cavalieri EL. Relative imbalances in estrogen metabolism and
conjugation in breast tissue of women with ca rcinoma: potential biomarkers of susceptibility to
cancer. Carcinogenesis. 24(4):697-702, 2003). In contrast to humans, cattle do not efficiently
metabolize estradiol to catechols. The latter explains the very low levels of catechols in meat. Thus,

the lack of catechols in meat does not imply that me at from estrogen-treated cattle is without risk for
genotoxicity.
18. Please comment on the US argument that the European Communities fails to
demonstrate through scientific evidence that oestradiol-17 β is genotoxic. Would your
reply have been different at the time of adoption of the EC Directive in September 2003?
If so, why? [see paras. 118-119 of EC Rebuttal Submission (US case), paras. 123-124 of
EC Rebuttal Submission (Canada case), paras. 87-91 and 153-156 of US First
Submission, paras. 35-40 and 46 of US Rebuttal Submission, and paras. 90-97 of Canada
Rebuttal Submission]

Dr. Boisseau

177. This issue regarding the genotoxic potential of oestradiol-17 β has been already adressed in
my reply to the question 13. In addition, I would like to comment the content the para 118 and 119 of
the EC Rebuttal Submission (US case). It is true that JECFA, considering the outcome of its
assessment, did not think necessary in 1988 to establish an ADI for the three natural hormones. Later
on, not because JECFA has amended its assessment re garding these three hormones but in order to
present in a more convincing way the outcome of its assessment, it decided, in its fifty second session
held in 1999, to establish an ADI for each of th e three natural hormones and to indicate that the
estimated intake of residues accounts respectively for 2%-4% of the ADI for oestradiol-17 β, 0,03% of
the ADI for progesterone and 0,05% of the ADI for testosterone. On the other hand, taking into
consideration my reply to the question number 8, it h as to be reminded that this theoritical estimated
intake of residues is all the more conservative that it disregards the very poor oral bioavailability of
these hormones.
178. My reply would not have been different at the time of adoption of the EC Directive in
September 2003.
Dr. Boobis

Genotoxicity of oestradiol-17β

179. This issue has been discussed in detail in res ponse to question 15. To reiterate, whilst there
are reliable studies demonstrating the genotoxicity of o estradiol in certain in vitro tests, the evidence
is against any genotoxicity in vivo. Some, if not all, of the genotoxicity observed in vitro would be
expected to exhibit a threshold, particularly that involving reactive oxygen species. My reply to this
question would have been the same at the time of adoption of the EC Directive in September 2003.
Dr. Cogliano

180. The EC does demonstrate through scien tific evidence that oestradiol-17 β is genotoxic. The
issue, though, is whether this genotoxicity would occur at levels found in meat residues. The EC's last
argument (in paragraph 124 of the EC's Rebuttal Submission, Canada case) that oestradiol-17 β is
carcinogenic by a combination of both genotoxicity and cell proliferation is not contradicted by earlier
arguments made by Canada and the US. On the othe r hand, it has not been established by the EC that
genotoxicity and cell proliferation would be induced by levels found in meat residues added to the
pre-existing levels occurring in exposed humans.
Dr. Guttenplan

181. There was scientific evidence cited by the EC in 2003 that oestradiol-17 β is genotoxic, "17 β
oestradiol induces mutations in various cultu red mammalian cells. The reactive metabolite,

oestradiol-3,4-quinone, also induces mutations in mouse skin in vivo . The catechol oestrogen-
quinones form DNA adducts in cultured cells and in mouse skin" (footnote 82, Rebuttal Submission
(US case). This evidence was stronger compared to previous reports. However the evidence now is
much stronger. (Rogan EG. Cavalieri EL. Estrogen metabolites, conjugates, and DNA adducts:
possible biomarkers for risk of breast, prostate, and other human cancers. Advances in Clinical
Chemistry. 38:135-49, 2004.)
19. The European Communities states that "... it is generally recognized that for substances
which have genotoxic potential (as is the case with oestradiol-17 β) a threshold can not be
identified. Therefore it cannot be said that there exist a safe level below which intakes
from residue should be considered to be safe. Therefore the fact that doses used in
growth promotion are low is not of relevance" . Does the scientific evidence referred to
by the European Communities support these co nclusions? Would your reply have been
different at the time of adoption of the EC Directive in September 2003? If so, why? [see
para. 201 of EC Rebuttal Submission (US case), paras. 120-122 of EC Rebuttal
Submission (Canada case), paras. 73 and 86-98 of Canada Rebuttal Submission,
paras. 87-91 and 153-156 of US First Submission and paras. 35-40 and 46 of US
Rebuttal Submission]

Dr. Boisseau

182. The issue regarding the genotoxic potential of oestradiol-17 β has been already adressed in my
reply to the question 13. The statement of the European Communities according which "it is
generally recognized … is not of relevance" is corr ect as long as it refers to the assessment of residues
of xenobiotics. The scientific evidence refe rred to by the European Communities does not
demonstrate that this statement can also apply in the case of oestradiol-17 β, progesterone and
testosterone as these three natural hormones are produced by both humans and food producing
animals. Therefore, even in the absence of an y consumption of food coming from animals treated by
growth promoting hormones, humans are natura lly and continuously exposed to these natural
hormones through, among others, (1) their own pr oduction of these hormones which may be very
high, for exemple in the case of pregnant women, (2) the consumption of meat from non treated cattle,
(3) the consumption of meat from other food produc ing animals, (4) the consumption of milk and
eggs. To my knowledge, there is no epidemiological survey indicating that this continuous exposure
of humans to these natural hormones results in any identified risk for health.
183. My reply would not have been different at the time of adoption of the EC directive in
September 2003.
Dr. Boobis

Genotoxic potential and the absence of a threshold

184. This issue has been addressed in part in r esponses above. Generally, in risk assessment
within the EU and JECFA, for compounds that ar e carcinogenic by a genotoxic mechanism (or mode
of action), it is assumed that there is no threshold and that there is no level below which exposure is
considered without risk. Hence, in such circumst ances no ADI would be set (as this would imply that
there was a "safe" level). However, the important point here is that it is the carcinogenic effect that is
of concern, not in vitro genotoxicity. Whilst in vivo genotoxicity without carcinogenicity may be of
concern, carcinogenicity by a mode of action other than genotoxicity, for which there is a
demonstrable and biologically plausible threshold, would not fall into this category. Hence, whilst
oestradiol may be genotoxic in certain in vitro assay s, whether this requires a no-threshold approach
to risk assessment depends critically upon a) the me chanism for genotoxicity and b) the relevance of
the in vitro findings for the in vivo effects. Th e EC has accepted that for some mechanisms of

genotoxicity, such as inhibition of spindle assembly, there is a threshold (EC, 2005a). Redox active
compounds also show a threshold in their genotoxic effects (Brusick, 2005). In addition, the EC has
accepted that on occasion kinetic factors in vivo ma y be such that the genotoxic potential of a
compound that is positive in vitro is not expressed in vivo at normal exposure levels, and hence there
is a de factor threshold (e.g. oral exposure to phenol; European Chemicals Bureau, 2006). There is no
good evidence that oestradiol is genotoxic in vivo or that it causes cancer by a genotoxic mechanism.
Indeed, the evidence is against this. Hence, th e scientific evidence does not support the EC on this
issue, that the levels of the hormones in meat from treated cattle are not of relevance.
185. My reply to this question would have been th e same at the time of adoption of the EC
Directive in September 2003.
 EC (2005a). Official Journal L 241 , 17/09/2005 P. 0051 – 0056

 European Chemicals Bureau (2006). Eur opean Union Risk Assessment Report on phenol.
CAS No. 108-95-2. EINECS No. 203-632-7. 1st Priority List, Volume 64. EUR 22229 EN

Dr. Cogliano

186. The EC's statement that a threshold cannot be identified reflects their view of genotoxic
mechanisms, just as the contrary statement that there is a threshold and that this threshold is above the
levels found in meat residues reflects how Canada and the US view genotoxic mechanisms. Neither
statement has been demonstrated by the scientific ev idence, rather, they are different assumptions that
each party uses in their interpretation of the available evidence.
Dr. Guttenplan

187. The data referred to by the EC supports a genotoxic mechanism as well as a hormonal
mechanism. It is true that there is no reason to e xpect a threshold to exist for a genotoxic chemical.
Although DNA repair can occur, it presumably is occurring at all doses and the fraction of DNA
damage repaired probably does not change at physiological levels , because the repair enzymes are
unlikely to be saturated. The statement that, "the f act that doses used in growth promotion are low is
not of relevance" is not necessarily true. (par a. 118-119 of EC Rebuttal Submission (US case). For
any toxin the dose determines the risk. When exposure is very low risk will be very low. However,
one can argue about the definition of "low". It s hould also be noted that at very low levels of
genotoxic carcinogens the decrease in risk is more than proportional than the decrease in applied dose.
188. The opinion about genotoxic effects would be less sure in 2003, but the opinion about the
existence and significance of thresholds would not change.
20. In your view, how do the European Communities' conclusions above relate to the
conclusion by Codex that "establishing an ADI or MRL for a hormone that is produced
endogenously in variable levels in human beings was considered unnecessary"? To
what extent, in your view, has JECFA's conclusion that oestradiol "has genotoxic
potential" affected its recommendations on this hormone?

Dr. Boisseau

189. The European Communities' conclusions referred to in question 19 relate obviously to the
conclusion by Codex that "establishing an ADI or MRL for a hormone that is produced endogenously
in variable levels in human beings was consid ered unnecessary". The reply given to the question
No 19 explains how it does and why these European Communities' conclusions are questionable.

190. The reply given to question 13 applies also to the second question of this question. JECFA's
conclusions that oestradiol-17 β "has genotoxic potential" did not affect its recommendations on this
hormone.
Dr. Boobis

Relevance of endogenous occurrence of oestradiol-17β in its risk assessment

191. The EC's conclusions depend somewhat on the conc ept of incremental risk. This holds that
whether an exogenous exposure is of concern de pends on the magnitude of the underlying
endogenous or background exposure. Some argue that for a compound with no threshold, even a very
modest increment is of concern, whilst others woul d argue that a small percentage change would not
materially affect risk (e.g. ICRP, 2003) . However, before consideri ng the question of incremental
risk, it is pertinent to ask whether low levels of expo sure impact on circulating hormone levels at all.
The production of oestradiol is under homeostatic control, that regulates its synthesis and degradation
(reviewed by Fotherby, 1996). In addition, the bioavailability of orally ingested oestradiol is very low
(<10%), due to presystemic metabolism ( Kuhnz et al, 1993) . Hence, there should be a range of
exposures for which there are compensatory alterati ons in endogenous levels, thereby maintaining the
oestradiol level in the body. There is evidence th at low exposures to oestradiol, though above those
that are found in meat from treated animals, do not r esult in any measurable change in the circulating
levels of oestradiol (Mashchak et al, 1986). Endogenous levels of oestradi ol vary with physiological
state. Hence, the endocrine effects of a given con centration of oestradiol will vary with the specific
physiological state. As a consequence, a modest incremental increase in oestradiol concentration
from exogenous exposure (above the ADI) might conceivably perturb endocrine effects, depending on
the physiological state. However, non-endocrine e ffects, such as genotoxicity, will depend on the
circulating concentration of oestradiol and will not vary with physiological state. Hence, the natural
variations in circulating oestradiol levels should have a much greater effect on any genotoxic response
than the much more modest change that could arise from the hormone in meat from treated animals, at
any conceivable level arising from its use as a grow th promoter. Indeed, this would be the case,
regardless of the mechanism of carcinogenesis. Hence, the EC conclusions on the absence of safety at
any level of exposure is somewhat at odds with the underlying basis of the Codex conclusion
regarding the need for an ADI or MRL.
192. I do not believe that JECFA's conclusion that o estradiol has "genotoxic potential" affected its
recommendations on this hormone, which were based on the conclusion that there was a threshold for
its carcinogenic effects. JECFA's conclusion rega rding genotoxicity was based on positive results in
certain in vitro tests, but the evidence was against a mutagenic response in vivo.
 Fotherby K (1996). Bioavailability of orally administered sex steroids used in oral
contraception and hormone replacement therapy. Contraception, 54:59-69

 ICRP (2003). The evolution of the system of radiological protection: the justification for the
new ICRP recommendations. J Radiol Prot, 23:129–142

 Kuhnz W, Gansau C and Mahler M (1993). P harmacokinetics of estradiol, free and total
estrone, in young women following single intravenous and oral administration of 17 beta estradiol.
Arzneimittelforschung (Drug Res), 9:966-973

 Mashchak CA, Lobo RA, Dozono-Takano R, Eggena P, Nakamura RM, Brenner PF and
Mishell DR Jr (1986). Comparison of pharmacodynamic properties of various estrogen formulations.
Am J Obstet Gynecol, 144:511-518

Dr. Cogliano

193. In my view, the EC's conclusions seem to refl ect a concern that endogenous hormone levels
are variable (the variability of e ndogenously produced hor mone levels is recognized by Codex). In
my view, the argument that the EC seems to be maki ng is that a threshold cannot be established for
the incremental human exposures that would be f ound in meat residues, because these additional
exposures may not be safe for some parts of the population.
Dr. Guttenplan

194. The European Communities' conclusions above are at variance with those of Codex.
Probably JECFA's conclusion that oestradiol "h as genotoxic potential" had some effect on the
European Communities' conclusions. However as also noted by the EC (SCVPH 2002 Opinion)
newer methods of analyses have identified areas of concern, such as developmental effects, since
levels of hormones in meat may represent a signi ficant increase in endogenous levels of prepubescent
children.
21. Does the scientific evidence referred to by the European Communities demonstrate that
the five hormones other than oestradiol-17 β, when consumed as residues in meat have
genotoxic potential? Does your answer depend on whether good veterinary practices are
followed? Would your reply have been different at the time of adoption of the Directive
in September 2003? If so, why? [see, inter alia, the SCVPH Opinions and paras. 63, 83,
89-91 and 93 of US First Submission, paras. 131-136 of Canada Rebuttal Submission]

Dr. Boisseau

195. My reply to question 16 applies also to the first question of this question.
196. The fact that good veterinary practices are followed or not has no impact on the genotoxic
potential of these hormones.
197. My reply would not have been different at the time of adoption of the directive in
September 2003.
Dr. Boobis

Genotoxicity of the five hormones other than oestradiol-17β

198. There is no evidence that the hormones testosterone or progesterone have genotoxic potential.
There is no convincing evidence that trenbolone acetat e, MGA and zeranol are genotoxic. They were
negative in a range of tests for genotoxicity. They were very weakly positive in a micronucleus test,
at high (potentially cytotoxic) concentrations. Tr enbolone also produced a low level of DNA adducts
measured by 32P-post-labelling (Metzler and Pfeiffer, 2001) . As indicated above, micronuclei can
arise via a non-genotoxic mechanism, particularly at concentrations that may have caused some
toxicity. In addition, the 32P-post-labelling assay is not specific, and data cited above suggest that
DNA adduction can arise by mechanisms other than direct interaction with DNA. In no case did any
of the compounds produce a mutagenic response. These data are insufficient to support the
conclusion that these hormones have genotoxic potentia l in vivo. Thus, there is no evidence that any
of the hormones are genotoxic in vivo at the levels found in meat from treated animals. Even if GVP
were not followed, the levels of exposure to the hormones would be such that no genotoxicity would
be anticipated in vivo.

199. I would have replied the same to this question at the time of adoption of the EC Directive in
September 2003.
 Metzler M and Pfeiffer E (2001). Genotoxic potential of xenobiotic growth promoters and
their metabolites. APMIS, 109:89-95

Dr. Guttenplan

200. There is no conclusive evidence presented by the EC that the five hormones other than
oestradiol-17β, when consumed as residues in meat have genotoxic potential. There is some evidence
that certain of the hormones have genotoxic pote ntial, but generally the potential is weak.
Testosterone and progesterone are negative in genotoxic assays. Zeranol can induce transformation of
breast epithelial cells in culture with efficiency simi lar to that of estradiol, but the mechanism is not
known, and it is negative or marginally active in other assays. Trenbolone is either negative or
marginally active in in vitro genotoxic assays. MGA is negativ e in genotoxicity assays. Any
genotoxic effects of the five hormones are likely to be minimized by good veterinary practice. My
reply for the hormones would not have been different in September 2003 (SCVPH 2002 Opinion).
22. How would you define in vivo DNA repair mechanisms? How effective or relevant are
in vivo DNA repair mechanisms with respect to po tential genotoxic effects from residues
of the growth promoting hormones at issue when consumed in meat? Does your answer
depend on whether good veterinary practices ar e followed in the administration of these
hormones? To what extent does the scienti fic material referred to by the European
Communities take into account these mechan isms in its evaluation of potential
occurrence of adverse effects from residues of growth promoting hormones? Would
your reply have been different at the time of adoption of the Directive in September
2003 and if so, why? [see paras. 40 and 46 of US Rebuttal Submission, footnote 107 of
US First Submission, and para. 89 of Canada Rebuttal Submission]

Dr. Boobis

DNA repair mechanisms

201. DNA repair mechanisms comprise a series of enzymatic systems that recognize different
types of damage to DNA and repair it. There are different systems for different types of chemical
modifier of DNA (e.g. small alkyl groups, bulky aromatic groups, oxidative damage), the site at
which the damage occurs (e.g. O6 or N7 of guani ne) and the nature of the damage (e.g. covalent
modification, inter-strand cross-linking, single strand breaks) (reviewed in Dip et al, 2004; Huffman
et al, 2005; Sharova, 2005) . It has been estimated that endogenous processes are responsible for
considerable oxidative DNA damage, but this rarely causes heritable changes to the cell (Shigenaga et
al, 1989; Pollycove and Feinendegen, 1999) . To a large extent this is because of the evolution of a
flexible and very efficient DNA repair pro cess. DNA damage caused by exogenous agents
(genotoxins) is repaired by similar mechanisms. Hence, adduction of DNA is detectable at much
lower doses that mutation (Williams et al, 2004) . DNA repair capacity needs to be overwhelmed
before mutagenicity increases as a linear function of dose, the lower end of the dose response showing
non-linearity (Williams et al, 2004) . A major difficulty in the risk assessment of such compounds
however, is the identification of the threshold for such effects. This is because they occur with low
incidence, and experimental studies do not have the statistical power to determine the location of the
threshold with any confidence. Thus, whilst re cognizing the likelihood for a threshold for even
genotoxic effects (Williams et al, 2004), the risk assessor if faced with the impossibility of locating it.
The conservative solution is to assu me that the response is linear and that there is no dose below
which exposure is safe (e.g. UK Committee on Carcinogenicity, 2004).

202. As indicated above, the evidence is against direct modification of DNA in vivo by hormones
in meat from treated animals, or by their metabolite s produced in vivo. In direct modification could
conceivably come about by products of active oxygen. The DNA repair processes for this are
amongst the most efficient (Arai et al, 2006; Russo et al, 2004) and even if such modification did
occur, it is anticipated that no heritable change would result, because of DNA repair (Arai et al,
2006). This would be true even at the levels of exposure that could arise should GVP not be followed.
203. My reply would have been the same at the time of adoption of the EC Directive in
September 2003.
 Dip R, Camenisch U, Naegeli H (2004). Mechanisms of DNA damage recognition and strand
discrimination in human nucleotide excision repair. DNA Repair (Amst), 3:1409-1423

 Huffman JL, Sundheim O, Tainer JA (2005) . DNA base damage recognition and removal:
new twists and grooves. Mutat Res, 577:55-76

 Sharova NP(2005). How does a cell repair damaged DNA? Biochemistry (Mosc),
70:275-291

 Shigenaga MK, Gimeno CJ, Ames BN (1989). Urinary 8-hydroxy-2'-deoxyguanosine as a
biological marker of in vivo oxidative DNA damage. Proc Natl Acad Sci U S A, 86:9697-9701

 Pollycove M and Feinendegen LE (1999). Molecu lar biology, epidemiology, and the demise
of the linear no-threshold (LNT) hypothesis. C R Acad Sci III, 322:197-204

 UK Committee on Carcinogenicity (2004). Guid ance on a Strategy for the Risk Assessment of
Chemical Carcinogens, Department of Health, London
(http://www.advisorybodies.doh.gov.uk/COC/guideline04.pdf)

 Williams GM, Iatropoulos MJ and Jeffrey AM (2004). Thresholds for the effects of 2-
acetylaminofluorene in rat liver. Toxicol Pathol, 32, Suppl 2:85-9.

Dr. Guttenplan

How would you define in vivo DNA repair mechanisms

204. DNA repair in vivo refers to the ability of the organism to remove damaged or chemically
modified portions of DNA and replace them non-damaged DNA.
How effective or relevant are in vivo DNA repair mechanisms with respect to potential genotoxic
effects from residues of the growth promoting hormones at issue when consumed in meat?

205. The repair processes involved in DNA dama ge produced by estrogen metabolites are no
different then those involved in DNA damage by many other DNA damaging agents. Most DNA
damage by any agent is repaired and there is considerable redundancy is DNA repair, insuring that
repair is effective. However, a small fraction of damage inevitably escapes repair. In essence, there is
a race for repair and cell division. If cell division occurs before repair then a mutation or cell death
may arise. Most DNA repair processes are not saturated in whole animals (including humans) as such
high levels would likely accompany extremely toxi c levels of carcinogens. Some DNA repair
processes are faulty (error-prone). They enable th e cell to survive potentially fatal DNA damage, but
they increase the levels of mutations in the cell. Increased levels of mutations increase the risk of
cancer. There is no reason to assume that DNA re pair processes involved in DNA damage produced

by estrogen metabolites are any more or less effective than those involved in repair of other
carcinogens.
Does your answer depend on whether good veterinary practices are followed in the administration of
these hormones?

206. NO
To what extent does the scientific material referred to by the European Communities take into account
these mechanisms in its evaluation of potential occu rrence of adverse effects from residues of growth
promoting hormones?

207. The scientific material referred to by the Eu ropean Communities for the most part doesn't
address DNA repair. However, since it is not likely to be different for estrogen derived damage than
other types of damage it is not really relevant. There is some evidence referred to in the SCVPH
Opinions that error-prone DNA repair of certain estrogen derived damage can occur.
Would your reply have been different at the time of adoption of the Directive in September 2003 and
if so, why?

208. NO
Lindahl T. Wood RD. Quality control by DNA repair. Science. 286(5446):1897-905, 1999, para. 40
and 46 of US Rebuttal Submission, footnote 107 of US First Submission, and para. 89 of Canada
Rebuttal Submission.

23. To what extent is it necessary or possible to take into account the "long latency period"
of cancer in the conduct of a risk assessme nt, which is supposed to assess carcinogenic
effects of these hormones when consumed in meat? Have the hormones in dispute been
used as growth promoters over a sufficient number of years for an assessment of their
long-term effects on human health to be made? [see para. 149 of EC Rebuttal
Submission (US Case), para. 143 of EC Rebuttal Submission (Canada case)].

Dr. Boisseau

209. I don't think possible/useful to take into account the "long latency period" of cancer in order
to assess properly and specifically the carcinogenic effects of residues of natural hormones only
resulting from the treatment of food producing animal s by growth promoting hormones. In my view,
epidemiological studies carried out in humans during long enough in order to take into account this
"long latency period" will not be able to discriminate, in the case of a possible but limited increase of
tumours, between the responsabilities of (1) horm one residues resulting from the treatment of food
producing animals by growth promoting hormones, (2) hormone residues resulting from the
endogenous production of these animals, (3) other components of the diet including other food
additives and contaminants. That is the reason fo r which, to my knowledge, even the hormones in
dispute have already been used as growth prom oters over a sufficient number of years, the
epidemiological studies in humans already carried out in this domain have failed to identify any
relation between the occurence of hormonally de pendent tumours and the consumption of meat
containing hormonally active residues resulting from the treatment of cattle with growth promoters.

Dr. Boobis

Latency period for cancer

210. The latency period is an important consideration in risk assessment, both in the design and in
the interpretation of studies. Thus, the duration of exposure, either of experimental animals or in
epidemiology studies, should be su fficiently long to permit assessment of effects with a long latency
period. Most forms of cancer come into this cate gory. The observational studies of humans (e.g. on
HRT or oral contraceptives) and the experimental studies in animals covered a sufficiently long
period to encompass the latency period for an y carcinogenic effects of the hormones (see
IARC, 1999).
211. The long term studies of the hormones undertaken in experimental animals and in humans,
involved much higher doses than would be encounter ed on consumption of meat from animals treated
with growth promoting hormones. The maximum risk from such low levels of exposure, even
assuming a linear dose-response relationship for cancer , would be such that it would be necessary to
study extremely large populations to detect any incr ease in cancer incidence, particularly as the most
likely cancers are quite common. This is because the lower the risk the greater the number of subjects
that are required to detect it, a function of the pow er of the study which takes account the magnitude
of the risk and the difference from the background rate (Hunter, 1997). Hence, in the risk assessment
of the hormones used as growth promoters, it is ques tionable whether an increase in risk, even if it
existed, could be detected in exposed populations. However, it is still necessary to protect against
such a risk. The risk assessment of the hormones conducted by JECFA suggested that there would be
no risk at exposure levels up to the respective ADI. Even if duration of exposure were for a
sufficiently long period (usually 20-25 years for solid tissue tumours), any increase in risk would
probably not be detectable. Hence, a negative result from such an observational study would not
resolve the issue.
212. A second issue with respect to the latency is th e significance it has for interpretation of the
exposure pattern. Where there is a long latenc y, and regular exposure is necessary before a
carcinogenic response is manifest, as appears to be the case for the hormones in question (Coombs et
al, 2005), occasional exposures above the ADI will not pose any additional risk (Larsen and Richold,
1999). Hence, latency is of value in assessing the risks from different exposure scenarios.
 Coombs NJ, Taylor R, Wilcken N, Fioric a J and Boyages J (2005). Hormone replacement
therapy and breast cancer risk in California. Breast J, 11:410-415

 Hunter DJ (1997). Methodological issues in the use of biological markers in cancer
epidemiology: cohort studies. IARC Sci Publ, 142:39-46

 IARC (1999). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans,
Vol 72. Hormonal Contraception and Post-menopausal Hormonal Therapy, IARC, Lyon, France

 Larsen JC and Richold M (199). Report of workshop on the significance of excursions of
intake above the ADI. Regul Toxicol Pharmacol, 30:S2-12.

Dr. Cogliano

213. It is definitely necessary to take into account the latency period of cancer in the conduct of a
risk assessment. In this rega rd, the guidelines for developing IARC Monographs state, "Experience
with human cancer indicates that the period from first exposure to th e development of clinical cancer
is sometimes longer than 20 years; latent periods substantially shorter than 30 year cannot provide

evidence for lack of carcinogenicity." [Internationa l Agency for Research on Cancer, Preamble to the
IARC Monographs, http://monographs.iarc.fr]
Dr. Guttenplan

214. When epidemiological data is used in perfo rming a risk assessment, the latency period is
extremely important. Usually a latent period of 20 years is taken for cancer, but this varies with the
carcinogen. It is indeed necessary to determine in cidence or prevalence at different times after the
onset of exposure. Attempting to perform a risk assessment based on epidemiological data obtained
to soon after the onset of exposure can seriously underestimate risk. With respect to hormones in
meat, it appears they have now been consumed for a sufficient number of years to observe strong or
moderate increases in risk. However, if the risk increase is small, a large enough identifiable long-
term exposed population may not be available.
Lagiou P. Trichopoulou A. Trichopoulos D. Nu tritional epidemiology of cancer: accomplishments
and prospects. [Lectures] Proceedings of the Nu trition Society. 61(2):217-22, 2002, para. 149 of
EC Rebuttal Submission (US Case), para. 143 of EC Rebuttal Submission (Canada case).

24. To what extent is it possible to identi fy possible co-founding factors causing cancer and
attribute them to identified sources? What are the implications of these factors for the
conduct of a risk assessment evaluating the adverse affects caused by residues of growth
promoting hormones in meat? Would your reply have been different at the time of
adoption of the EC Directive in September 2003? If so, why?

Dr. Boisseau

215. Generally, it is very difficult to identify possible co-founding factors causing cancer and
attribute them to identified sources. The reply gi ven to the question No 23 applies also to this
question No 24.
216. My reply would not have been different at the time of adoption of the EC Directive in
September 2003.
Dr. Boobis

Confounding factors in cancer attribution

217. Although the causes of many cancers remain to be identified (though they are likely to be
multi-factorial), strong risk factors have been identified for a number of cancers. The main
hormonally-related cancers are breast, ovarian and endometrium in women, testes and prostate in men
(IARC, 1999). In females, genetic factors, particularly BRCA1 and 2, have a strong influence on a
small number of breast cancers ( Wooster and Weber, 2003) . Breast and ovarian cancer are affected
by a number of lifestyle factors, which in ge neral influence circulating oestrogen levels (Amant et al,
2005; Henderson and Feigelson, 2000; Vogel and Taioli, 2006) . These include parity (number of
children), age at first birth, age at menarche, menopausal state. In addition, there are associations with
diet, such as fat and meat consumption, fo r male and female hormonally-related cancers (Colli and
Colli, 2006; Gonzalez, 2006; Kushi and Giovannuc ci, 2002; Rieck and Fiander, 2006; Shirai et al,
2002; Wakai etal, 2005) . Exogenous exposure to high levels of hormones, such as oestradiol, can
cause cancer (JECFA, 2000b; IARC, 1999) . However, though HRT (hormone replacement therapy)
and oral contraceptive steroids (OCS) represent e xposures orders of magnitude greater than those
encountered in consuming meat from treated animals, the relative risk is still relatively modest (RR of
1.3 for oestrogen-only HRT (Beral, 2003) ; RR of 1.24 in women taking combined OCS

(Collaborative Group on Hormonal Factors in Breast Cancer, 2006)) . These risks were detectable
only because of the very large populations involved.
218. A number of such factors could confound a study of the effects of growth promoting
hormones in meat (they should not affect the risk asse ssment, but the interpretation of the data used in
the risk assessment). For example, socioeconomic and demographic differences in lifestyle choices
and diet may utterly confound exposure comparisons of growth promoting hormones in meat. Meat
consumption, regardless of whether it is from animals treated with growth promoting hormones, is an
independent risk factor for a number of hormonally-related cancers, including breast in females and
prostate in males (Colli and Colli, 2006 ; Gonzalez, 2006) . The average age at first pregnancy, a
lifestyle choice, is appreciable greater in some countries than in others (Beets, 1999; United Nations
Economic Commission for Europe, 2003), as is socioeconomic status (Robert et al, 2004 . Thus any
apparent effect of growth hormone exposure fro m meat of treated animals may be confounded by
other known risk factors. Whilst it is sometimes possible to correct for confounding, when the risk
from the confounder is appreciably greater than the ri sk from the exposure of interest, which is likely
to be the situation here, it is very difficult to account for all of the confounding (for example, see
Toledano et al, 2005). Residual confounding may still bias the result, obscuring a null difference.
219. My reply would not have been different at the time of adoption of the EC directive in
September 2003.
 Amant F, Moerman P, Neven P, Timmerman D, Van Limbergen E and Vergote I (2005).
Endometrial cancer. Lancet, 366:491-505

 Beets G (1999). Education and age at first birth. DEMOS, 15 (Special Issue)

 Beral V (2003). Million Women Study Co llaborators. Breast cancer and hormone-
replacement therapy in the Million Women Study. Lancet, 362:419-427. Erratum in: Lancet, 2003,
362:1160

 Collaborative Group on Hormonal Factors in Breast Cancer (1996). Breast cancer and
hormonal contraceptives: collaborative reanalysis of individual data on 53 297 women with breast
cancer and 100 239 women without breast cancer from 54 epidemiological studies. Lancet,
347:1713-1727

 Colli JL and Colli A (2006). International co mparisons of prostate cancer mortality rates
with dietary practices and sunlight levels. Urol Oncol, 24:184-194

 Gonzalez CA (2006). The European Prosp ective Investigation into Cancer and Nutrition
(EPIC). Public Health Nutr, 9:124-126.

 Henderson BE and Feigelson HS (2000). Hormonal carcinogenesis. Carcinogenesis,
21:427-433

 JECFA (2000b). Toxicological Evaluation of Certain Veterinary Drug Residues in Food:
Who Food Additives Series 43, WHO, Geneva, Switzerland

 Kushi L and Giovannucci E (2002). Dietary fat and cancer. Am J Med, 113, Suppl 9B:63S-
70S

 Rieck G and Fiander A (2006). The effect of lifestyle factors on gynaecological cancer.
Best Pract Res Clin Obstet Gynaecol, 20:227-251

 Robert SA, Strombom I, Trentham-Dietz A, Hampton JM, McElroy JA, Newcomb PA and
Remington PL (2004). Socioeconomic risk factors for breast cancer: distinguishing individual- and
community-level effects. Epidemiology, 15:442-450

 Shirai T, Asamoto M, Takahashi S and Imai da K (2002). Diet and prostate cancer.
Toxicology, 181-182:89-94.

 Toledano MB, Nieuwenhuijsen MJ, Best N, Wh itaker H, Hambly P, de Hoogh C, Fawell J,
Jarup L and Elliott P (2005). Relation of trihalomet hane concentrations in public water supplies to
stillbirth and birth weight in three water regions in England. Environ Health Perspect, 113:225-232

 United Nations Economic Commission for Eur ope (2003). Trends in Europe and North
America

 Vogel VG and Taioli E (2006). Have we found the ultimate risk factor for breast cancer? J
Clin Oncol, 24:1791-1794

 Wakai K, Tamakoshi K, Date C, Fukui M, Suzuk i S, Lin Y, Niwa Y, Nishio K, Yatsuya H,
Kondo T, Tokudome S, Yamamoto A, Toyoshima H and Tamakoshi A; JACC Study Group (2005).
Dietary intakes of fat and fatty acids and risk of brea st cancer: a prospective study in Japan. Cancer
Sci, 96:590-599

 Wooster R and Weber BL (2003), Breast and ovarian cancer, N Engl J Med 348:2339–2347

Dr. Cogliano

220. It is generally possible to identify confounding factors in epidemiological studies. [Please
note that the question mentions "co-founding factors. "] It is often difficult, however, to determine
whether the observed tumours can be attributed to the agent under study or to a confounding factor.
When a causal interpretation is credible but confou nding factors cannot be ruled out, IARC considers
this to provide limited evidence of carcinogenicity.
Dr. Guttenplan

221. Although the question mentions co-founding, from the documents submitted, what is
probably meant is "confounding" factors. These are factors other than the one investigated which may
also correlate with the disease endpoint. For instan ce, if meat eaters are also obese, the observed
effects may result from obesity and not meat-eating (although the two may be related to each other).
It would be very difficult to identify all of the confounders with meat eating and cancer, although
there are many models for the effects of dietar y agents on cancer incidence. One would want a
perfectly matched group of consumers of hormone -treated meat with consumers of non-hormone-
treated meat, where both groups have the same "lifest yle". This is probably difficult to achieve, since
individuals consuming non-hormone-treated meat are probably very health conscious or from a
different geographical region. A number of confound ers can indeed be identified (e.g., age, race, sex,
medications), but one is never sure if all or even most have been identified. These are important
considerations for risk assessment of adverse affects caused by residues of growth promoting
hormones in meat, as the effects of the hormones (if any) are likely to be small and might be obscured
by confounders. The reply would not be different in 2003.
Potter JD. Colorectal cancer: molecules and popul ations. [Review] Journa l of the National Cancer
Institute. 91(11):916-32, 1999

25. To what extent do the three recent studi es referred to by the European Communities
confirm a risk to human health from the co nsumption of meat from cattle treated with
growth promoting hormones? Please also comment on the EC statement that one of the
studies "was carried out after the introducti on of the ban on the use of hormones for
growth promotion in Europe, which means th at the subjects should have been exposed
to hormone-free meat in their diet. This m ay further imply that it cannot be excluded
that the risk of cancer may be further increased if meat treated with hormones for
animal growth promotion were to be cons umed". [see paras. 145-148 of EC Rebuttal
Submission (US case) and paras. 139-142 of EC Rebuttal Submission (Canada case),
footnote 97 in para. 147 of EC Rebuttal Subm ission (US case), and Exhibits EC-71, 72,
73]

Dr. Boisseau

222. My comment concerning zeranol in the reply give n to question 13 can apply to the first study
about zeranol reported in this question 25.
223. My reply to question 23 can be used as the requested comment regarding the second study
about colorectal cancer risk reported in this question 25.
224. Comments to be made on the EC statement that one of the studies " was carried out … to be
consumed" can be derived from the very careful conclusions drawn by SCVPH in its 1999 report
which say that " the link, if any, with consumption of hormone-treated meat cannot, at present, be
confirmed nor refuted …there is moderately consistent evidence th at higher meat consumption … is
associated with higher risk of breast cancer … it w as therefore concluded that diets high in meat
possibly increase the risk of prostate cancer … there is a weakly consistent evidence that total meat
consumption is associated with the risk of prosta te cancer. For red meat, the evidence is moderately
consistent". In conclusion, this EC statemen t just expresses a concern but does not provide any
scientific evidence supporting this concern.
225. The third study about the treatment of postmenopausal women reported in this question No 25
is out of scope.
Dr. Boobis

The three recently published studies referred to by the EC (note that the relevant EC exhibits are EC-
062, EC-071 and EC-072, respectively)

 (a) Liu S and Lin YC (2004). Transforma tion of MCF-10A human breast epithelial cells
by zeranol and oestradiol-17beta. Breast J, 10:514-521.

226. This paper reports the effects of oestradiol-17 β and zeranol on the breast cancer derived cell
line MCF-10A. This cell line is devoid of ER α and has little or no ER β. The data show that both
compounds produced changes in the cells following multiple exposures that were characteristic of
malignant transformation. However, it should be noted that the malignancy of the cells was not tested
by inoculation into animals, which is the final ev idence that complete transformation has occurred.
The doses of the compounds used were high, partic ularly for the effects observed in MCF-7 (ER α
positive) cells, in which the EC50 for proliferati on is around 2 pM (cf LOEC of 15 nM in present
study). There was no change in the response with concentration in the MCF-10A cells. There was no
demonstration by the authors or by others of th e metabolic capacity of the cells. The two compounds
were equi-potent in both the ER α negative (MCF-10A) and ER α positive (MCF7) cells. This is
somewhat surprising given the known difference in both oestrogenic ( Le Guevel and Pakdel, 2001)
and genotoxic (Metzler and Pfeiffer, 2001; Stopper et al, 2003) potency of the two compounds.

227. Nevertheless, the two compounds do appear to produce a positive response in the cells, which
is consistent with previous studies in this cell line (Russo et al, 2002). However, it is well established
that oestrogens can be genotoxic in certain in vitr o test systems, most likely a consequence of redox
cycling with generation of reactive oxygen species. Evidence for this mech anism has been reported
recently by Cuendet et al (2004). As indicated above, genotoxicity by this mechanism should exhibit
a threshold and is also militated against in vivo by antioxidant defence systems and efficient repair of
oxidant-damaged DNA. Hence, the study by Liu and Lin (2004) does not confirm a risk to human
health from the consumption of meat from cattle treated with growth promoting hormones.
 Cuendet M, Liu X, Pisha E, Li Y, Yao J, Yu L and Bolton JL (2004). Equine estrogen
metabolite 4-hydroxyequilenin induces anchorage-ind ependent growth of human mammary epithelial
MCF-10A cells: differential gene expression. Mutat Res, 550:109-121

 Le Guevel R and Pakdel F (2001). Assessment of oestrogenic potency of chemicals used as
growth promoter by in-vitro methods. Hum Reprod, 16:1030-1036

 Russo J, Lareef MH, Tahin Q, Hu YF, Slater C, Ao X and Russo IH(2002). 17Beta-estradiol
is carcinogenic in human breast epithelial cells. J Steroid Biochem Mol Biol, 80):149-16.

 (b) Norat T, Bingham S, Ferrari P, Slimani N, Jenab M, Mazuir M, Overvad K, Olsen A,
Tjonneland A, Clavel F, Boutron-Ruault MC, Kesse E, Boeing H, Bergmann MM,
Nieters A, Linseisen J, Trichopoulou A, Trichopoulos D, Tountas Y, Berrino F, Palli
D, Panico S, Tumino R, Vineis P, Bueno-de-Mesquita HB, Peeters PH, Engeset D,
Lund E, Skeie G, Ardanaz E, Gonzalez C, Navarro C, Quiros JR, Sanchez MJ,
Berglund G, Mattisson I, Hallmans G, Palm qvist R, Day NE, Khaw KT, Key TJ, San
Joaquin M, Hemon B, Saracci R, Kaaks R and Riboli E (2005). Meat, fish, and
colorectal cancer risk: the European Pr ospective Investigation into cancer and
nutrition. J Natl Cancer Inst, 97:906-916.

228. This paper reports the results of a prospectiv e epidemiological study into the relationship
between diet and colorectal cancer. The study, in a large number of subjects, confirms previous
reports that there is a statistically significant associ ation between the consumption of red meat and the
risk of developing colorectal cancer. This associ ation has been know for some time (for example see
Modan, 1977) . Moreover, it is a relatively consistent observation, regardless of geographical area
(subject to allowance for any conf ounding by known risk factors) (Marques-Vidal et al, 2006) . A
number of possible explanations have been proposed for this association, including the formation of
mutagens during the cooking of meat (Sinha et al, 2005) and the generation of nitroso compounds in
the colon through the effects of heam iron from meat (Cross et al, 2003) . Although the geographical
variation in risk is consistent with a role of m eat consumption in coloreactal cancr, it provides little
support for a contribution from hormones present in meat from their use as growth promoters. This is
because the association is just as strong in regi ons where hormones are not used as where they are
used. Age standardized rates for colorectal cancer in males and females are 48.2 and 36.9 in
Australasia, 44.4 and 32.8 in North America, 37.5 and 26.4 in Northern Europe and 35.9 and 23.5 in
Southern Europe (IARC, GLOBOCAN 2002) . In comparison, meat consumption as protein is as
follows: Australia 40.3 g/day, USA 40.2 g/day and Europe 25.2 g/day (FAO, 2003).
229. Hence, the study by Norat et al (2005) does not confirm a risk to human health from the
consumption of meat from cattle treated with growth promoting hormones.
 Cross AJ, Pollock JR, Bingham SA (2003). Haem, not protein or inorganic iron, is
responsible for endogenous intesti nal N-nitrosation arising from red meat. Cancer Res, 63:2358-
2360

 FAO (2003). FAOSTAT (http://faostat.fao.org/)

 IARC (2002). GLOBOCAN (http://www-dep.iarc.fr/)

 Marques-Vidal P, Ravasco P and Ermelinda Camilo M (2006). Foodstuffs and colorectal
cancer risk: a review. Clin Nutr, 25:14-36

 Modan B (1977). Role of diet in can cer etiology. Cancer, 40 (4 Suppl):1887-1891

 Sinha R, Peters U, Cross AJ, Kulldorff M, Weissfeld JL, Pinsky PF, Rothman N and Hayes
RB (2005). Meat, meat cooking methods and preservati on, and risk for colorectal adenoma. Cancer
Res, 65:8034-8041

 (c) Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML,
Jackson RD, Beresford SA, Howard BV, J ohnson KC, Kotchen JM and Ockene J;
Writing Group for the Women's Health Initiative Investigators (2002). Risks and
benefits of oestrogen plus progestin in healthy postmenopausal women: principal
results From the Women's Health Initiativ e randomized controlled trial. JAMA,
288:321-333.

230. This paper reports the results of a randomised controlled trial on the use of a combination of
oestrogen and progestin as hormone replacement ther apy. The hazard ratio in the group receiving the
hormones for breast cancer was 1.26 (95% CI: 1.00-1 .59). There was a reduction in the risk of
colorectal cancer (0.63; 0.43-0.92) and no change in the risk of endometrial cancer (0.83; 0.47-1.47).
This study confirms a number of previous repor ts, that exposure of postmenopausal women to an
oestrogen-progestin combination increases th e risk of breast cancer (see above; Beral, 2003). This
was recognised by JECFA in its ri sk assessment of these hormones ( JECFA, 2000b) . However,
whether the finding is relevant to the risk from resi dues of the hormones in meat due to their use as
growth promoters depends upon the conclusions of the risk assessment. Hence, as explained above,
the weight of evidence is such that the hormones cause cancer by a mechanism exhibiting a threshold.
As long as exposure does not consistently exceed th e ADI, there should be no appreciable risk to
human health. Related to this, the doses to whic h the women receiving hormone replacement therapy
were exposed in this study were many times tho se to which consumers would be exposed from meat
from cattle treated with growth promoting hormones.
231. Hence, the study by Rossouw et al (2002) do es not confirm a risk to human health from the
consumption of meat from cattle treated with growth promoting hormones.
232. With respect to the EC statement on the signi ficance of the fact that one of the studies (Norat
et al, 2005) was performed after a ban on the use of hormones for growth promotion in Europe, this
will depend on the interpretation of the risk assessme nt. If there is no risk from the consumption of
meat from animals treated with the hormones, as was the view of JECFA, then it would be immaterial
whether the study was conducted before or after the ban, the risk would have been the same. If the
risk is non-zero, as suggested by the EC, then certainly there might be an incrementally greater risk if
such meat were consumed. However, as indicated elsewhere in my responses, the evidence is against
an increased risk from such exposures. In addition, the EC statement is not scientifically defensible.
The risk of cancer when eating meat from treated animals has not been measured. Hence, it is
impossible to infer anything from the risk in the ab sence of such exposure. The EC could have made
such a statement in the absence of any study as it is based entirely on conjecture.

Dr. Cogliano

233. The study by Norat et al (2005) indicates a ri sk to human health from the consumption of
meat. The other two studies suggest a risk to human health (the term "suggest" is used rather than
"indicate" because the exposure levels in these studies are higher than those found in meat residues).
When a dietary study includes exposures to hormone -free meat, this would reduce the observed level
of risk. As a result, the risk from exposure to m eat containing hormones would likely be higher than
what was indicated in the study.
Dr. Guttenplan

234. The first of the studies suggests a risk from zer anol. That observation was not previously
reported. However, the results were obtained in cultured cells and the relevance to human exposure to
hormone-treated cannot be extrapolated from this st udy because of a myriad of uncertainties in such
extrapolation. The study does suggest that additional tests of zeranol should be carried out. There is
also some evidence that a metabolite of zeranol (zearalenone) induces oxidative damage in cultured
cells. This is a possible genotoxic effect, but again it cannot be extrapolated to meat consumption.
The other two studies do not confirm a risk from hormone-treated meat. The statement that one of the
studies was carried out after the introduction of the ban on the use of hormones for growth promotion
in Europe, negates any relevance to the possible connection of hormone-treated meat consumption
and cancer.
26. Does the scientific evidence referred to by the European Communities, in particular any
epidemiological studies, identify a relationship between cancer and residues of hormonal
growth promoters? In its risk assessm ent of 1999, the European Communities makes
reference to the higher rates of breast and pr ostate cancer observed in the United States
as compared to the European Communities. Can a link be established between these
statistics and the consumption of meat from animals treated with the hormones at issue?
Would your reply have been different at the time of adoption of the EC Directive in
September 2003? If so, why? [see pages 17-19 of 1999 Opinion of the SCVPH and
related Tables A4-A5 on pages 83-91]

Dr. Boisseau

235. My replies to questions 23 and 25 apply also to this question 26.
236. My reply would not have been different at the time of adoption of the directive in
September 2003.
Dr. Boobis

Significance of epidemiological studies for risk from the hormones

237. Information relevant to this issue can be found in my responses to questions 24 and 25 above.
238. There is no scientific evidence demonstrating any association between consumption of meat
from animals treated with growth promoting hormon es and the risk of cancer in humans. There are
some studies that are consistent with such an association, but there are several other possible
explanations for the findings, some of which are more plausible than hormones in meat as being
causal. In addition, there is some lack of cons istency in the epidemiological studies between the
associations observed and use of the growth promoting hormones.

239. There are an appreciable number of studies showing an association between the risk of certain
cancer types, including breast and prostate and the consumption of meat (Colli and Colli, 2006; Norat
et al, 2005; see also SCVPH Opinion, 1999) . For breast, the incidence is similar is developed
countries such as Western Europe, North America and Australasia. The correlation is strongest with
meat consumption and shows little relationship with whether the meat is fro m animals treated with
growth promoting hormones or not. For example rates in Iceland (87.2 per 100,000), where such
hormones are not used, are not dissimilar to those in the USA (101.1 per 100,000), where they are
used. Prostate cancer rates are 124.8/100,000 in the USA and 90.9 per 100,000 in Sweden (IARC,
2002). For comparison, average daily consumption of meat (as protein) in 2000 was as follows: USA
40.2 g/day; Iceland 29.5 g/day; Sweden 24.8 g/day (FAO, 2003) . Hence, there is a much better
association with meat consumption and risk of breast or prostate cancer than there is with the use of
growth promoting hormones to treat cattle. It is also important not to infer too much from
geographical differences in cancer incidence rates with respect to causation. This is because of what
is known as the ecological fallacy. This has been defined as the inference that a correlation between
variables derived from data grouped in social or other aggregates (ecological units) will hold between
persons (individual units) (Society for Risk Assessment, 2004). The difficulty is that many factors will
vary between populations, including ethnicity, gene tics, health and socioeconomic status, diet,
lifestyle and environment. Without considering the possibility of confounding, such ecological data is
really only of value in generating hypotheses (Morgenstern, 1995). These would need to be evaluated
in more structured investigations, with better control of confounding variables.
 Morgenstern H (1995). Ecologic studies in epid emiology: concepts, principles, and methods.
Annu Rev Public Health, 16:61-81

 Society for Risk Assessment (2004). Glossary of Risk Analysis Terms.
(http://www.sra.org/resources_glossary.php)

240. My reply to this question would have been th e same at the time of the adoption of EC
Directive in September, 2003. Although some of the studies cited above were not published at that
time, there was still sufficient information to iden tify the clear trend between meat consumption and
the risk of breast and prostate cancer, independent of the pattern of use of the growth promoting
hormones.
Dr. Cogliano

241. The difference between the US and the EC in rates of breast cancer and prostate cancer
almost certainly has multiple causes. It is po ssible that differences in exposure to exogenous
hormones can be one cause, but the data are not su fficiently specific to establish a link between these
observations.
Dr. Guttenplan

242. The epidemiological studies do not identify a relationship between cancer and residues of
hormonal growth promoters. The references to the higher rates of breast and prostate cancer observed
in the United States as compared to the Europ ean Communities are not very convincing as there is
considerable variation in rates in different geograp hical locations. Also, the differences in rates of
breast and prostate cancer observed in the United States as compared to th e European Communities
are relatively small. There is no way to definite ly establish a link between these statistics and the
consumption of meat from animals treated with the hormones at issue as there are many possible
confounders, and the differences in cancer rates are sma ll. However, the results are at least consistent
with a possible effect of hormones on breast and prostate cancer. My reply would not have been
different in 2003.

Pages 17-19 of 1999 Opinion of the SCVPH and related Tables A4-A5 on pages 83-91.

(b) Residue analysis
27. How do the residues in meat from cattle treated with the three synthetic growth
promoting hormones differ from residues in meat from cattle treated with the three
natural growth promoting hormones at issue?

Dr. Boisseau

243. Residues in meat from cattle treated with the three synthetic growth promoting hormones
differ from residues in meat from cattle treated with the three natural growth promoting hormones at
issue because the parent substances being diffe rent chemical entities associated with specific
toxicological and physiological properties, the r esidues deriving from these different substances will
be also different chemical entities associated with specific toxicological and physiological properties.
Dr. De Brabander

244. The residues of the three synthetic growth promoting hormones are substances which are
exogenous: they do not occur in the body of a healthy human being or animal.
245. The structures of the synthetic hormones are different from those of the natural hormones.
Melengestrol and trenbolone could be considered as being derived from respectively progesterone and
testosterone while zeranol has a totally other structure.
28. How do the hormones naturally present in an imals, meat, or human beings differ from
the residues in meat of the three natural hormones used for growth promotion
purposes?

Dr. Boisseau

246. The definition of residues encompasses both th e parent substance and all the metabolites
derived from this parent substance. Therefore, in the case of the part of residues of the natural
hormones which consists of parent substances, there is no difference between hormones naturally
present in food producing animals, meat or human beings. Metabolites of these natural hormones
existing in cattle and meat are, obviously, the same. To my knowledge, there is no scientific evidence
showing that the main metabolites of the three na tural hormones existing in cattle and humans are not
similar.
Dr. De Brabander

247. At first sight there are no differences between r esidues in meat of the three natural hormones
used for growth promotion purposes and the hormon es naturally present in animals, meat and human
beings. However …
• The use of the three natural hormones u sed for growth promotion purposes will
trigger a mechanism of reactions in the body of animals and human beings which
may lead to the presence of other substances which are not naturally present. The
conversion of testosterone to boldenone is an example. Boldenone is a very potent
hormone used by "body-builders"

• The "natural" hormones used for growth promotion purposes are synthetised
(prepared) from plant material. In plant material the 13C/12C ratio is different from the

13C/12C ratio of animals. Research is ongoing which demonstrates that the pattern of
hormones changes by the application of the "natural" hormones used for growth
promotion purposes. (see further question 32)

• The residues of the natural hormones in cattle are in the 17α form (inactive) while the
use of "natural" hormones used for growth promotion purposes may lead to residues
in the ß form (active form)

• The residues of (the esters of) the natural hormones are incoporated in hair of the
animals.

29. To what extent do the SCVPH Opinions eval uate evidence on the actual residue levels of
the synthetic hormones found in meat in their assessment of the risks from such
residues? Are specific references provided as to how the evidence on residues relates to
the observance of good veterinary practices or lack thereof? How do they compare with
the MRLs set by Codex? [see para. 165-176 of EC Rebuttal Submission (US case);
pages 55-68 of the Opinion of the SCVPH of 30 April 1999 in US Exhibit 4, para. 144 of
US First Submission, Exhibits US-6 and 7, footnote 46 of US Rebuttal Submission]

Dr. Boisseau

248. Levels of residues are taken into considerati on at the third step, exposure assessment, of the
risk assessment procedure after an ADI has been established at the end of the second step, hazard
characterisation, of this procedure. As, in its 1999 report, SCVPH conclude d "that no threshold level
and, therefore, no ADI can be established for any of the six hormones" (including the three synthetic
ones), there was no need for SCVPH to conduct a quantitative assessment of the exposure of
consumers to the residues of hormona l growth promoters including the determination of the levels of
residues in food from treated animals, the impact of the non observance of good veterinary practices
on these levels and the comparison between these levels and the MRLs set up by Codex.
Dr. De Brabander

249. In the case of the 3 synthetic hormones the assess ment of risk as evaluated by the SCVPH is
in terms of actual residue levels is less complex th an in the case of the natural hormones: since these
hormones does not occur naturally the endogenous leve ls in humans and in the environment are to be
considered as zero.
250. Each administration of these 3 hormones will incr ease the levels of these hormones in human
causing a number of effects, cited by the SCVPH and leaving the doubt about a number of still
unknown effects. Moreover, for the risk assessment some "old" data for residue concentrations are
used.
251. In my experience, extreme care should be taken regarding publications of concentrations of
residues. These concentrations cited (e.g. Table 8 : residue levels of alpha and beta trenbolone in
tissues of treated cattle ; p 56 ; exhibit US-4) ar e extremely low (e.g. 10 ng/kg (= 10 ppt)) and
serious doubts about their accuracy can be made. At the time they were produced (1987) there were
no analytical methods available to quantify these resi dues at that concentration level in a correct way
(methods as GC-MS-MS or LC-MS-MS). The de tection capability and way of validation of
analytical method have changed a lot in the last 20 years. The concentrations may seriously be
underestimated making the risk assessment even more risky.
252. Moreover, all studies are too much focused on the direct effect on human health only (NOEL,
ADI, MRL). As demonstrated in several documents a major part of the horm ones used are excreted

through the faeces (for MGA ca. 75 %) from which they enter into the environment causing a number
of uncontrolable effects. In the case of the 3 sy nthetic hormones there are no enough data on their
metabolites, possible transition products and effe cts on the large number of aquatic life in our
environment.
253. The MRLs set by the codex are high in relation to modern analytical limits
(normally ≤ 1 µg/kg)
Trenbolone and zeranol Cattle - Muscle - 2 µg/kg
 Cattle - Liver - 10 µg/kg

Melengestrol acetate Cattle-liver 5 µg/kg
 Cattle-fat 8 µg/kg

254. Both from their relation to human health in all of its aspects and from an analytical point of
view these MRLs are not acceptable.
30. To what extent do the SCVPH Opinions eval uate evidence on the actual residue levels of
the three natural hormones in meat in their a ssessment of the risks from such residues?
Is it possible to compare these to the AD Is recommended by JECFA in 1999? Are
specific references provided as to how the ev idence on residues relates to the observance
of good veterinary practices or lack thereo f? [see paras. 120-123 and 155-164 of EC
Rebuttal Submission (US case), pages 33-54 of 1999 Opinion in Exhibit US-4, para. 144
of US First Submission, and 52
nd JECFA Report in Exhibit US-5]

Dr. Boisseau

255. My reply to question 29 applies also to this question.
Dr. Boobis

Exposure assessment in the SCVPH Opinions

256. In the SCVPH Opinions of 1999 and 2002, the Committee did not itself evaluate evidence on
the incurred residue levels of the three natural hor mones in meat in their assessment of risks from
such residues. In their 1999 Opinion, the Comm ittee cited the data tabulated by JECFA but then
calculated an exposure based on the US tolerances, which resulted in higher estimates of exposure
than the theoretical maximum daily intakes (TMDIs) calculated on the basis of incurred residue levels
by JECFA in 1999. In this Opinion, there is also some confusion between ADIs and tolerances
(which are equivalent to MRLs). Tolerances ar e set to enable compliance to be determined.
However, exposure at the tolerance level cannot be e quated with risk to health . It is the ADI that
determines whether there is a health risk at a gi ven exposure. In their 2002 Opinion, the Committee
did not revisit exposure following use according to GVP. Rather, the Committee considered potential
exposure following several inappropriate use scenarios. This was based on a series of experimental
studies, to determine the consequences of a number of defined misuses on hormone levels in meat.
However, whilst of potential value in any risk assess ment, these data are limited in the absence of any
information on the frequency of occurrence of such misuse in the use of the products in question in
normal veterinary practice. It would have been possible to compare the SCVPHs estimates of
exposure with the ADIs derived by the JECFA but this was not done. The ADI would have exceeded
the exposure estimates for the three hormones. In both Opinions, comparison was with estimated
intake at the US tolerances.

257. References are provided to a series of studies on the effect of various misuse scenarios on
residue levels. However, no refere nces are provided that the effects GVP or the lack thereof has on
residue levels in normal veterinary practice.
 SCVPH (2002). Opinion of the Scientific Committee on Veterinary Measures Relating to
Public Health: Review of previous SCVPH opinions of 30 April 1999 and 3 May 2000 on the potential
risks to human health from hormone residues in bovine meat and meat products
(http://ec.europa.eu/food/fs/sc/scv/out50_en.pdf)

Dr. De Brabander

258. In the case of the three natural hormones the assess ment of risk as evaluated by the SCVPH is
in terms of actual residue levels and is more complicated than in the case of the synthetic hormones.
Different data of concentrations of endogenous levels of the 3 hormones in humans and in farm
animals could be found in literature.
259. The argument used in favour of the allowan ce of natural hormones is often that the
contribution of the residues of these hormones in me at is only a small part of the naturally produced
hormones in the body of a human being (JECFA,1987) . Again, for the risk assessment some "old"
data for residue concentrations are used and their accuracy could be doubted.
260. As for the three synthetic hormones, all studies are focused too much on the direct effect on
human health only (as measured witt a NOEL, ADI, MR L etc.). As demonstrated in several studies a
major part of the hormones used are excreted through urine and faeces and the administration of
natural hormones to a herd increases the concentr ation of these hormones in the environment.
Recently it was demonstrated in our laboratory that maggots of flies were able to convert high
concentrations of natural hormones to strongly anabolic agents as boldenone and boldione
261. Moreover it should not be overlooked that these hormones may act as pheromones. The best
known example is androstenone (the pheromone of the boar). But on Thursday, 10 February, 2005
BBC news mentioned that: a spray that helps increase women's enjoyment of sex has undergone
successful trials. The spray, developed by Australian company Acrux, contains the male sex hormone
testosterone. It was initially designed with post-menopausal women in mind, but has also been shown
to work for young women with a low libido. The spra y was tested over four months in three doses on
261 women with a low sex drive and low testosterone levels.
262. The tests with this type of spray illustrated the impact of hormones on human behaviour. Dr
Geoff Hackett, of the British Society for Sexual Me dicine, said it was important that the spray was
only given to women who had been thoroughly assessed, and shown to have low testosterone. Dr
Hackett also warned that raising testosterone levels too high was linked to side effects such as beard
growth, hair loss, greasy skin and acne.
263. In addition to the above there is also ZMA (sold by a well known lab in the US and on the
internet). ZMA is a scientifically designed an abolic mineral formula. It contains Zinc
Monomethionine Aspartate plus Magnesium Aspartate and vitamin B-6, and is an all-natural product
that has been clinically proven to significantly in crease anabolic hormone levels and muscle strength
in trained athletes. In tests the ZMA group had 30% increases in free and total testosterone levels
compared to 10 percent decreases in the placebo group. The ZMA group also had a slight increase in
insulin-like growth factor-1 (IGF-1) levels compared to a 20 percent decrease in the placebo group.
264. In all studies on residue levels of natural hormones I have found no indication of the influence
of such ZMA formulations (and s ynergism with implantation). Howe ver, elements such as Zn and
Mg are known to play an important role in enzymatic reactions.

31. Please comment on the US statement that "concentrations of oestradiol-17 β in meat
from treated cattle do not vary significantly fr om concentrations in untreated cattle, i.e.,
residue levels in meat from hormone-treate d cattle are well within the physiological
range of residue levels in untreated cattle. While tissue concentrations of oestradiol-17 β
in treated cattle may be slightly higher than those in untreated cattle, this increase is
much smaller than the large variations observed in (reproductively) cycling and
pregnant cattle and is thus well within the ra nge of naturally observed levels." In your
reply please take into account the US 11
th Carcinogenesis Report where it is stated that
"Meat and milk may contain estrogens. Ve terinary use of steroidal estrogens (to
promote growth and treat illness) can increase estrogens in tissues of food-producing
animals to above their normal levels" and the statement by the European Communities
that "meat consumption from pregnant heif ers is exceptional as usually these animals
are not slaughtered. [see para. 51 and 144 of US First Submission and Exhibits US-6
and 7, para. 98 of EC Replies to Panel Quest ions, Exhibit EC-101, and para. 2.3.2.3 of
the 1999 Report of SCVPH]

Dr. Boisseau

265. The US statement that " concentrations …natura lly observed levels " is certainly right if one
considers included among the physiological range of oestradiol-17 β and of progesterone in cattle the
levels of these hormones occurring during pregnancy. It is also true that meat and milk from non
treated cattle contain residues of these two natu ral hormones. The comment of the European
Communities that "meat consumption from pregnant heifers is exceptional as usually these animals
are not slaughtered " is also true. Even if, accep ting the substance of the EC comment, it is possible
to limit the physiological range of oestradiol-17 β and of progesterone in cattle, it has nevertheless to
be recognized that (1) consumers are exposed to these two natural hormones through their
consumption of meat and milk from the different non treated food producing animals and, mainly as
least for women, through their endogenous produc tion, (2) this exposure cannot be avoided.
Therefore, the use of the concept of threshold in the risk assessment of the natural hormone residues is
legitimate and the additional intake of residues of these natural hormones from the meat from treated
cattle has to be considered in this context and not according to a theoretical " no additional intake of
residues is acceptable".
Dr. De Brabander

266. The US statement that "concentrations of oestradiol-17 β in meat from treated cattle do not
vary significantly from concentrations in untreated cattle, i.e., residue levels in meat from hormone-
treated cattle are well within the physiological range of residue levels in untreated cattle" may at first
sight be correct. However, pregnant animals are normally not slaughtered and even if one is, the
consumption of meat from those animals is small. On the contrary, if all animals are treated with
estradiol-17ß there could be a significant increase of estradiol in human food. Meat and milk may
contain estrogens (as also other foods which may contain estrogens but which don't pose problems at
a normal food consumption level) and therefore there is no need to add more by artificial ways.
267. Moreover, the use of hormones is not only questioned from the point of view of the risk of
food:
• There is also the influence on animal welfar e, which is an important item. There are
a lot of publications indicating that the use of hormones will influence behaviour in
animals (and humans). Animals treated with hormones may become more aggressive
or feel unconfortable. Nowadays this is not tolerated.

• There is the influence on the environmen t. All natural hormones as well as the
synthetic ones end up in the environment. If the load of hormones is too high this
may influence the life and behaviour of some fish and invertebrates and changing the
normal pathway of life. There are a lot of investigations in that area

• Finally, most consumers in Europe don't want that the meat they eat is derived from
animals treated with hormones (and want to also keep the level of veterinary drugs
used as low as possible). This tendency of the consumer, increasing over the years is
taken over by distribution chains who finally are deciding what is coming into the
market.

32. Please comment on the conclusions of the EC risk assessment (Opinion of the SCVPH of
April 2002) that ultra sensitive methods to detect residues of hormones in animal tissues
have become available but need further validation. What is the significance of this with
regard to identifying whether the natural ho rmones in meat are endogenously produced
or are residues of hormones used for growth promotion purposes?

Dr. Boisseau

268. It is a general requirement that any analytical method, used to detect residues among others,
be validated before being used. This validation mu st be carried out in compliance with well defined
and internationally accepted criteria. As these u ltrasensitive methods, referre d to by the European
Communities, are not yet properly validated, there is a need for additional work in this domain.
Nevertheless, it has to be reminded that, when MR Ls have been established for a given substance,
there is not any more a need for highly sensitive an alytical methods but for a validated analytical
method the sensitivity of which must be consistent with the values of the established MRLs. In
addition, if it is true that ultrasensitive analyti cal methods remain useful to control the use of
forbidden veterinary drugs, such as for example grow th promoters in EU, they are less useful in the
case of the three natural hormones, which are endogenously produced by food producing animals.
Dr. De Brabander

269. Methods for the determination of hormones ar e improving constantly and it can be forseen
that they will do so for some time. When I started -a s a chemist- in the faculty of veterinary medicine
(1973) the method of choice was TLC (Thin Layer Chromatography) with fluorescence detection.
That method has been used (with succes) for some time untill GC-MS (gas chromatography with mass
spectrometric detection) was coming up (in practi ce end of the 80's). Later MS-MS and even MS n
could be used as a very selective detector. In th e middle of the 90's affordable LC-MS systems came
on the market.

Fig 32.1: Evolution of the availability of analytical techniques in function of time.

270. LC-MS is still strongly in evolution. As an illustration Fig 32.2 shows the evolution of limits
of detection of GC and LC-MS. As can be seen the LOD's has decreased considerably in the last
years.

0,01
0,1
1
10
100
1000
10000
100000
1985 1990 1995 2000 2005 2010 2015 2020
Year LC-MSGC-MS
fg

Fig 32.2: Evolution of the limits of detection in function of time

271. The improvement of limits of detection is more pronounced in LC than in GC-MS and will
continue with the introduction of new methods of separation and detection. GC and LC-MS should
also be used complementary. An illustration with an example: the main metabolite of stanozolol (a
synthetic anabolic steroid) in cattle is 16-hydro xystanozolol. This was only observed when samples
are also analysed with LC-MS next to GC-MS. I ndeed, according to its structure the determination of
16-hydroxystanozolol with GC-MS is very difficult or nearly impossible.
Multi-laboratory study of the analysis and kinetics of stanozolol and metabolites in treated calves.
H.F. De Brabander, K. De Wasch, L.A. van Ginkel, S.S. Sterk, M.H. Blokland, Ph. Delahaut, X.
Taillieu, M. Dubois, C.J.M. Arts, M.J. van Baak, L. G. Gramberg, R. Schilt , E.O. van Bennekom,
D. Courtheyn, J. Vercammen, R.F. Witkamp The Analyst, 123, 12 (1998) 2599-2604.

1970 1980 1990 2000
LC-MSGC-MSTLC
?? MSn
HPLC

272. Consequently all data on sta nozolol metabolism before the use of LC-MS (1998) have little
value because the major metabolite was not detected . There are a number of analogous cases where
"older" analytical data should be used with cau tion. Moreover, when apparatus allowing better
separation and/or lower limits of detection become available it is not always possible to repeat
expensive animal experiments to update the data.
273. Here is a strong difference between Europa a nd the US. A difference which can be clearly
measured in the citation of literature. Since little re searchers in the US work on the determination of
residues of banned substances (since they are not banned) publications on these matters (the
development of methods) are not cited very much in the US and this could be measured on the
citation index.
274. This question refers also to the use of GC -C-IRMS (gas chromatography combustion isotope
ratio mass spectrometry). As mentioned in my reply to question 28 the "natural" hormones used for
growth promotion purposes are synthetised (prepared) from plant material. In plant material the
13C/12C ratio is different from the 13C/12C ratio of animals. There are now new data available
demonstrating that the pattern change of hormones by the application of the "natural" hormones used
for growth promotion purposes.
275. The methods are still in strong evolution –as well in doping control laboratories as in residue
analysis. In practice new methods and apparatus need some time to improve technical details in order
to improve the robustness of the apparatus. Theref ore validation of the methods take considerable
time.
33. What were the reasons for the re-evaluati on by JECFA of the three natural hormones in
1999? Were the residues data used for the thre e natural hormones in 1999 the same as
those used in 1988? What additional information was used for the JECFA evaluation in
1999 of the three natural hormones which wa s not available in 1988? How did the
conclusions differ? What led JECFA to est ablish ADIs for the three natural hormones?
What are the implications of establishi ng ADIs? Why were JECFA's more recent
recommendations not considered by the Code x Committee on Residues of Veterinary
Drugs in Food? What is the status of these recommendations? [see paras. 96-97 of EC
Rebuttal Submission (US case), para. 79-80 of EC Rebuttal Submission (Canada case)]

Dr. Boisseau

276. As already mentioned in my reply to the ques tion 29, it is useless to establish MRLs in the
absence of ADIs. As, in its 1988 session, JECFA concluded that, given the outcome of its risk
assessment for the three natural hormones, there w as no need to establish ADIs in order to protect
human heath, it was therefore useless to establish MRLs (cf the reply given to the question No 18
which applies also to this question No 33). For the reasons explained in this reply to the question
No 18, JECFA decided in 1999 to establish ADI s for these three natural hormones. Usually,
establishing ADIs leads to establishing MRLs. In the case of these three natural hormones, the
outcome of the assessment of the exposure of c onsumers by JECFA in 1999 having shown in the
reply to question 18 that the highest estimated intake of residue was so low compared with the values
of the corresponding ADIs, that there was no need to establish MRLs to protect human health.
277. The residues data considered in 1999 for assessing the exposure were those already used in its
thirty second session.
278. If the wording of the conclusions adopted by JECFA has been formally different, the
substance of these conclusions remained unchanged.

279. Establishing such ADIs had no specific implications as no MRLs have been established.
280. These new recommendations have not been considered by CCRVDF because CCRVDF did
not request JECFA to reassess these hormones an d because the new proposals of JECFA did not
change the substance of the previous ones.
Dr. Boobis

Reasons for JECFA re-evaluation of the natural hormones

281. The hormones were re-evaluated by the 52
nd JECFA on the suggestion of the JECFA
Secretariat, due to the availability of new inform ation that had appeared in the published literature
since the last evaluation (FAO, 2000). This was endorsed by the 11 th session of the Codex CCRVDF
in their revision of the priority list of substances for review (CCRVDF, 1998).
 CCRVDF (1998). Report of the Eleventh Session of the Codex Committee on Residues of
Veterinary Drugs in Foods, 15-18 September, 1998, Washington, DC: Alinorm 99/31
(http://www.fao.org/docrep/meeting/005/X0203E/x0203e00.htm#Contents)

282. I am unable to comment on whether the residues data were the same as evaluated in 1988.
283. There were a number of additional studies on the toxicology and human (including
epidemiological) evaluation of therapeutic exposures to the hormones (e.g. in the form of oral
contraception or for hormone replacement therapy) that were not available in 1988.
284. The conclusion of the 1999 JECFA was to establish ADIs for the hormones evaluated
whereas in 1988 it was considered unnecessary to allocate ADIs or indeed to prepare toxicological
monographs on the hormones. This was presumably because at the time, the view was that it was
unnecessary to conduct a detailed evaluation of the toxicology of substances produced endogenously.
However, in the intervening time from the first to the second evaluation, it became clear that exposure
to the natural hormones, albeit at levels appreci able higher that found in meat from treated cattle,
could have adverse effects in humans. Hence, the implicit conclusion was that it was necessary to
establish ADIs, to serve as health based guidance values. These coul d then be used as a benchmark
for comparison with exposure via the diet. In JECFA's opinion, exposure at levels up to the ADI daily
over a lifetime would be without appreciable risk (IPCS definition of ADI; this is also the CVMP
definition of the ADI for a veterinary drug residue (EMEA, 2005)).
285. "Acceptable daily intake (ADI): the estimate of the residue, expressed in terms of micrograms
or milligrams per kilogram of bodyweight, that ca n be ingested daily over a lifetime without any
appreciable health risk."
 EMEA (2005). Volume 8: Notice to applicants and Guidance: Veterinary medicinal products.
Establishment of maximum residue limits (MRLs ) for residues of veterinary medicinal products in
foodstuffs of animal origin
(http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/vol-8/pdf/vol8_10-2005_.pdf)

286. The data used to establish ADIs for oestradiol and for progesterone in 1999 were from studies
in humans that had been undertaken since 1988.
287. According to their report, the CCRVDF did not consider these recommendations because they
had not requested the review and found no basis to change their previous decision that MRLs need not
be specified for the natural hormones.

"Recognizing that this Committee had not requested the re-evaluation of these
substances and that the new MRLs recommended by the 52nd JECFA did not differ
significantly from the current MRLs, the Co mmittee decided not to consider these
new recommendations" (CCRVDF, 2000)
 CCRVDF (2000). Report of the Twelfth Session of the CCRVDF (ALINORM 01/31)
 Washington, D.C., 28-31 March 2000
 (http://www.codexalimentarius.ne t/download/report/217/Al01_31e.pdf)

288. JECFA establishes ADIs but recommends MR Ls. CCRVDF endorsed the recommendation
that MRLs for the natural hormones did not need to be specified.
289. The consequence of this decision is that the cu rrent status of the Codex MRLs for the three
natural hormones is that they are listed as "unnecess ary" for tissues from cattle (the species in which
the hormones are used).
Dr. De Brabander

290. The driving force after the re-evaluation by th e JECFA of the 3 natural hormones can only be
guessed (by me). Obviously scientific thinking has evolved in such a way that in 1988 the JECFA
found it "unneccessary" to establish ADI's and MRL's for the 3 natural hormones while in 1999 this
has changed.
291. The residue data used by the JECFA in 1999 were according to my knowledge the same as
those in 1998. Data on residues were generated with radioimmunoassays, which are according to the
2002/657/EC only permitted as screening methods and the validation procedure of the methods can be
doubted. The additional information should be from tests on experimental animals and human beings.
However, no references to the open literature are given in the 52th JECFA report.
292. The conclusions are different in the fact that ADI s were established: this is recognition of the
danger of hormones to human health and welfare in all of his aspects.
293. The last question: Why were JECFA's more recent recommendations not considered by the
Codex Committee on Residues of Veterinary Drugs in Food? What is the status of these
recommendations? It is out of my power to answer.
34. Please comment on the EC argument that the 1999 JECFA report based its findings on
(a) outdated residues data and (b) not on evid ence from residues in meat but on studies
with experimental animals and on general st udies of IARC. If the data were not new,
did JECFA take this into account in its evaluation? What are the implications of using
such data for the purpose of conducting a risk assessment? How reliable are
extrapolations from animal studies to possi ble adverse effects on humans? How does
this compare with the kind of data and studi es used with respect to other veterinary
drugs? [see para. 120 of EC Rebuttal Submis sion (US case), para. 102 of EC Rebuttal
(Canada case)]

Dr. Boisseau

294. As mentioned in my reply to question 33, it is true that (1) the residues data considered by
JECFA in 1999 for assessing the exposure were those al ready used in its thirty second session held in
1987, (2) some of them have not b een published in peer-reviewed scientific journals. It is just a
banality to say that JECFA is provided with new data when it is requested to assess veterinary drugs
recently placed on the market and older data in the ca se of veterinary drugs already marketed since a

long time ago. Anyway, the quality and the number of the available data are more important than the
dates at which these data have been produced. In order to carry out the assessment of the residue
exposure and to establish the appropriate MRLs for the substances under review, JECFA has to
consider the available data regarding residues. If it considers that the quality and the number of these
data, the validation of the analytical methods u sed to provide these data are satisfactory, JECFA
assesses the exposure and establishes MRLs. If it think that it is not the case, it does not establish
MRLs and ask for additional data. In the case of hormones, JECFA has considered that the quality
and the number of the available residue data were satisfactory and therefore the fact that these data
were not new had no specific impact on its evaluation. In addition, the very wide margin of safety
between the exposure of consumers to hormones re sidues and the value of the established ADIs did
not raise an acute problem in this case (see my reply to question 18).
295. I don't really understand the meaning of the second EC comment stating that "the
toxicological findings in the 1999 JECFA report … are not based on evidence from residues in meat
from animals treated with these hormones for growth promotion purposes". As already mentioned in
my reply to question 13, toxicological studies are, practically, always carried out with amounts of
substances always larger than those corresponding to residues and it is assumed that residues have the
same toxicological potential like the tested substa nces. In the case where there is, among these
residues, a metabolite associated with a specific toxicological potential which is of concern with
regard to public health, JECFA can base its risk assessment on this metabolite if appropriate and
technically possible. Moreover, it is the normal way for assessing the toxicological potential of a
substance to take into consideration in vivo studi es with experimental animals, in vitro studies and
also reports already published by internationally recognized scientific organisations such as IARC.
296. Extrapolation from animals to humans regarding the toxicological potential of the substances
under review is not avoidable as it is not possible to carried out experiments in humans. It is the
responsability of JECFA to assess to what extent this extrapolation is meaningful for the products
under review.
297. For assessing the growth promoters, JECFA has us ed the same procedure it has used for all
the other veterinary drugs.
Dr. De Brabander

298. I think that the EC argument that the JECFA report is based on outdated residue data is
correct. I was unable to find in the open scientific literature much modern residue data. Moreover,
the laboratories which were able to produce such data and publish them are scarce. Because
hormones are banned in Europe and animal experiments on farm animals as cattle are more and more
complicated by all kinds of regulations the frequency of these experiments faded out since the 1980's.
In the US the intensity of recent data on residue analysis is even more scarce. Only a few US
researcher participate in the leading residue conferences which are held in Europe regularly.
299. I haven't seen any impact of studies of the IARC in the JECFA report. However, there are
such studies available: a.o. on the website of IARC a recent press release (No 167 of 29 July 2005)
could be found: IARC monographs programme finds combin ed estrogen-progestogen contraceptives
and menopausal therapy are carcinogenic to humans. This press release illustrates the danger of
hormones and also the problematic of synergism: less studies involve combinations of hormones as
they are used as production aids (e.g the combination of estradiol and progesterone).
300. Hereunder a citation of the IARC press release
"Breast cancer and endometrial cancer a re increased. Epidemiological studies
consistently demonstrate an increased risk of breast cancer in women who used

combined menopausal therapy. Largely con fined to current or recent users, the risk
increases with duration of use and exceed s that in women taking estrogen-only
therapy. Endometrial cancer risks depend on the number of days that progestogens
are included in the combined therapy. Wh en progestogens are taken fewer than 10
days per month, the risk of endometrial c ancer is increased, but when progestogens
are taken daily, the risk is similar to that in women who never used hormonal
therapy. There was not sufficient evidence to conclude that hor monal therapy has a
protective effect at any cancer site.
Overall risks and benefits should be weighe d carefully. Both beneficial and adverse
effects other than cancer have been estab lished for combined estrogen-progestogen
menopausal therapy. As for oral contra ceptives, a rigorous risk/benefit analysis
would be useful to put the different e ffects in perspective and assess the overall
consequences for public health."
301. The implications of not using such (modern) da ta are that the results of the risk assessment
are biased in favour of the "allowence" of hor mones. Giving an answer on the reliability of
extrapolations from animal studies to possible adve rse effects on humans is and remains difficult.
Therefore a safety factor is used.
302. For the comparison with other veterinary drugs (MRLs) the following reasoning could be
held. Veterinary (and human) drugs are not used on each animal (as hormones as production aids are)
but only in case that they are needed (desease). This is a totally different situation.
35. Please comment on the European Communiti es claim that nearly all the studies referred
to in the 2000 JECFA report on MGA date from the 1960s and 70s. Is this correct?
Have subsequent reports of JECFA, prior or subsequent to the adoption of the
Directive, also relied on the same studies? [see para. 171 of EC Rebuttal Submission
(US case), para. 161 of EC Rebuttal Submission (Canada case), para. 55, including
footnote 60 of US First Submission and Exhibits CDA-20, 33, 34, and 35]

Dr. Boisseau

303. It is correct to say that nearly all the studies referred to in the 2000 JECFA report on MGA
date from the 1960s and 70s. The comment to be made on this issue is similar to the comment already
made in the reply to the question 34. JECFA considered a wide series of toxicological studies in its
assessment, used as end point a non hormonal effect dose by far more conservative than a NOAEL
based on tumorigenic effect and adopted a 200 safety factor to derive an ADI from this NOAEL.
Dr. De Brabander

304. I think that the claim of the European Commun ities that nearly all the studies referred to in
the 2000 JECFA report on MGA date from the 1960s and 70s is correct. I scanned the literature on
"melengestrol acetate" on the web of science and fou nd only 257 hits. Of these 213 are published in
1980 and later.
305. Of these publications only a few refer to the use of melengestrol acetate as a growth promotor
in cattle and therefrom only a few originate from US authors.
 Hereunder a selection could be found:

 Tepfer AJ, McFee RM, Bott RC, et al.

 Feeding melengestrol acetate (MGA) to bulls during the peri or pre-pubertal period induces
differences in endocrine profiles which may lead to alterations in testis size.
 BIOLOGY OF REPRODUCTION: 225-225 Sp. Iss. SI 2005

 Merritt DA, Wilson EM, Martin RA, et al.
 Metabolism of melengestrol acetate (MGA) in the bovine: Biological activity assessment of
tissue residues and implications for human food safety.
 ABSTRACTS OF PAPERS OF THE AMERICA N CHEMICAL SOCIETY 228: U102-U102
118-AGRO Part 1 AUG 22 2004

 Schiffer B, Totsche KU, Jann S, et al.
 Mobility of the growth promoters trenbolone a nd melengestrol acetate in agricultural soil:
column studies
 SCIENCE OF THE TOTAL ENVIRONM ENT 326 (1-3): 225-237 JUN 29 2004

 Lange IG, Daxenberger A, Hageleit M, et al.
 Non-invasive screening for treatment of heifers with the anabolic steroid melengestrol acetate
(MGA) by feces analysis
 JOURNAL OF IMMUNOASSAY & I MMUNOCHEMISTRY 24 (3): 265-272 AUG 2003

 Schiffer B, Daxenberger A, Meyer K, et al.
 The fate of trenbolone acetate and melengestrol acetate after application as growth
promoters in cattle: Environmental studies
 ENVIRONMENTAL HEALTH PERSPECTIVES 109 (11): 1145-1151 NOV 2001

 Hageleit M, Daxenberger A, Meyer HHD
 A sensitive enzyme immunoassay (EIA) for the de termination of melengestrol acetate (MGA)
in adipose and muscle tissues
 FOOD ADDITIVES AND CONTAMINANTS 18 (4): 285-291 APR 2001

 Daxenberger A, Meyer K, Hageleit M, et al.
 Detection of melengestrol acetate residues in plasma and edible tissues of heifers
 VETERINARY QUARTERLY 21 (4): 154-158 OCT 1999

 Karg H, Meyer HHD
 Update evaluation of trenbolone acetate, zeranol and melengestrol acetate as growth
promoters (considerations concerning the "hor mone issues" between EU and USA at the
WTO)
 ARCHIV FUR LEBENSMITTELHYGIENE 50 (2): 28-37 MAR-APR 1999

 Henricks DM, Brandt RT, Titgemeyer EC, et al.
 Serum concentrations of trenbolone-17 beta an d estradiol-17 beta and performance of heifers
treated with trenbolone acetate, melengestrol acetate, or estradiol-17 beta
 JOURNAL OF ANIMAL SCIENCE 75 (10): 2627-2633 OCT 1997

 CAMPBELL HM, SAUVE F
 LIQUID-CHROMATOGRAPHIC DETERMIN ATION OF MELENGESTROL ACETATE IN
FEEDS
 JOURNAL OF AOAC INTERNATIONAL 76 (6): 1163-1167 NOV-DEC 1993

 NEIDERT EE, GEDIR RG, MILWARD LJ, et al.
 DETERMINATION AND QUALITATIVE CONFIRMATION OF MELENGESTROL
ACETATE RESIDUES IN BEEF FAT BY ELECTRON-CAPTURE GAS-

CHROMATOGRAPHY AND GAS-CHROMATOGRAPHIC CHEMICAL IONIZATION MASS-
SPECTROMETRY
 JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 38 (4): 979-981 APR 1990

 CHICHILA TMP, EDLUND PO, HENION JD, et al.
 DETERMINATION OF MELENGESTROL ACETATE IN BOVINE-TISSUES BY
AUTOMATED COUPLED-COLUMN NORMAL-PHASE HIGH-PERFORMANCE LIQUID-
CHROMATOGRAPHY
 JOURNAL OF CHROMATOGRAPHY-BI OMEDICAL APPLICATIONS 488 (2): 389-406
MAR 24 1989

 KRZEMINSKI LF, COX BL, GOSLINE RE
 FATE OF RADIOACTIVE MELENGESTROL ACETATE IN THE BOVINE
 JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 29 (2): 387-391 1981

306. Also: If you look at the "International Portal on Food Safety, Animal & Plant Health"
http://www.ipfsaph.org/servlet/CDSServlet?status=ND1jdGh0dHB3d3dmYW9vcmdhb3NpcGZzYX
BoaW5mb3JtYXRpb25zb3VyY2VqZWNmYS5KRUNGQUVWQUxtZWxlbmdlc3Ryb2xhY2V0YX
RlJjY9ZW4mMzM9Zm9ybWFsX3RleHQmMzc9aW5mbw~~ the three pdf files relating
melengestrol acetate, which can be downloaded contain only "old" references (before 1980).
(c) Dose-response relationship
36. How would you describe a dose-response assessme nt? Is it, as suggested by Canada in
para. 78 of its Rebuttal Submission, "widely, if not universally, accepted that adverse
effects arising from hormonal activities are dose-dependent"? Is dose-response
assessment a necessary component of hazard characterization? Or, is there an
alternative approach which can replace the dose-response assessment. Is a dose-
response assessment feasible/nece ssary for substances that are found to be genotoxic or
to have genotoxic potential? [see para. 153 of EC Replies to Panel Questions, para. 200
of EC Rebuttal Submission (US case); paras. 143, 154, and 156 of US First Submission,
paras. 70-74 of US Replies to Panel Questio ns, and paras. 34 and 37-40 of US Rebuttal
Submission; paras. 76-82 of Canada Rebuttal Submission]

Dr. Boisseau

307. The comment made by Canada regarding the dose response relationship is correct. Dose-
response assessment is a necessary component of hazard characterisation and there is, to my
knowledge, no alternative approach which can re place this dose- response assessment. A dose-
response assessment is not feasible for substances th at are found to be genotoxic or to have genotoxic
potential if, as it has been already said in the re ply to the question No 19, these substances are
xenobiotics and if it is thought that this genotoxic potential can be expressed in in vivo conditions.
Dr. Boobis

308. Dose-response assessment is analysis of the rela tionship between the total amount of an agent
administered to, taken up or absorbed by an or ganism, system or (sub)population and the changes
developed in that organism, syst em or (sub)population in reaction to that agent, and inferences
derived from such an analysis with respect to the entire population (IPCS, 2004). In a dose-response
assessment, information that is sought includes wh ether there is evidence for a compound-dependent
effect, if so, the dose range over which these effect s occur, the quantitative relationship between dose
and the magnitude or incidence of the effect, the st eepness of the dose-response curve, the severity or
incidence of the maximum effect observed, whether th ere is a threshold and if so its location and the

spacing between the no observable adverse effect le vel (NOAEL) and the lowest observable adverse
effect level LOAEL).
309. It is generally accepted that adverse eff ects arising from hormonal activities are dose-
dependent (IUPAC, 2003) . The caveat is that this is when the response is mediated by the
physiological mechanisms of the hormone, via receptor occupancy. However, even should this not be
the case because of a genotoxic mode of action, th e response would depend on dose. The difference
would be in the shape of the dose response curve. In the former, th ere would be a threshold, a dose
below which there would be no effect (NOAEL). In the latter, there would not necessarily be a such a
threshold. Hence, although risk would diminish as dose decreases, it may never reach zero.
 IUPAC(2003). Special Topic Issue on the Imp lications of Endocrine Active Substances for
Humans and Wildlife, Pure Appl. Chem., Vol. 75, Nos. 11–12

310. In risk assessment, dose-response assessment is an essential part of hazard characterization, as
it forms the basis for deriving health-based guidan ce values such as the ADI, with which dietary
intake can be compared. Th e only exception would be a h azard-based approach, that is
recommendations as to potential safety based on intrin sic capacity to cause harm rather than on the
probability of harm occurring. The former can be based on hazard identification whilst the latter
requires hazard characterization and exposure assessme nt. The most widely used instance of hazard-
based safety guidance is that for substances that are genotoxic or that have genotoxic potential.
However, this needs to be qualified, in that not all su ch substances would be treated in this way. This
is discussed in my reply to question 19 above. There may be kinetic (e.g. phenol following oral
exposure, European Chemicals Bureau, 2006 ) or dynamic (e.g. spindle inhibitors such as
thiophanate-methyl, EC, 2005a) reasons why a genotoxic compound exhibits a threshold in its dose-
response relationship. Similarly, compounds that are genotoxic via the formation of reactive oxygen
species also exhibit a threshol d in their dose-response curve (Brusick, 2005). Such effects would be
treated as would non-genotoxic endpoints, i.e. an ADI would be established using the threshold dose
(NOAEL) as a starting point, and appropriate uncertainty factors. Where the mechanism of
genotoxicity is not known, DNA reactivity is assumed. However, for compounds that are known or
assumed to be genotoxic via DNA reactivity, genotoxic potential would normally have to be
confirmed in vivo before this endpoint would be used as the basis for a risk assessment (CVMP,
2004). For such compounds, unless there is good evidence to the contrary, it is assumed that there is
no threshold for the dose-response curve.
311. The approach for such compounds that are known or assumed to exhibit no threshold in their
dose-response curve, varies from one region to anothe r. In Europe and generally within JECFA, once
a compound is identified as an in vivo DNA-reactive mutagen, or as causing a carcinogenic response
via a genotoxic mode of action, no exposure is cons idered without risk, and hence a recommendation
of maintaining intake as low as reasonable practicable (ALARP) may be made, without any
consideration of the dose-response relationship. In the case of a veterinary drug residue the simplest
way to achieve ALARP would be not to use the drug in veterinary practice. In other regions, such as
the USA, appropriate dose-response assessment will be undertaken, using an approach that assumes
no threshold (linear, low dose extrapolation). In this approach, if intake is below that associated with
a very low risk (often an incidence of 1 in 10
6 of the population), exposure may be considered
acceptable (US EPA, 2005; US FDA, 2005).
US FDA, Guideline No. 3, 2005. General Principles for Evaluating the Safety of Compounds Used in
Food-Producing Animals (http://www.fda.gov/cvm/Guidance/GFI003.htm)

US EPA, 2005. Guidelines for Carcinogen Risk Assessment
(http://oaspub.epa.gov/eims/eimscomm.getfile?p_download_id=439797)

Dr. Cogliano

312. Dose-response assessment is a qua ntitative characterization of the relationship between the
dose of an agent and the occurrence of adverse effect s. In my view, it is widely accepted that adverse
effects arising from hormonal activities depend on the do se; that is, the level of effect depends on the
level of exposure. Dose-response assessment is not, however, a necessary component of hazard
characterization. Without a dose -response assessment, it is possible to conclude that an agent can
alter the risk of one or more adverse effects. With a dose-response assessment, it may also be
possible to estimate how much the risk may be altered for a given level of exposure.
37. Do JECFA or Codex materials confirm Cana da's statement in para. 80 of its Rebuttal
Submission that "...while international risk assessment techniqu es suggest that a dose-
response assessment is optional for biological or physical agents when the data cannot be
obtained, a dose-response assessment should always be conducted for chemical
agents..."? [see Exhibit CDA-25]

Dr. Boisseau

313. JECFA has always established ADIs for veterinary drugs on the basis of a dose-response
assessment. As already said in the reply to the question No 7, JECFA did not establish any ADI when
it was not possible to carry out a dose-response asse ssment such as with xenobiotic compounds such
as chloramphenicol and nitroimidazoles.
Dr. Boobis

Is dose-response assessment optional?

314. Codex and JECFA materials certainly require th at a dose-response assessment should always
be conducted as part of the risk assessment of a chemical agent (CAC, 2005; IPCS: EHC 70, 1987
and EHC 104, 1990; IPCS, 2005; WHO, 1996 and 2001) . JECFA would be unable to recommend
MRLs (or conclude that they need not be speci fied) unless it had quantitative information on the
levels of exposure that were considered not to cause harm (from either observations in humans and/or
studies in experimental animals) and an estimate of actual human exposure. If JECFA concluded that
the toxicity of a compound was without threshold because it was a genotoxic carcinogen of potential
relevance to humans (the default assumption), it may well conclude th at it would be inappropriate to
recommend MRLs, and hence in this specific circumstance a dose-response assessment might be
considered unnecessary. However, this is a very un likely occurrence for a veterinary drug because, in
general, producers tend to screen out genotoxic co mpounds during the development process. This is
not to say that such drugs will never be encountered, but simply to point out that there are procedures
in place that are such that they will substantially reduce the probability of this occurring.
(d) Sensitive populations
38. Please describe the range of physiological (o r background) levels of the sex hormones in
humans and identify the variations in these levels on the basis of age, sex group, and
physiological stages.

Dr. Boisseau

315. According to several scientific publications reported by the SCVPH in its 1999 Opinion, the
endogenous steroid hormone levels in human females and males are the following:

Hormone Women
Prepubertal
Women
Follicular-Luteal
Women
Postmenopausal Men Prepubertal Men Adult
Oestradiol pg/ml 8-23 10-375 0-28 5-14 6-44
Estrone pg/ml 19 15-250 15-55 16 15-65
Progesterone ng/ml 0.1-0.4 0.2624 0.1-0.3 0.3-0.5
Testosterone ng/ml 0.1-0.2 0.4-0.8 0.4-0.8 0.1-0.2 3-9

316. According to the data reported by JECFA, in its 32 nd session held in 1987, and by the SCVPH
in its 1999 Opinion, the daily production rates fo r hormones in human females and males are the
following:
Hormone Women
Prepubertal Women Follicular Women
Pregnancy Men Prepubertal Men Adult
Oestradiol μg/day 445 37.800 6,5 48
Progesterone μg/day 418 94.000 150 416
Testosterone μg/day 32-65 140-240 32-65 6.500

317. According to the SCVPH in its 1999 Opinion, new ultrasensitive bioassays, 100 fold more
sensitive than RIA methods used to provide the data reported in the a bove tables, would lead to lower
values, respectively 0.6 pg/ml and 0.08 pg/ml of oestradiol-17 β for prepubertal girls and boys. It
would be important to know whether these new bioassays have been properly validated as this
SCVPH Opinion says nothing about that and wether the data obtained with these methods for both
men and women are also totally different from those obtained with the RIA methods.
39. Please comment on the SCVPH opinion st ating that "any excess exposure towards
oestradiol-17β and its metabolites resulting from the consumption of meat and meat
products presents a potential risk to public he alth in particular to those groups of the
populations which have been identified as particularly sensitive such as prepubertal
children" [see para. 147 of the EC Replies to Panel Questions]

Dr. Boisseau

318. As already said in my reply to question 31, the comment of SCVPH needs to integrate a
quantitative assessment of the risk associated with this excess exposure for the groups of populations
which have been identified as particularly sensiti ve such as prepubertal children. This excess
exposure of these sensitive populations needs to be assessed and compared with the exposure
resulting from the daily consumption of meat from cattle which have not been treated by growth
promoters, from other food and products of animal origin and from their own production of hormones.
Dr. Sippell2

319. The SCVPH opinion that "any excess exposure towards estradiol-17ß … prepubertal
children" is supported by increasing evidence from more recent scientific data in the international
literature, both from Europe and from America. Du e to the almost 100 times lower estradiol-17ß (E
2)
serum levels found by modern ultrasensitive assay t echniques (Klein et al 1994, Larmore et al 2002)
in prepubertal children as compared to conventional E 2 assays, the resulting potential E2 exposure risk
from consumption of meat and meat products has grea tly increased by a factor of at least 160 times, if
one compares the maximum acceptable daily intake (ADI) estimates of E 2 of 65 ng/day (old) and 0.4
ng/day (new) in prepubertal boys (Andersson & Sk akkebaek, 1999). This revised ADI threshold
would be reached already after ingestion of as little as 10 g of E 2 –treated meat from cattle

2 A full list of references cited in responses from Dr. Sippell can be found in Attachment 2.

(Daxenberger et al 2001). Moreover, in this comparison only E 2 but not its numerous estrogenic
metabolites, glucosidic conjugates and fatty acid esters (Maume et al 2001) were taken into account.
320. It has been shown in numerous scientific publicatio ns in vitro, in vivo and in the human that
infants and prepubertal children are highly sensitive to increased E 2-levels, resulting in premature
breast development (Schmidt et al 2002), growth acceleration (Lampit et al 2002), earlier sexual
maturation in girls, in particular in the USA (Sun et al 2002, WU et al, 2002) and less in Europe
(Muinck-keizer & Mul 2001), and the well known significantly higher incidence of precocious
puberty in girls than in boys (Teilmann et al 2005) . Accidental exposure of prepubertal boys to
estrogens has resulted in gynecomastia and advanced bone maturation (Felner & White 2000).
321. Late effects: There is now increasing epidemiological eviden ce that exposure to elevated
estrogen levels during early life (pre- and postnatally) carries an increased risk of breast cancer in
adult life (Ekbom et al 1997, Swerdlow et al 1997, Weiss et al 1997, Halakivi-Clarke et al 2000),
whereas conditions with low E 2 levels, such as preeclampsia, seem to have a protective effect (Innes
& Byers 1999). Moreover, indirect evidence sugg ests that male reproductive disorders such as
testicular cancer, cryptorchidism, hypospadias and poor sperm quality may also have their origin in
hormonal disturbances induced by E 2 and/or estrogenic substances dur ing fetal life (Skakkebaek et al
2001) and also during childhood (Higuchi et al 2003, Ramaswamy 2005).
 Andersson AM & Skakkebaek NE (1999) Exposu re to exogenous estrogens in food: possible
impact on human development and health. Eur. J. Endocrinol. 140, 477-485.

 Daxenberger A, Ibarreta D & Meyer HH (2001) Po ssible health impact of animal oestrogens
in food. Hum. Reprod. Update. 7, 340-355.

 Ekbom A, Hsieh CC, Lipworth L, A dami HQ & Trichopoulos D (1997) Intrauterine
environment and breast cancer risk in women: a popul ation-based study. J. Natl. Cancer Inst. 89,
71-76.

 Felner EI & White PC (2000) Prepubertal gynecomastia: indirect exposure to estrogen
cream. Pediatrics 105, E55.

 Halakivi-Clarke L, Cho E, Onojafe I, Liao DJ & Clarke R (2000) Maternal exposure to
tamoxifen during pregnancy increases carcinogen-induced mammary tumorigenesis among female rat
offspring. Clin. Cancer Res. 6, 305-308.

 Higuchi TT, Palmer JS, Gray LE, Jr. & Veeramachaneni DN (2003) Effects of dibutyl
phthalate in male rabbits following in utero, adolescent, or postpubertal exposure. Toxicol. Sci. 72,
301-313.

 Innes KE & Byers TE (1999) Preeclampsia and breast cancer risk. Epidemiology 10,
722-732.

 Klein KO, Baron J, Colli MJ, McDonnell DP & Cutler GB, Jr. (1994) Estrogen levels in
childhood determined by an ultrasensitive recombinant cell bioassay. J. Clin. Invest 94, 2475-2480.

 Lampit M, Golander A, Guttmann H & Hochberg Z (2002) Estrogen mini-dose replacement
during GnRH agonist therapy in central precocious puberty: a pilot study. J. Clin. Endocrinol.
Metab 87, 687-690.

 Larmore KA, O'Connor D, Sherman TI, Funanage VL, Hassink SG & Klein KO (2002) Leptin
and estradiol as related to change in pubertal status and body weight. Med. Sci. Monit. 8, CR206-
CR210.

 Maume D, Deceuninck Y, Pouponneau K, Paris A, Le Bizec B & Andre F (2001) Assessment
of estradiol and its metabolites in meat. APMIS 109, 32-38.

 Muinck-Keizer SM & Mul D (2001) Trends in pubertal development in Europe. Hum.
Reprod. Update. 7, 287-291.

 Ramaswamy S (2005) Pubertal augmentation in juvenile rhesus monkey testosterone
production induced by invariant gonadotropin stimula tion is inhibited by estrogen. J. Clin.
Endocrinol. Metab 90, 5866-5875.

 Schmidt IM, Chellakooty M, Haavisto AM, Bo isen KA, Damgaard IN, Steendahl U, Toppari
J, Skakkebaek NE & Main KM (2002) Gender differen ce in breast tissue size in infancy: correlation
with serum estradiol. Pediatr. Res. 52, 682-686.

 Skakkebaek NE, Rajpert-De Meyts E & Main KM (2001) Testicular dysgenesis syndrome: an
increasingly common developmental di sorder with environmental aspects. Hum. Reprod. 16, 972-
978.

 Teilmann G, Pedersen CB, Jensen TK, Skakkebaek NE & Juul A (2005) Prevalence and
incidence of precocious pubertal development in De nmark: an epidemiologic study based on national
registries. Pediatrics 116, 1323-1328.

40. The European Communities states that "the levels of endogenous production of the
hormones by prepubertal children is much lower than previously thought and this
finding, which is subsequent to the 1999 JECFA report, casts serious doubts about the
validity of JECFA's findings on the dose-resp onse relationship..." Please comment on
the methodology used by the SCVPH to support the conclusion that hormone levels are
lower than previously thought, and in particular comment on the validity of these
methodologies and their conclusions. Would your conclusions have been the same at the
time of adoption of the Directive in September 2003?

Dr. Boisseau

322. My comment given at the end of question 38 replies to this question.
323. My reply would not have been different at the time of adoption of the EC Directive in
September 2003.
Dr. Boobis

Hormone production in prepubertal children

324. There is certainly some evidence that endogeno us levels of hormones in children are lower
than previously thought. However, the suggestio n that this is by orders of magnitude is not
substantiated by the data. One group has reported ve ry low levels of oestradiol in male children,
0.08 pg/ml (Klein et al, 1994), but in a later study (Klein et al, 1998), the same group reported mean
levels somewhat higher, at 0.27 pg/ml. The reliability of the Klein et al assay has yet to be
determined. The assay is particularly sensitive to oestradiol, but there is no obvious explanation for
this, as it relies upon affinity for the oestrogen receptor. Diethylstilbestrol is a potent oestrogen yet is

much less sensitive than oestradiol in the assay. Klein et al (1994) have reported that there are
unidentified factors in plasma and in blood collection tubes that can interfere in the assay. In contrast,
using a similar yeast-based assay, Coldham et al (1997) found that oestradiol and DES had similar
potency, and other have found that, if anything, DES is more potent that oestradiol in such assays
(Folmer et al, 2002). At the very least, this shows that resu lts with the yeast reporter assay are not
consistent, and use of such data in risk assessment requires that the assay be adequately validated.
 Coldham NG, Dave M, Sivapathasundaram S, McDonnell DP, Connor C and Sauer MJ
(1997). Evaluation of a recombinant yeast cell est rogen screening assay. Environ Health Perspect,
105:734-742

 Folmar LC, Hemmer MJ, Denslow ND, Kroll K, Chen J, Cheek A, Richman H, Meredith H
and Grau EG (2002). A comparison of the estrogenic potencies of estradiol, ethynylestradiol,
diethylstilbestrol, nonylphenol and methoxychlor in vivo and in vitro. Aquat Toxicol, 60:101-110

 Klein KO, Baron J, Colli MJ, McDonnell DP and Cutler GB Jr (1994). Estrogen levels in
childhood determined by an ultrasensitive recombinant cell bioassay. J Clin Invest, 94:2475-2480

 Klein KO, Baron J, Barnes KM, Pescovitz OH and Cutler GB Jr (1998). Use of an
ultrasensitive recombinant cell bioassay to determin e estrogen levels in girls with precocious puberty
treated with a luteinizing hormone-releasing hor mone agonist. J Clin Endocrinol Metab, 83:2387-
2389

325. However, there are studies from two other gro ups using more specific methods than the
original radioimmunoassay, reporting that leve ls were somewhat higher than this. Ikegami et al
(2001) used a very sensitive, 2-stag e immunoassay technique. This was shown to be specific and
sensitive. In this assay, mean levels of oestradiol in prepubertal males were 1.85 pg/ml (6.8 pmol/ml).
Paris et al (2002) used a recombinant oestrogen receptor assay in a mammalian cell line, a similar
principle to the assay of Klein et al. In this study, estogenic levels in prepubertal males were found to
be 1.44 pg/ml. There are many issues affecting such measurements. These include the presence of
binding proteins, relative specificity and sensitivit y. None of the assays is entirely specific for
oestradiol. Both the oestrogen receptor and the an tibodies used could cross-react with structurally
related compounds. Depending on how the assay is performed, protein binding could reduce the
concentration of hormone detectable in the assay by sequestering hormone from the assay target.
However, it should be noted that whilst binding to protein in plasma my reduce clearance it will also
reduce the biologically active dose. In general, it is the free concentration that determines biological
activity (Teeguarden and Barton, 2004) . Hence, if SHBG is elevated in children this would tend to
reduce the effect of an equivalent total plasma concentration by reducing the free concentration.
326. The advantage of the recombinant assays is th at they measure biologically active material,
whereas the immunoassays may in clude cross-reacting less or in active metabolites. Whilst the
recombinant assays may include hormonally active materi al other than the speci fic analyte, this does
provide an indication of to what the body is expo sed in vivo. Hence, on balance, the data of Paris et
al (2002) may be the most meaningful to date. Th is presumably reflects circulating total active
oestrogenic material, but not that bound to proteins.
 Ikegami S, Moriwake T, Tanaka H, Inoue M, Kubo T, Suzuki S, Kanzakili S and Seino Y
(2001). An ultrasensitive assay revealed age- related changes in serum oestradiol at low
concentrations in both sexes from infancy to puberty. Clin Endocrinol (Oxf), 55:789-795

 Paris F, Servant N, Terouanne B, Balaguer P, Nicolas JC and Sultan C (2002). A new
recombinant cell bioassay for ultrasensitive determination of serum estrogenic bioactivity in children.
J Clin Endocrinol Metab, 87:791-797

 Teeguarden JG and Barton HA (2004). Com putational modeling of serum-binding proteins
and clearance in extrapolations across life stages and species for endocrine active compounds. Risk
Anal, 24:751-770

327. Assuming a plasma concentration of 1.44 pg/ml (Paris et al, 2002), this would be equivalent
to a daily oestradiol production of 2 µg/day. These da ta suggest that exposure to oestradiol at levels
near the JECFA ADI of 50 ng/kg, equivalent to 1. 3 µg/day (assuming a body weight of 26 kg) could
result in intakes close to the daily production of o estradiol in prepubertal males, the group suggested
to be most at risk. However, this exposure is via the oral route, and bioavailability by this route is
very low (<5%) (Fortherby, 1996). In addition, very little of the absorbed hormone will be free, over
95% being bound to plasma proteins such as SHBG. Such binding reduces the biological activity of
the hormone (Teeguarden and Barton, 2004). Hence, the JECFA ADI would appear to be appropriate
for all groups of the population. This conclusion woul d have been the same at the time of adoption of
the Directive by the EC in September 2003.
Dr. Sippell

328. There is no doubt that the development of an ultrasensitive recombinant cell bioassay
(RCBA) of E2 by Karen Klein, Gordon Cutler and co-workers at the N.I.H. in Bethesda, USA (Klein
et al 1994) represented a quantum leap in E 2 assay methodology. It opened a new door on our
understanding of basic physiological phenomena, e.g. why normal puberty starts so much earlier in
girls than in boys or why bone maturation in childre n differs so much between the sexes. The validity
of the N.I.H.-RCBA has now been confirmed by another RCBA of E 2 which was developed by
Charles Sultan's group at the University of Montpellier, France (Paris et al 2002). Unfortunately, the
complexity of the RCBA so far prevents its wide r use for routine measurements in small serum
samples from infants and prepubertal children.
329. Since pediatric endocrinologists and other researchers in the field had already been hearing
and discussing these breakthrough findings since 19 93/94, and again in 2001/02, I would certainly
have come to the same conclusions in September 2003.
 Klein KO, Baron J, Colli MJ, McDonnell DP & Cutler GB, Jr. (1994) Estrogen levels in
childhood determined by an ultrasensitive recombinant cell bioassay. J. Clin. Invest 94, 2475-2480.

 Paris F, Servant N, Terouanne B, Balaguer P, Nicolas JC & Sultan C (2002) A new
recombinant cell bioassay for ultrasensitive determination of serum estrogenic bioactivity in children.
J. Clin. Endocrinol. Metab 87, 791-797.

41. Why would individuals with the lowest endoge nous hormone levels be at greatest risk?
How would the risks for these individuals ar ising from hormones naturally present in
meat differ from the risks arising from the residues of hormone growth promoters?

Dr. Boisseau

330. Individuals with the lowest endogenous hormone levels have been considered as being at
greater risk by JECFA because the rationale followed by JECFA was, in the case of natural hormones,
to avoid that any excess intake of hormonally active r esidues be significant with regard to the rate of
their daily production of hormones in order to protect them from any physiological disturbance. In
this respect, the target was that the hormonal residue intake does not account for more than 1% of the
daily production rate of any group of the human popul ation. Therefore, the lower is the endogenous
production of hormones in a given human group, the lower must be the excess intake of hormonally
active residues for the individuals of this group.

331. From a qualitative point of view, the risks for these individuals arising from residues resulting
from the use of hormones growth promoters in cattle does not differ from the risks arising from the
residues of hormones naturally present in meat. The potential problem which may exist is only a
quantitative one.
Dr. Boobis

Relevance of endogenous hormone levels to risk

332. The reason that those with the lowest levels of hormone production are considered at the
greatest risk is because for a given exposure this gr oup will experience the greatest percentage change
in their circulating hormone levels. There is ev idence that the levels are normally low in such
subjects to ensure a biological effect that is no great er than that appropriate to their gender and age.
Hence, the extent of the biological response is lik ely to reflect the percentage increase in hormone
levels. In addition, prior to puberty, some biological systems are more sensitive to hormonal
perturbation than after puberty (Caruso-Nicoletti et al, 1985; Miyamoto and Burger, 2003).
 Caruso-Nicoletti M, Cassorla F, Skerda M, Ross JL, Loriaux DL and Cutler GB Jr (1985).
Short term, low dose estradiol accelerates ulnar grow th in boys. J Clin Endocrinol Metab, 61:896-
898

333. There is no basis to think that the effect of hormone growth promoters would be different in
any way whatsoever from hormones naturally present in meat, at equivalent internal exposure levels.
Dr. Sippell

334. This question relating to the specific vulnerability of young children having the lowest levels
of endogenous hormones (estradiol, testosterone, pr ogesterone and their metabolites) has largely been
answered above (see my reply to question no. 39).
335. The risk to children arising from hormones whic h are naturally present in meat as compared
to that from residues of hormonal growth promot ers has, to my knowledge, been estimated for E 2
only and only in beef (Daxenberger et al 2001). The average E 2 content of 500 g of meat (standard
daily consumption of 300 g muscle, 100 g liver, 50 g kidney and 50 g fat according to JECFA) was
4.3 ng and 20 ng of E2 in untreated and E2-treated cattle, respectively. The new threshold of 0.4 ng E2
/day would thus be reached after the ingestion of 47 g of untreated meat and of as little as 10 g of
hormone treated meat. These authors also estimated that eating meat from E 2 –treated cattle increased
the daily intake of E2 from food by 38% compared to non-treated meat. This percentage, and thus the
potential health risk, will be considerably higher if the food intake from pork, poultry, eggs and dairy
products derived from E2 –treated farm animals are taken into account.
336. Synthetic hormone growth promoters such as Ze ranol and its metabolites have been shown to
be as potent as E 2 and diethylstilbestrol (DES) in increasing the expression of estrogen-related genes
in human breast cancer cells (Leffers et al 2001). On the other hand, the synthetic androgen
Trenbolone and the gestagen Melengestrol bind w ith high affinity to the human androgen and
progesterone receptors, respectively (Bauer et al 2000). Exposure during pregnancy might result in
severe transplacental virilisation of a female fetus.
 Bauer ER, Daxenberger A, Petri T, Sauerwein H & Meyer HH (2000) Characterisation of the
affinity of different anabolics and synthetic horm ones to the human androgen receptor, human sex
hormone binding globulin and to the bovine progestin receptor. APMIS 108, 838-846.

 Daxenberger A, Ibarreta D & Meyer HH (2001) Po ssible health impact of animal oestrogens
in food. Hum. Reprod. Update. 7, 340-355.

 Leffers H, Naesby M, Vendelbo B, Skakkebaek NE & Jorgensen M (2001) Oestrogenic
potencies of Zeranol, oestradiol, diethylstilboestro l, Bisphenol-A and genistein: implications for
exposure assessment of potential endocrine disrupters. Hum. Reprod. 16, 1037-1045.

42. To what extent, in your view, has JECFA taken into account the particular situation of
sensitive populations, in particular prepubertal children, in its risk assessments with
respect to oestradiol-17 β? Please compare the original data concerning endogenous
production of natural hormones by prepubertal children upon which JECFA based its
assessment and those used by the European Communities in its risk assessment. In your
view, does the scientific material referred to by the European Communities require a
revision of the Codex recommendation with respect to oestradiol-17β?

Dr. Boisseau

337. Individuals with the lowest endogenous hormone levels have been considered as being at
greater risk by JECFA because the rationale followed by JECFA was, in the case of natural hormones,
to avoid that any excess intake of hormonally active r esidues be significant with regard to the rate of
their daily production of hormones in order to protect them from any physiological disturbance. In
this respect, the target was that the hormonal residue intake does not account for more than 1% of the
daily production rate of any group of the human popul ation. Therefore, the lower is the endogenous
production of hormones in a given human group, the lower must be the excess intake of hormonally
active residues for the individuals of this group.
338. From a qualitative point of view, the risks for these individuals arising from residues resulting
from the use of hormones growth promoters in cattle does not differ from the risks arising from the
residues of hormones naturally present in meat. The potential problem which may exist is only a
quantitative one.
Dr. Boobis

Consideration of sensitive populations by JECFA

339. In keeping with its risk assessment principl es, the ADI established by JECFA would have
been designed to protect all segments of the population, including prepubertal children ( IPCS: EHC
70, 1987 and EHC 104, 1990; IPCS, 2005; WHO, 1996 and 2001). For this reason, in establishing
the ADI from the NOAEL in a human study, JECFA u sed a 10-fold safety factor to protect sensitive
populations, in addition to a 10-fold factor to allo w for interindividual variation within the adult
human population. For an evaluation of the impact of differences in assumed endogenous production
of hormones by JECFA and the EC on the risk assessment see my reply to question 40 above.
340. In my view, there is no requirement for any revision in the Codex recommendation with
respect to oestradiol-17β on the basis of the material referred to by the EC.
Dr. Sippell

341. To my knowledge, the particular situation of sensitive populations, in particular infants and
prepubertal children, has not been adequately taken into account by JECFA in respect of E2.
342. The original data on prepubertal children used by JECFA have been questioned in a number
of more recent publications (Andersson & Skakke baek 1999, Maume et al 2001, Partsch & Sippell

2001) in view of the new ADI based on the ultrasensitive RCBA determination of E 2 in children. The
data have already been compared in the answer to questions 39 and 41.
343. In my view, the scientific material referred to by the European Communities definitely
requires a revision of the codex recommendation with respect to E2, as outlined above.
 Andersson AM & Skakkebaek NE (1999) Exposu re to exogenous estrogens in food: possible
impact on human development and health. Eur. J. Endocrinol. 140, 477-485.

 Maume D, Deceuninck Y, Pouponneau K, Paris A, Le Bizec B & Andre F (2001) Assessment
of estradiol and its metabolites in meat. APMIS 109, 32-38.

 Partsch CJ & Dr. Sippell WG (2001) Pathogenesi s and epidemiology of precocious puberty.
Effects of exogenous oestrogens. Hum. Reprod. Update. 7, 292-302.

[For the questions in this section, see paras. 121-122 of EC Rebuttal Submission (US case),
paras. 103-104 of EC Rebuttal Submission (Canada case), Exhibits EC-88, 99, paras. 42-45 of
US Rebuttal Submission, paras. 84 and 159 of US First Submission, and for JECFA's work
Exhibits CDA 11, 16, 17, 18, 39]

(e) Bioavailability
43. Please define bioavailability, comment on the significance of bioavailability to
assessments of risk, and on the degree of bi oavailabilitiy of the residues of the hormones
at issue when consumed in meat, taking into account parties' differing views on this
matter. [see paras. 123-124 of EC Rebuttal Submission (US case), para. 105-106 of EC
Rebuttal Submission (Canada case), paras. 100, 155-159 of the EC Replies to Panel
Questions, paras. 32 and 41-42 of US Rebuttal Submission, paras. 69, 71, 88-89 and 146
of US First Submission, and para. 134 of Canada Rebuttal Submission]

Dr. Boisseau

344. Bioavailability is the capacity of a substance to enter the general blood circulation and to
diffuse into the whole body of th e animal or the human being administered this substance. The
physico-chemical characteristics of substances and their route of administration to humans and
animals are of a paramount importance regarding the ra te at which they are bioavailable. Oral route,
which is the route for the ingestion of residues, is no t the most efficient. It is even very poor for the
three natural hormones.
345. The bioavailability of residues has to be taken into consideration in the risk asessment, in
particular at the third step regarding the exposur e assessment of residues. Residues which are not
bioavailable are not of concern for the public health if the toxicological potential of the substance is a
systemic one. The residues, the bioavailability of which has not been determined, are considered as
totally bioavailable. Those which have been recognised as non bioavailable can be discarded from the
exposure assessment.
346. Natural hormones are known to be poorly bioavailable in humans : oestradiol-17 β is inactive
orally, the bioavailability of prog esterone and of testosterone are respectively less than 10% and
around 4%.
347. The bioavailability of melengestrol, trenbol one and zeranol residues have not been
determined.Therefore all their residues have been considered as being totally bioavailable.

Dr. Boobis

348. Bioavailability can be defined as that fraction of a dose that is available to the systemic
circulation. It can take values from 0, no systemic availability, to 1, 100% availability. It is normally
estimated by comparing the dose-corrected area under the plasma concentration time curve (AUC)
after dosing by the route of interest, for exam ple orally, with the dose-corrected AUC after
intravenous administration (assumed to be completely available) (Rowland and Tozer, 1995). T h e
bioavailability reflects both the extent of absorption and any presystemic metabolism that occurs. In
the case of the oral route, this could be in the intestinal tract and/or the liver
 Rowland M and Tozer T (1995). Clinical Phar macokinetics: Concepts and Applications,
Lippincott Williams and Wilkins, London

349. In general, only that fraction of the dose that is bioavailable is toxicologically relevant. The
exceptions would be when some or all of the non- bioavailable dose is in the form of a bioavailable
metabolite that is biologically active, or when the effects of concern o ccur presystemically, for
example local effects on the gastrointestinal muco sa. In the case of the natural hormones, low
bioavailability is due to presystemic metabolism to products with substa ntially reduced hormonal
activity and their bioavailability is <5-10% ( Christiaens et al, 2005; Hoogenboom et al, 2001;
JECFA, 2000; Jockenhövel, 2002;Kuhnz et al, 1993; Stanczyk, 2003) . The effects of concern are
systemic. Hence, the toxicological significance of the low bioavailability of hormones is that the risk
is less than from the equivalent dose produced endo genously or administered by some other route
with higher bioavailability (e.g. subcutaneous implant).
 Christiaens V, Berckmans P, Haelens A, W itters H and Claessens F (2005). Comparison of
different androgen bioassays in the screening for envi ronmental (anti)androgen ic activity. Environ
Toxicol Chem, 24:2646-2656

 Hoogenboom LAP, de Haan L, Hooijerink D, Bor G, Murk AJ and Brouwer A (2001).
Estrogenic activity of estradiol and its metabolites in the ER-CALUX assay with human T47D breast
cells. APMIS, 109:101-107

 Jockenhövel F (2002). Practical aspects of testosterone substitution. Aging Male,
5 (Suppl 1):21–46

 Stanczyk FZ (2003). All progestins are not created equal. Steroids, 68:879-890

350. However, low bioavailability does not necessarily increase the margin of safety (the ratio of
ADI to actual exposure). This is because the e ffects of concern are usually determined following
exposure by the route of interest, in this case oral . Hence, the ADI represents a "bioavailability
adjusted" dose, just as the TMDI does. The conse quence of this is that anything that increases
bioavailability will reduce the margin of safety whilst anything that reduces bioavailability will
increase the margin of safety. In the case of the natural hormones, changes in bioavailability are
likely to be a consequence of changes in the en zymes of metabolism in the liver and/or small
intestine.
351. The bioavailability of the non-natural hormon es (melengestrol acetate, trenbolone acetate and
zeranol) in humans is, to my knowle dge, not known. Whilst it is likel y that it will be less than 100%
there is no specific information available. However, it should be noted that in the risk assessment of
these hormones by JECFA, the risk characterizati on involved comparison of the theoretical maximum
daily intake with the ADI. No correction was made for bioavailability. Hence, the situation is likely
to be similar to that for the natural hormones, in that changes in bioavailability from the normal value
would change the margin of safety.

Dr. Guttenplan

352. Bioavailability is defined as the fraction of a ch emical that enters the general circulation by
oral administration compared with that entering the circulation by iv. Injection.
353. Presumably, only the bioavailable chemical can produce adverse (or any) effects, thus in
terms of risk assessment, only the portion of the dose of chemical that is bioavailable is significant.
(Toxicokinetics in the National Toxicology Progr am. NIDA Research Monograph. 173:273-304,
1997.)
354. The US and Canada maintain that orally ing ested estradiol (the major potentially harmful
hormone) is essentially inactive, because of poor bioavailability.
355. The US states (para. 41, US rebut. Sub.) "The EC also asserts that the U.S. argument that
estradiol 17β is generally inactive when given orally, while "well known", is "still controversial and
not consensually accepted by the scientific communi ty." To the contrary, estradiol's low oral
bioavailability has found international support in Codex and JECFA ("[i]n general, estradiol 17 β is
inactive when given orally because it is inactivated in the gastrointestinal tract and liver"), as well as
support within the EC from the CVMP, which noted that "the bioavailability of 17β-oestradiol esters
after oral administration is low (3% as unchanged oestradiol) , but might be higher if estrone, an
estrogenic metabolite, is included." Also, included is a reference that in a cell culture model
(immortalized human intestinal cells (Caco-2 ce lls)) estrogen does not enter the cell although the
estradiol convertible metabolite was detected in th e cells. The US states that this supports the
hypothesis that estradiol cannot cross the intestin al wall. This latter finding seems paradoxical,
because somehow estradiol was converted to estrone, so it must have entered the cell.
356. On the other hand the EC maintains (EC Rebut, para 123) that "recent developments put in
doubt the findings of the 1999 JECFA report concer ns the bioavailability of residues of these
hormones. The 1999 and 2002 SCVPH reports ha ve found that data on which JECFA based its
findings are incorrect or insufficient." For instan ce, "Metabolic studies of orally administered 17ß-
oestradiol indicate that as much as 20 percent of a 2 mg dose of micronized E2 is absorbed, with a
serum half-life in the range of 2 to 16 hour s (Zimmermann et al., 1998; Vree and Timmer, 1988:
Ginsburg et al., 1998) and in it is stated that the above reports indicate, the bioavailability of fine-
particle 17β-oestradiol administered orally was determined to be 5% compared to a dose administered
intravenously. It is also pointed out (EC Rebut, para 124) lipoidal esters (m etabolites of estradiol)
have high bioavailab ility and may accumulate in adipose tissue. No data accompany the
accumulation hypothesis, although it seems reasonable.
357. It appears that the bioavailability of estrogen is low but not insignificant (probably between 5
and 20%, if estrone is also taken into account. (Est rone is readily inter- convertible with estrogen).
Calculations are presented in the above reference th at suggest that even with low percentages of
bioavailability of estrogen, the levels in meat c ould result in bioavailable estrogen approaching the
daily production rate of oestradiol in pre-pube rtal children (EC Rebut, para 122). This would
represent a risk factor (EC Rebut, para 122).
(f) Good veterinary practice (GVP)
44. Please define "good veterinary practice" (GVP) and/or "good practice in the use of
veterinary drugs" (GPVD). What are th e relevant Codex standards, guidelines or
recommendations relating to GVP/GPVD? Please comment on the statement by the
European Communities that the definition of the GPVD is "circular and hence
problematic." [see para. 88 of the EC Replies to Panel Questions]

Dr. Boisseau

358. There are several definitions for the good ve terinary practice (GVP) and the following one
adopted by Codex is satisfactory : " GVP in the use of veterinary drugs is the officially recommended
or authorized usage, including withdrawal periods , approved by national authorities, of veterinary
drugs under practical conditions "
359. Nevertheless, to my knowledge , the Codex Commission has only adopted, in July 2005, a
guideline on GVP intended to minimize and put under control the microbial resistance. It did not
adopt any guideline on GVP aimed at minimizing the occurrence of veterinary drug residues in animal
derived food.
360. The comment of the Europan Communties about GVP is not clear. I suppose that the
European Communities means that the conditions of used of veterinary drugs, even officially
approved, may differ in a very significant way from one country to another one.
Dr. De Brabander

361. Most probably, several definitions could be f ound for defining GVP. Hereunder I give one of
the Veterinarians of Europe:
 Federation of Veterinarians of Europe: Code of good veterinary practice

Veterinarians play an important role in protecting animal welfare, animal health, public health as
well as the environment and provide a wide range of services.

This Code of Good Veterinary Practice is a standard specifying the European veterinary ethics and
principles of conduct as well as the requirements relating to the quality management system within a
veterinary organisation, when the latter:

1. Wishes to improve its ability to give services in conformity with:

• The legislation in force,
• The Professional Code of Conduct in force,
• The requirements of the clients,
• The ethics principles relating to the service s provided and/or the animals under its
care.

2. Must demonstrate its ability to deliver service s, which are constantly in line with customer
requirements and the legislation in force.

362. A selection of paragraphs on Public Health and the environment is also given below:
2. G Veterinarians and Public Health

• Veterinarians shall seek to ensure the best protection of public health.

• Veterinarians shall, whenever appropriate, advise their customers about measures to
minimise the risk of exposure to zoonotic agents, food borne pathogens, residues,
contaminants (biological and chemical agents) and antimicrobial resistance.
• Veterinarians shall make animal owners aware of their responsibilities to the public.

2. H Veterinarians and the Environment

• Veterinarians shall attempt to reduce pollution of the environment by waste
avoidance, recycling, using re-usable articles when appropriate, and correct disposal
of waste.

• Veterinarians shall endeavour to redu ce environmental pollution by careful and
appropriate use of disinfectants, medicinal products and other chemicals.

• Veterinarians shall aim to be environmen tally responsible by the economical use of
energy and water.

• Veterinarians shall organise facilities for se parate collection of different types of
waste so that they can be sent to the appropriate recycling points.

• Veterinarians shall encourage customers to dispose of veterinary waste in a safe
manner.

363. This selection illustrates the importance of the veterinarians in reduction of pollution of the
environment and the link between residue analysis a nd environmental sciences. This applies also to
the use of hormones
364. Good practice in the use of veterinary drugs (GPVD), as defined by the CCRVDF, is the
official recommended or authorized usage includi ng withdrawal periods, approved by national
authorities, of veterinary drugs under practical conditions ( document: Recommended international
code of practice for control of the use of veterinary drugs cac/rcp 38-1993 ; cxp_038e).
365. The statement by the European Communiti es that the definition of the GPVD is " somewhat
circular and hence problematic" refers to the fact that the national authorities have a large impact on
this so-called international standard and can influence the application of it.
366. Neither GVP neither GPVD will reduce the risk of "using hormones" for several reasons.
Also education plays an important role. Two years ago we had some American students in veterinary
medicine in an exchange program : their knowledge of "hormones" their use in the USA and the risks
involved was almost zero.
45. In conducting a risk assessment of specific veterinary drugs, what assumptions are made
concerning GVP, if any? How, if at all, a re risks that might arise from the failure to
follow good veterinary practice in the administration of veterinary drugs addressed?

Dr. Boisseau

367. Residue data considered by all the committ ees such as JECFA, CVMP etc… conducting risk
assessments of veterinary drugs are always obtai ned from studies in which veterinary drugs under
review have been admnistered to the target food producing animals according to the officially
approved conditions of use of these veterinary drug s. Therefore, MRLs adopted by Codex are only
meaningful in countries where GVP are effectivel y implemented. In addition, it would not be
appropriate for the risk assessors to address the cas e where the veterinary dr ugs under review could
not be used in practice according to the GVPs. It would not be appropriate because it would not be
possible for the risk assessors to identify all the possible misuses/abuses and to get the residue data
derived from these misuses/abuses. It would not be appropriate also because it would not be ethical
for the case where such data, being available, would lead to the conclusion of the risk assessment that,

given a possibly wide margin of safety for a ve terinary drug under review, the excess intake of
residues associated with these misuses/abuses does not raise any problem of public health. That
would encourage all theses misuses/ abuses. Therefore, this issue of GVP, misuses/abuses must be
taken into consideration not by the risk assessors but by the risk managers.
Dr. De Brabander

368. In conducting a risk assessment of specific ve terinary drugs, it is assummed that GVP is
followed. However can this be guaranteed?
369. In "A Primer on Beef Hormones" Date : 02/26/99 text released by the U.S. Interagency Task
Force on Beef Hormones. the following could be read:
"Furthermore, the prescribed dosage is the level which produces the maximum
economic response in the animal -- the law of diminishing returns -- so that there is
no economic incentive for a farmer to use additional implants. A U.S. control system
ensures that animals taken to slaughter have normal hormone levels. Thus, farmers
have no incentive, economic or otherwise, to misuse the implants."
370. However, arround the same date studies could be found on the use of Zilpaterol (a powerful
beta-agonist of the 3th generation) on top of re valor (both control animals and test animals are
implanted with revalor). This illustrates that fa rmers (and vets) have indeed economic incentives to
misuse growth promoting substances (implants or others).
371. I am not aware of any study on public health of the combination of zilpaterol with the
substances in revalor. Are the animals of this experiment destroyed?
372. Proceedings, Western Section, American So ciety of Animal Science Vol. 50, 1999
INFLUENCE OF THE beta-AGONIST, ZILPATEROL, ON GROWTH PERFORMANCE AND
CARCASS CHARACTERISTICS OF FEEDLOT STEERS
A. Plascencia
1, N. Torrentera1, and R.A. Zinn2
1Universidad Autónoma de Baja California, Mexicali (México)
2University of California, Davis

ABSTRACT: One hundred forty crossbred steers (373 kg) were used in a randomized complete block
design experiment (14 pens, 10 steers/pen) to evaluate the influence of supplementation of a steam-
rolled wheat-based finishing diet with 6 mg/kg (as-fed basis) zilpaterol during the final 6 weeks of the
finishing period on growth performance and carcass characteristics. Supplemental zilpaterol did not
influence (P > .20) DM intake (8.55 vs 8.45 kg/d), but enhanced (P < .01) ADG (27%, 1.42 vs 1.94
kg/d), and feed efficiency (28%, 6.08 vs 4.37). Based on observed NE intake, ADG of the non-
supplemented steers was 99% of expected. In contrast, with zilpaterol supplemented steers ADG was
29% greater (P < .01)

Experimental Procedure:
One hundred and forty crossbreed yearlings steers (373 kg) were used in a 42-d finishing trial. Steers
were blocked by weight and randomly assigned, within weight groupings, to 14 pens (10 steers/pen).
Pens were 510 m with 64 m overhead shade, auto matic waterers, and 17 m fence-line feed bunks.
The trial was initiated July 22, 1997. Treatments c onsisted of a steam-flaked wheat-based finishing
diet (Table 1) supplemented (as fed basis) with 0 or 6 mg/kg zilpaterol (Zilmax , Hoechst Roussel
Vet, D.F., Mexico). Steers were implanted with Revalor (Hoechst Roussel Vet, D.F., Mexico) upon
initiation of the trial. Steers were allowed ad libitum access to experimental diets.

46. To what extent were risks from misuse or abuse assessed by JECFA in its evaluation of
the hormones at issue? In terms of the three synthetic hormones at issue, how is GVP
relevant to the establishment of MRLs by JECFA?

Dr. Boisseau

373. My reply to question 45 above applies also to this question. It covers also the three synthetic
hormones which do not raise any specific problem in this domain.
Dr. Boobis

Assessment of risks from misuse and abuse by JECFA

374. Where the conclusion of a risk assessment is such that it is considered appropriate to establish
an ADI to protect health (i.e. the critical effects e xhibit a threshold), no consid eration is given to the
likely levels of exposure. The ADI is driven entirel y by the toxicology and other relevant biological
effects of the substance, which determine the point of departure (usually the NOAEL), and an
appropriate uncertainty (or safety) factor. The un certainty factor used has to account for any inter-
species extrapolation, interindividual differences, sensitive sub-populations, absence of a NOAEL and
any non-critical gaps in the database (see my repl y to question 8 above). In establishing MRLs
(relevant here only for the three synthetic horm ones), the appropriate residues studies are those
obtained after the normal use of the hormones, i.e. in accordance with GVP. This is the policy of all
agencies and organisations involved in such activities (EEC, 1990; EMEA, 2005; FAO, 2006) (see
response to question 62 below under "Multiple implanting, multiple dosing"). The point at which
misuse and abuse are relevant in the risk assessme nt is at the risk characterisation stage, when
potential exposure is compared with the ADI. Henc e, whilst the TMDI is estimated following use of
the hormones according to GVP, it would also be po ssible to consider other exposure scenarios, in
which the hormones were misused or abused. Wh ere exposure exceeded the ADI, the toxicological
implications of this would depend on a number of factors (see respon se to question 62 below). These
are as follows:
• The likelihood of violations or off-label use
• The residue levels occurring after such misuse or abuse
• The extent to which exposure to such residues will result in an exceedance of the ADI
• The likely frequency or period over which the ADI will be exceeded
• The acute consequences of exceeding the ADI
• The severity of the endpoint upon which the ADI is based
• The steepness of the dose-response curve for the endpoint upon which the ADI is
based

375. JECFA did consider misuse of zeranol when it was evaluated in 1988 (JECFA, 1988b) .
JECFA gave some consideration of the effects of doses of up to 20 times those approved for the use of
MGA on residue levels (JECFA, 2000a) . JECFA does not appear to have explicitly considered
misuse or abuse of trenbolone. The implications of misuse and abuse of these hormones for human
health are considered below (question 62). Ther e would have been no implications for the MRLs
recommended. It would have been a decision of Codex as to how it would deal with any exceedances
of the ADIs as a consequence of misuse or abuse, as this involves risk management decisions.
 EEC (1990). Council Regulation (EEC) No 2377/90 of 26 June 1990 laying down a
Community procedure for the establishment of maximum residue limits of veterinary medicinal
products in foodstuffs of animal origin. OJ L 224, 18.8.1990, p. 1–8

 FAO (2006). Updating the Principles and Methods of Risk Assessment: MRLs for Pesticides
and Veterinary Drugs, Rome Italy
(http://www.fao.org/ag/AGP/AGPP/Pesticid/JMPR/DOWNLOAD/bilthoven_2005.pdf)

 JECFA (1988b). Residues of some veterinary drugs in foods and animals, Vol. 41/1, FAO,
Rome, Italy

Dr. De Brabander

376. Up to my knowledge, risks from misuse or a buse are not assessed (even denied) by JECFA in
its evaluation of the hormones at issue. As was illustrated by the example of the experiments with
Zilpaterol (question 45), GVP is very relevant to the establishment of MRLs by JECFA. If other
substances (like zilpaterol or ZMA etc…) or incorrect use of implants are used the principle of the
establishment of MRLs by JECFA is certainly unvalid.
47. How significant are any differences in GVP in the European Communities, the United
States, and Canada? Does the EC risk asses sment take into account relevant control
mechanisms with respect to GVP in place in the United States and/or Canada? If so,
what are their conclusions?

Dr. Boisseau

377. In my e-mail of 26/04/06, I have indicated that I did not think that I am in the position to
answer this question. Nevertheless, I think that, as far as growth promoters are concerned, the main
problem for the European Communities is that these products are, in the USA and Canada, sold over
the counter without any veterinary prescription.
378. As, as it has already been said, the Europ ean Communities did not conduct any quantitative
risk assessment for growth promoters, it is not po ssible to say that the European Communities took
into account relevant control mechanisms with respect to GVPs in place in the USA and/or Canada.
Dr. De Brabander

379. The interpretation of the rules of GVP can be different from country to country. Concerning
the dispute on hormones THE difference is of course that in Europe the use of hormones as
production aids is not allowed (and thus not included in GVP).
380. As to the relevant control mechanisms with respect to GVP in place in the United States
and/or Canada: any control mechanism, that is onl y based on audits and paper work will not prevent
farmers to use either uncorrect use of legal produc tion aids either the use of other illegal growth
promotors which are readily available in the US and Canada through the internet.
381. An example: on 2006-17 April 21, 2006 (thu s very recently) Health Canada advises
consumers not to use unauthorized produc ts containing anabolic steroids. http://www.hc-
sc.gc.ca/ahc-asc/media/advisories-avis/2006/2006_17_e.html
382. OTTAWA - Health Canada is advising consumers not to use five products containing illegal
anabolic steroids, as they can potentially cause serious health issues such as liv er disorders and heart
problems. The five products are: Anabolic Xtreme Superdrol, Methyl-1-P, Ergomax LMG,
Prostanozol and FiniGenX Magnum Liquid. They ar e not authorized for sale in Canada as either
drugs or natural health products . Canadians using any of these pr oducts or any other supplements
containing anabolic steroids are advised to stop ta king these products immediately and consult with a
health care professional.

383. All these products are available through the In ternet, maybe mostly for use in bodybuilding
but they can also be used in cattle fattening.
384. Are there methods and laboratories to control the abuse of these substances in the US and
Canada?
385. Note that "Health Canada" (correctly) recognises the danger of these substances.
48. To what extent does the scientific evid ence referred to by the European Communities
assess risks to human health from residues of misplaced implants or improper
administration, i.e. when administered differently than indicated on the label of the
manufacturer or contrary to GVP, of any of the six hormones? Would your reply have
been different at the time of adoption of the EC Directive in September 2003? What are
the potential hazards, if any, to human heal th of the use of large quantities, or doses
higher than recommended, of any of the six hormones in dispute?

Dr. Boisseau

386. Once again, the reply given to the question No 45 applies also to this question. The outcome
of a risk assessment of veterinary drug residues, including the six hormones at issue, cannot apply in
the case of misuse/abuse such as use of dosages higher than those approved, use of not approved
combinations, repeated administrations, deep intr amuscular injections instead of implantation of
pellets etc…In this respect, the European Communities is right to state that, in case of these different
misuses/abuses, the exposure of consumers may be tota lly different. Once again, this situation is not
specifc to hormones as it applies also to all the veterinary drugs already assessed by JECFA, EU, USA
or anywhere else in the world.
387. Having said that, as the European Communities did not conduct any quantitative risk
assessment for growth promoters, it is not possible to say that the scientific evidence referred to by the
European Communities assesses th e risk to human health from residues resulting from these
misuses/abuses.
388. My reply would not have been different at the time of adoption of the EC directive in
September 2003.
389. ADIs are established in order to prevent the t oxicological/physiological effects, associated
with the corresponding NOAEL, to occur. In case of misuses/abuses, the exposure of consumers to
residues may increase to such an extent that the intake of these residues may exceed the established
ADI and the toxicological/physiological effects intended to be avoided may occur.
Dr. Boobis

Assessment of risk to human health from misuse or abuse of the hormones in the scientific evidence
referred to by the EC

390. The evidence itself is discussed in detail under qu estion 62 below. There was no attempt to
evaluate the risks from the resultant exposures on misuse or abuse, either in the papers cited or by the
SCVPH (2002) in their evaluation of these studies. Indeed, the SCVPH (2002) simply noted that
"Therefore, these data have to be considered in any quantitative exposure assessment exercise",
without undertaking such an exercise. However, it should perhaps be pointed out that the EC had
previously taken the view that there was no threshol d for some of the critical effects of the hormones
and that it was therefore not appropriat e to conduct a quantitative risk assessment (SCVPH, 1999) .
The SCVPH did not change their view on this in their 2002 evaluation of the new information.

391. My reply would not have been different at the time of adoption of the EC directive in
September, 2003.
392. In my view, the potential hazards from the u se of large quantities of the six hormones in
dispute are those dependent on their endocrine activity, including cancer in hormonally responsive
tissues. However, I should stress that this is th eir potential hazard. The potential risk, i.e. the
probability that effects would occur, would depe nd on a number of factors. These include the
magnitude of the exposure, the duration of the expos ure and the life stage of the exposed individual.
From the range of exposures likely from anticipated mi suse or abuse the risks are likely to be very
low (see question 62).
Dr. De Brabander

393. The answer to this question is already give n in question 47. Improper administration of
implants or misplaced implants create potential hazards to human health. Moreover, as already
mentioned the potential hazards to human health are not the only factor in the debate of the use of
hormones as production aids. There are also:
 – animal welfare: how do animals feel at improper administration of implants or
misplaced implants?

 – the environment: excretion of an excess of hormones at improper administration of
implants or misplaced implants disturbs the hormonal balance in surface water.

 – transformation of hormones: enzymatic reactions are equilibrium reactions (the
enzyme is just a catalyst for the reaction) ; excess of hormones can drive a enzymatic
reaction in another direction (e.g. formati on of boldenone out of testosterone). Little
is still known about this phenomenon.

394. If my reply would have be different at the time of adoption of the EC Directive in September
2003 is a difficult question. Fact is that my reply now is certainly different: more and more scientific
data sustain the ban on the use of hormones: the economical profits resulting from using hormones do
not balance the potential danger in all of its aspects.
49. What analytical methods, or other technical means, for residue detection in tissues exist
to control the use of the six hormones in dispute for growth promotion purposes in
accordance with good animal husbandry practice and/or good veterinary practice?
What tools are available to control the use by farmers of the six hormones in dispute for
growth promotion purposes in accordance with good animal husbandry practice and/or
good veterinary practice?

Dr. De Brabander

395. There are a large number of analytical methods available to control the use of the six
hormones in dispute for growth promotion purposes. New methods are regularly presented in
international conferences and in the open literature . In Europe a system of community reference
(CRL) and national reference laboratories (NRL) is in stalled so that the analysis carried out by the
field laboratories are kept up to the standards of the moment. If necessary I can provide the panel
with a large number of methods but I don't think that is the purpose.
396. To control the use by farmers of the six hormones in dispute for growth promotion purposes a
number of tools can be used:

 – Analysis for legal and illegal hormones in urine, faeces, hair, animal feed and
drinking water.
 – Audit of the farms on the presence of le gal and illegal substa nces in all kind of
formulations.
 – Control of the weight gain of the anim als in the farm in comparison with normal
weigth gains.
 – Inspection of the herd on certain symptoms (e.g. hypothyreosis).
 – Inspection of the animals at slaughter (e.g. for injection sites).

50. Are there other measures available to the European Communities (other than a
complete ban) which could address risks ar ising from misuse and failure to follow good
veterinary practice with respect to the use of the hormones at issue for growth
promotion purposes? Would your reply have been different at the time of adoption of
the EC Directive in September 2003? If so, why?

Dr. Boisseau

397. In my e-mail of 26/04/06, I have indicated that I did not think that I am in the position to
answer this question.
398. Nevertheless, I would like to say that, in a given country, the implementation of GVPs and the
elimination of misuses/abuses of veterinary drugs mu st be the responsability of the official authorities
of this country which should be able to demonstrat e that (1) veterinary drugs are effectively used in
compliance with GVP, (2) veterinary drugs associated with sensitive public h ealth issues are used
under veterinary control, (3) the official contro ls, including surveillance of residues, of the
implementation of these GVPs are efficient. In the case where it can be established that an
exportating country is not in the situation to guaran tee that veterinary drugs are effectively used in
compliance with GVP, any importating country shoul d have the freedom to take any appropriate
measure likely to protect the health of its population. Ban is the last possible measure if all the other
options have failed or have been proved ineffective. In the case of hormonal growth promoters, due
to the temptation for the farmers to use these produc ts in a way different from the approved ones in
order to expect some more economical profit, a agreement should be made between an exporting and
an importing country about the content of GVP regarding the use of these products, including a
possible involvement of veterinary supervision/ prescription and an appropriate scheme of residue
surveillance. In addition, if legally authorised a nd technically feasible, an appropriate information of
the consumers of the importating country through a clear labelling could be considered as there is
nowadays an increasing demand from consumers about the tracking of food with an informative
labelling even concerning legally approved food additives.
399. My reply would not have been different at the time of adoption of the EC directive in
September 2003.
Dr. De Brabander

400. To my knowledge, there are no other measures po ssible to the European Communities, other
than a complete ban, which could address risks arising from misuse and failure to follow good
veterinary practice with respect to the use of the hormones at issue for growth promotion purposes.
401. My reply now is even stricter than it would have been at the time of adoption of the EC
Directive in September 2003.
51. Does the material put forth by the Europe an Communities regarding misuse or abuse of
the hormones at issue in the United States and Canada call into question the potential

applicability of Codex standards with regard to imports of meat from cattle treated with
hormones from the United States and Canada?

Dr. Boisseau

402. The replies given to the questions No 45 and 48 apply also to this question.
Dr. De Brabander

403. The material put forth by the European Co mmunities regarding misuse or abuse of the
hormones at issue in the United States and Cana da calls indeed into question the potential
applicability of Codex standards with regard to imports of meat from cattle treated with hormones
from the United States and Canada. Since the "older" experiments on which the MRLs (for the
3 synthetic hormones) and the ADIs (for the 3 natu ral hormones) are based, the scientific knowledge
on residues, their link with animal welfare an d the impact on the environment has increased
considerably. These items are alr eady discussed in the answers on ot her questions and show clearly
that the economical profit of using hormones as production aids doesn't balance the present and
potential hazards. Moreover, most consumers aren't prepared to take this risk.
404. Regularly new findings on the residue domain ar e published. As example, the findings in our
own laboratory with maggots of Lucilia Sericata, a blowfly. We were able to demonstrate that
maggots of Lucilia Sericata , when exposed to the hormone are able to convert testosterone into
boldenone (2.2 %), boldione (or ADD, 1 %) and AED (15 %).
Boldenone formation by maggots of Lucilia Se ricata K. Verheyden, H. Noppe, J. Vercruyss ,
E. Claerebout, V. Mortier, C.R. Janssen, H.F. De Brabander Anal. Chim Acta 2006, submitted

405. This is only one example of a number of s till unknown reactions and illustrates that it is
dangerous to introduce substances which may disturb the equilibrium of enzymatic reactions in the
body of an animal or a human being (in this case equilibrium of hormones). I have serious doubts that
in all the "old" studies which have lead to the establishment of ADI's and MRL's for these substances,
these and analogous reactions are taken into account and for instance boldenone en ADD are
measured.
[For questions on GVP see the SCVPH Opinions in Exhibits US-1,4, and 17, para.125-127 of EC
Rebuttal Submission (US case), paras. 107-109 of EC Rebuttal Submission (Canada case), para.
154 of EC Replies to Panel Questions, Exhibits EC-12, 67, 68, 69, 70, 73, 96, 102, 103, paras. 32
and 54-65 of US Rebuttal Submission, para. 75 of US First Submission, paras. 107-111 of
Canada Rebuttal Submission, page 40 of Exhibit CDA-27]

(g) Other
52. Do the risk assessment of the European Communities or any other scientific materials
referred to by the European Communities de monstrate that a potential for adverse
effects on human health arises from the consumption of meat from cattle treated with
any of the six hormones in dispute for growth -promotion purposes? If yes, why? If not,
what kind of evidence would be required to demonstrate such potential adverse affects?
Would your response have been different at the time of adoption of the Directive in
September 2003?

Dr. Boisseau

406. As already said in the reply to the question No 16, the European Communities did not carry
out, strictly speaking, a risk assessment but pr ovided scientific data and hypothesis supporting its
worries regarding the safety of these six hormones for human health. Therefore, the European
Communities concluded that, given the genotoxic potential of oestradiol-17 β, it is not possible to
accept any excess intake of residues of this hormone as they are likely to raise a problem of health for
consumers and that the available data for the five other hormones were not sufficient to carried out a
risk assssment. For the three na tural hormones, the European Communities should have integrated in
its risk assessment the exposure of consumers to these hormones resulting from the consumption of
hormone residues from animals which have not been treated by hormonal growth promoters and the
from the daily production of these hormones by human s. So, to my view, the European Communities
did not demonstrate that a potential for adverse e ffects on human health arises from the consumption
of meat from cattle treated with any of the six hor mones in dispute for growth promotion purposes.
The kind of evidence required to demonstrate such potential adverse effects should be (1)
toxicological data indicating that the values of the ADIs established by JECFA are not conservative
enough, (2) data on residues in treated/non treated cattle and on daily production of hormones in
sensitive individuals indicating that the hormonal residue intake associated with the consumption of
meat from treated cattle is such that the establis hed ADIs would be exceeded in the case of use of
growth promoters.
407. My reply would not have been different at the time of adoption of the EC directive in
September 2003.
Dr. Boobis

Risk to humans from consumption of meat from animals treated with growth promoting hormones

408. This is a complex question to address. A ri sk assessment involves some interpretation of
data. For example, whether an effect is compound-re lated, whether an effect is adverse, the location
of any threshold, whether effects seen in vitro are apparent in vivo, whether associations reported in
epidemiological studies might be subject to bias or confounding (IPCS: EHC 70, 1987 and EHC 104,
1990; WHO, 1996 and 2001) . Hence, whether a risk assessment demonstrates that a potential for
adverse effects on human health arises from consump tion of meat from cattle treated with any of the
six hormones in dispute depends on those conducting the risk assessment. However, whether the
scientific information on which this risk assessment was based, or any other materials referred to by
the EC demonstrate such potential is another questio n. In my view, none of information provided by
the EC demonstrates the potential for adverse effects in humans of any of the six hormones in meat
from cattle in which they are used for growth promotion purposes at the levels to which those
consuming such meat would be exposed. The studies on genotoxicity provide no convincing evidence
of potential for harm in consumers. The weight of evidence is that the hormones are not genotoxic in
vivo even at doses well above those that would be present in meat from treated cattle. The
carcinogenic effects observed are entirely consistent with a hormonal mode of action that exhibits a
threshold that would be well above the intake aris ing from consumption of meat from treated cattle.
Other effects of the hormones that have been observe d either in experimental animals or in exposed
subjects occur at doses much higher than those to which consumers would be exposed via meat from
treated cattle. As such, there would be no risk of such effects in humans from such exposures. There
is much debate about the possible endocrine effe cts of low dose exposures to hormones such as
oestradiol. However, all of the major reviews in th is topic have concluded that whilst there are data
gaps, there is no evidence that low level exposure is causing harmful effects in humans.

409. "Analysis of the human data by itself, while ge nerating concerns, has so far failed to provide
firm evidence of direct causal associations between low-level (i.e., levels measured in the general
population) exposure to chemicals with EDCs and adverse health outcomes" (Damstra et al, 2002)
 Damstra, T., Barlow, S., Bergman, A., Kavlock, R., and Van der Kraak, G. (2002). Global
Assessment of the State-of-the-Science of E ndocrine Disruptors. WHO publication no.
WHO/PCS/EDC/02.2. World Health Organization, Geneva, Switzerland.

410. "However, it is somewhat reassuring that after s ubstantial research in the past decade, there
have been no conclusive findings of low-level environmental exposures to EASs [endocrine active
substances] causing human disease" (Miyamoto and Burger, 2003).
411. The question on what sort of evidence would be required to demonstrate such potential
adverse effects presupposes that they are demonstrable . In order to demonstrate whether or not such
effects occur it would be necessary to conduct studi es of human systemic exposure from consumption
of meat from treated cattle. Such studies would re quire very sensitive analytical methods, capable of
establishing whether there is any change in circula ting hormone levels in the first place. Studies
would need to be carried out in relevant sub-popul ations, such as prepubertal males. Epidemiological
studies, in which both exposure and outcome are carefully assessed would also be necessary.
However, it should be emphasised that on the basis of the information available, I would rate the risk
of adverse effects in humans consuming meat from treated cattle as minimal.
412. My response to this question would have been the same at the time of adoption of the EU
Directive in September 2003.
Dr. Guttenplan

413. Calculations are presented (EC Rebut, para 122) th at suggest that even with low percentages
of bioavailability of estrogen, the levels in meat could result in bioavailable estrogen exceeding the
daily production rate of oestradiol in pre-pubertal children (EC Rebut, para 122). Although the US
and Canada question the accuracy of the assay origin ally employed for estrogens at the low levels
found in children, recent reports (Wang, S., Paris, F., Sultan, C. S., Song, R. X., Demers, L. M.,
Sundaram, B., Settlage, J., Ohorodnik, S., and Santen, R. J. Recombinant cell ultrasensitive bioassay
for measurement of estrogens in postmenopausal women. J Clin Endocrinol Metab, 90: 1407-1413,
2005 and references therein), indicate that more recentl y reported levels used by the EC are accurate.
In addition, levels in post-menopausal women were also very low. However, other approximations
are used in that calculation. For pre-pubertal ch ildren, even with the low bioavailabilty of estrogen
along with and its low levels in meats, it appears pos sible that intake levels would be within an order
of magnitude of those of the daily production rate. This is greater than FDA's ADI and suggests some
risk to this population. If there genotoxic effects of estradiol in children, they may be reflected over a
lifetime, as mutations arising from DNA damage are permanent. It seems the more accurate methods
of analysis could now be used to measure the effect of eating hormone-treated beef on blood levels of
estrogen in children and post-menopausal women. If practical, this experiment would be important in
establishing or refuting the arguments of the EC.
414. My response would have been more uncertain in 2003, because the assay for serum levels of
estrogens was less validated then.
53. Please comment on the statement by th e European Communities that the natural
hormones progesterone and testosterone are used only in combination with
oestradiol-17β or other oestrogenic compounds in commercial preparations? Would the
systematic use of these and the synthe tic hormones in combination have any
implications on how the scientific experiments and the risk assessments are to be carried

out? If so, have the scientific materials referred to by the European Communities or
relevant JECFA reports taken into account the possible synergistic effects of such
combinations on human health? [see sections 4.2-4.3 of the Opinion of the SCVPH of
2002 in US Exhibit 1]

Dr. Boisseau

415. It is true, as it is written in the section 4.3. of the 2002 Opinion of SCVPH, that the data about
genotoxicity of hormones have obtained from tests conducted only with indi vidual substances as it
has been always the case for all the toxicity studies considered everywhere in the risk assessment of
veterinary drug residues. Considering that it has been established that progesterone and testosterone
are not genotoxic, it is not likely that the testing of combinations of progesterone or testosterone with
oestradiol-17β would have led to synergistic effects compared with those obtained from these
individual substances.
Dr. Guttenplan

416. From the data presented, it is true that pr ogesterone and testosterone are used only in
combination with oestradiol-17 β or other oestrogenic compounds in commercial preparations
(sections 4.2-4.3 of the Opinion of the SCVPH of 2002).
417. In principle the use of mixtures should complicat e risk assessments/scientific experiments, as
they would have to evaluate/investigate each compon ent alone and in combination. This is a major
undertaking as effects of individual agents may be additive, inhibitory, and synergistic or there may
no effect. It appears from the evidence submitted that, by far, estrogen is the major agent of risk and
because the concentrations of all of the hormones in beef are so low, that they would be unlikely to
affect the potency of estrogen. However, it appears that no experiments on effects of combinations
were performed, so some uncertainty exists there.
54. What is the acceptable level of risk ref lected in the Codex standards for the five
hormones at issue? How does this compa re to the European Communities' stated
objective of "no risk from exposure to unnecessary additional residues in meat of
animals treated with hormones for growth pr omotion". [see para. 149 of EC Rebuttal
Submission (US case)]

Dr. Boisseau

418. The acceptable level of risk reflected in the C odex standards for the five hormones at issue is,
as for all the other substances already assessed, expressed by the ADIs established for these
substances. The spirit of the risk assessment pro cedure adopted by the Codex and implemented by
JECFA is that the amount of residues expressed by an ADI represents the quantity of these residues
which can be ingested daily by consumers over life time without causing any problem of health. This
approach is obviously different from the European Communities' stated objective of "no risk from
exposure to unnecessary additional residues in meat of animals treated with hormones for growth
promotion" which implies, to be reached, that these five hormones should not be used. The idea
supporting this statement is probably that growth promoters raise a specific problem regarding the
benefit/risk assessment associated with their use. In the case of veterinary drugs used for therapy, the
risk is expressed in term of human health and the be nefit in terms of animal health and that can be
considered as ethical. For the growth promoters, th e benefit is "only" economical and for this reason,
the European Communities may not accept any risk, even theoritical, resulting from the use of these
growth promoters only intended to increase economical profits.

Dr. Boobis

Acceptable level of risk

419. The Codex standards for the hormones at issu e represent "no appreciable risk with daily
exposure over a lifetime" (Definition of ADI by WH O, 1996, 2001 and CVMP, 2005). This is based
on the JECFA conclusion that all of the potentially adverse effects of the hormones have thresholds.
By using a NOAEL or surrogate if necessary, such as the LOAEL, and appropriate uncertainty (or
safety) factors, a level of exposure is determined up to which the risk is considered minimal, i.e. the
ADI (see my replies to questions 7-12 above). The level of any residual risk has never been
quantified but is considered to be acceptable to society. The Codex st andard is equivalent to the EC's
stated objective of "no risk from exposure to unn ecessary additional residues in meat of animals
treated with hormones for growth promotion", and indeed is the dame as the CVMPs (2005) definition
of ADI. This is because the EC uses the same risk assessment paradigm as JECFA for establishing
ADIs and hence if the data are interpreted in the sa me way there should be no difference in the level
of risk identified in the risk assessment. From a scientific perspective, the difference arises from
differences in the way that the data are interpreted and in particular whether or not it is concluded that
there is a threshold for the effects of concern.
420. However, the distinction between risk assessmen t and risk management must be stressed (see
my reply to question 5 above). Whether to invoke the precautionary principle is a risk management
decision. It is beyond the scope of my responses to go into this in detail. One input to risk
management decisions is the output of the risk as sessment, part of which is an evaluation of the
uncertainty associated with that output. Risk management has to weigh the risk assessment and a
number of other factors in reaching a conclusion. Hen ce, the issue is in part not so much the level of
risk that is acceptable, but the level of concern should the risk estimates be incorrect.
Dr. Guttenplan

421. Codex has set ADI's for the hormones (except M GA, which, I couldn't locate) but states that
MRL's are not necessary as meat from animals ma intained with good animal husbandry practices
would not be likely to pose a threat to human health (CDA 22, Codex list of standards). This is not in
accord with the EC's stated objective. The EC maintains that for estroge n and possibly the other
hormones, some potential risk exists. The question of what level of risk has not been addressed by
the EC.
55. Do the Opinions of the European Communi ties or other scientific materials referred to
by the European Communities evaluate the extent to which residues of growth
promoting hormones in meat contribute to what the European Communities calls
"additive risks arising from the cumulative ex posures of humans to multiple hazards, in
addition to the endogenous production of some of these hormones by animals and
human beings"? Would your reply have been different at the time of adoption of the
EC Directive in September 2003? If so, why? [see para. 151 of EC Replies to Panel
Questions, paras. 43-44 of US Rebuttal Submission, paras. 83-85 of Canada's Rebuttal
Submission]

Dr. Boisseau

422. The European Communities did not assess quan titatively the extent to which residues of
growth promoting hormones in meat contribute to "additive risks arising from the cumulative
exposures of humans to multiple hazards, in additio n to the endogenous production of some of these
hormones by animals and human beings". Besid es the European Communiti es recognizes that in
saying, in the para 151 of its reply to Panel questions that " it is not so much necessary to compare (if

it is only possible) the two situations (residues of hormones in meat from cattle not treated with
growth-promoting hormones and residues in meat from cattle treated with growth-promoting
hormones) and then try to quantify how much one is more risky than the other one". Therefore, it can
be thought that the position of the European Commun ities is a position of principle. This position is
inflenced by risk management considerations and by the implementation of the so called
precautionary principle.
423. My reply would not have been different at the time of adoption of the EC Directive in
September 2003.
Dr. Boobis

Additive risks

424. Two different issues are represented here. One is so-called aggregate risk. This has been
defined as "the likelihood of the occurrence of an adverse health effect r esulting from all routes of
exposure to a single substance" (US EPA, 2001 ). Hence, at issue is the extent to which exposure to
the natural hormones present in m eat from treated cattle aggregat es with endogenous levels of the
same hormone, and exposure to that hormone fro m any other source, such as in the form of a
therapeutic agent.
425. The second is cumulative risk. This has been de fined as "the likelihood of the occurrence of
an adverse health effect resulting from all routes of exposure to a group of substance sharing a
common mechanism of toxicity" (US EPA, 2001). Here, the issue is the extent to which compounds
with similar effects should be cumulated, with each other and with other similar substances of either
exogenous or endogenous origin.
 US EPA Office of Pesticide Programs (2001). Ge neral Principles For Performing Aggregate
Exposure And Risk Assessments (http://www.epa.gov/pesticides/trac/science/aggregate.pdf

426. The EC Opinions and other materials referred to by the EC do not quantify the extent to
which residues of the hormones contribute to ag gregate exposures or cumulative exposures to
multiple hazards. Aggregate risk where one of the major exposures is from a substance found
endogenously, is most common for essential minerals and vitamins. However, here the exposures of
concern are mainly exogenous, as for most vitamins and minerals there is no endogenous production
(see SCF, 2000) . However, there are a few exceptions, such as vitamin D. In this case, the SCF
(2002) took into account the endogenous production rate in estimating a tolerable upper intake level.
An important consideration was the extent to which exogenous exposure changed circulating levels of
the active vitamin. This is somewhat analogous to the approach taken by JECFA (2000b) for the
natural hormones, in that exposur e was calibrated against doses that did or did not cause any change
in circulating levels of the hormone.
 SCF (Scientific Committee on Food) (2000). Guidelines of the fo r the development of
tolerable upper intake levels for vitamins and minerals. SCF/CS/NUT/UPPLEV/11 Final
(http://ec.europa.eu/food/fs/sc/scf/out80a_en.pdf)

 SCF (Scientific Committee on Food) (2002). Op inion of the Scientific Committee on Food on
the Tolerable Upper Intake Level of Vitamin D. SCF/CS/NUT/UPPLEV/38 Final
(http://ec.europa.eu/food/fs/sc/scf/out157_en.pdf)

427. The issue of cumulative risk is more complex. Indeed, such considerations are currently
applied in only very limited circumstances, for ex ample dioxins and organophosphates. Within the
EU, no such cumulative risk assessments have b een undertaken routinely for any residues of

pesticides or veterinary drugs in food. Indeed, th ere is currently no agreement as to the appropriate
methodology to use (see EC, 2005b) and such assessments do not appear to be foreseen in the near
future for residues of veterinary drugs. Hence, risk assessments are performed on the individual
compounds. However, it should be emphasized th at any risk assessment group would deal with
substances on a case by case basis, and the absence of agreed methodology would not necessarily
preclude some consideration of cumulative risk, if this was deemed to be a major and immediate
concern.
 EC (2005b). Regulation (EC) No 396/2005 of the European Parliament and of the Council of
23 February 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal
origin and amending Council Directive 91/414/EEC
(http://europa.eu.int/eur-lex/lex/LexUriServ/site/en/oj/2005/l_070/l_07020050316en00010016.pdf)

428. The importance of both aggregate and cumulative risk would depend critically on whether or
not there was a threshold for the dose-response relations hip. One of the arguments of the EC is that
hormones can cause cancer by a genotoxic mechanism which would have no threshold. If this were
true, it is certainly correct that any additional expos ure would have an incremental effect on risk, if
one assume a linear no-threshold dose-response relati onship. This would be the default under such
circumstances. The incremental risk would depend on the extent to which the additional exposure
from hormones in meat from treated cattle changed the overall exposure, with respect to the
endogenous levels present. In contrast, for com pounds with the same mechanism of action, if there
were a threshold for all of the biological effects of concern, additional exposures would only be of
concern when they resulted in a total potency-corr ected exposure that was above the threshold (the
ADI) (Silva et al, 2002).
429. My reply would have been the same at the time of adoption of the EC Directive in September
2003.
 Silva E, Rajapakse N, Kortenkamp A (2002). Something from "nothing"--eight weak
estrogenic chemicals combined at concentrations below NOECs produce significant mixture effects.
Environ Sci Technol, 36:1751-1756

Dr. Guttenplan

430. In general the EC do not attempt to evaluate "the additive risks arising from the cumulative
exposures of humans to multiple hazards, in additio n to the endogenous production of some of these
hormones by animals and human beings". However, as described in the answer to question 52, an
estimated comparison has been presented of amount of estrogen contributed by consumption of meat
from hormone-treated cattle and the amount normally produced in prepubescent children.
431. I am more comfortable with this estimation now , and then I would have been in 2003 because
of improved analytical techniques.
56. Has JECFA/Codex considered in its risk a ssessment of the five hormones such "additive
risks? Are there internatio nally recognized guidelines fo r conducting assessments of
"additive risks"?

Dr. Boisseau

432. JECFA/Codex considered in its risk assessment of the natural hormones such "additive risks"
and concluded that, given the wide margin of safety between the maximum estimated intake of
residues for the these hormones and the corresponding established ADIs, that there was no risk for
consumers' health associated with the estimated ingestion of these residues. JECFA /Codex did not

consider such "additive risks" in its risk assessment of the synthetic hormonal growth promoters. To
my knowledge, there is no internationally r ecognized guidelines for conducting assessment of
"additive risks".
Dr. Boobis

Consideration of additive effects by JECFA/Codex

433. JECFA/Codex did consider aggregate risk from exposure to the natural hormones where
present as residues in meat from treated cattle. Such exposures were considered to represent a trivial
increase in overall exposure to hormonally-activ e material from other exogenous sources and in
particular from endogenous sources (JECFA, 2000). JECFA/Codex did not use formal methodology
to assess cumulative risk from exposure to the hormone s. However, JECFA did consider that the
dose-response curves for potential adverse effects fro m the hormones all exhibited thresholds and that
there was a considerable margin of exposure for a ll of the hormones between the TMDI and the ADI.
Hence, it was concluded that ther e would be no additional risk over background by exposure to any
residues from meat from treated cattle. In a cumu lative risk assessment consideration needs to be
given to the exposure pattern. It is not appropriate to assume that exposure to each substance will be
at the TMDI, as this would require chronic exposure to each hormone at the maximum possible level
for all of them. To overcome such compounded conservatism in the evaluation, probabilistic
approaches to exposure assessment have been used when performing a cumulative risk assessment
(US EPA, 2002)
434. JECFA has developed specific methodology for performing a risk assessment of dioxins and
related substances that share a common mechanism of action (JECFA, 2002a) . Ad hoc approaches
have been applied to a very limited extent to certa in pesticide and veterinary drug combinations, from
example when they share a common metabolite. However, there are no agreed international
guidelines for conducting a full cumulative risk assessme nt. One of the difficulties is in obtaining
representative consumption data for substances used in food production. Some of the issues involved,
and the methodological approaches that have been developed have been reviewed by Wilkinson et al
(2000). However, it should be noted that these methodologies are applicable to compounds that share
what has been defined as a common mechanism, i. e. there is a cumulative risk. There is no
international agreement on how to undertake a combin ed risk assessment of compounds acting by the
carcinogenic mechanisms suggested by the EC for the hormones, i.e. genotoxicity via direct or
indirect interaction with DNA. If one were to as sume no threshold for the effect, and apply ALARP,
it would not be necessary to perform a cumulative risk assessment of such compounds.
 JECFA (2002a). Safety Evaluation of Certain Food Additives and Contaminants:
Polychlorinated Dibenzodioxins, Polychlorinated Dibenzofurans, and Coplanar Polychlorinated
Biphenyls. WHO Food Additives Series: 48, WHO, Geneva

 US EPA (2002). Organophosphate Pesticides: Revised Cumulative Risk Assessment
(http://www.epa.gov/pesticides/cumulative/rra-op/)

 Wilkinson CF, Christoph GR, Julien E, Kelley JM, Kronenberg J, McCarthy J and Reiss R
(2000). Assessing the risks of exposures to multiple chemicals with a common mechanism of toxicity:
how to cumulate? Regul Toxicol Pharmacol, 31:30-43

Dr. Guttenplan

435. I could find assessment of additive risks of the hormones in the documents, and I not aware of
any internationally recognized guidelines.

57. Canada comments that "one single molecu le that the European Communities considers
so dangerous from meat derived from animals treated with hormone growth promoters

is suddenly not at all that dangerous when consumed from meat from animals treated
for therapeutic or zootechnical purposes. The European Communities' concern about
the genotoxic potential of oestradiol-17 β suddenly and inexplicably disappears." To
what extent are hormone treatments of cattle for purposes other than growth
promotion, such as for therapeutic or zoot echnical purposes, taken into account by the
European Communities, if at all, in its assessment of the cumulative effects from the
consumption of meat containing residues of the hormones at issue? Would your reply
have been different at the time of adoption of the EC Directive in September 2003? If
so, why? [see para. 97 of Canada Rebuttal Submission; paras. 17-20 of US Opening
Statement]

Dr. Boisseau

436. The comment of Canada stating that " one singl e molecule …inexplicably disappears" refers
to the theoritical assumption that one single mo lecule of a genotoxic compound, coming in contact
with the human genetic material, could be likely to damage it and therefore to induce a carcinogenic
process. This worry supports to some extent th e position of the European Communities regarding
oestradiol-17β. Therefore, starting from this theoritical and somewhat extreme assumption, Canada
challenges the authorization, by the European Communities, of the use of oestradiol-17 β for
therapeutic and zootechnical purpose. It has to be noted that, according to the directive 2003/74/EC,
oestradiol-17β can only be used for three precise therapeutic indications and only until 16/10/2006 for
the induction of oestrus. The Eu ropean Communities thinks that, gi ven the conditions of theses uses
of oestradiol-17β (limited number of treated animals, limited use in the life of these animals and very
low probability to see these animals slaughtered after treatment), the exposure of consumers to
oestradiol-17β residues resulting from these uses can be c onsidered as negligeable. If this EC
asssumption can be accepted, it raises nevertheless a problem of principle as it represents an exception
regarding the very strict position of EC stating that it is not possible to accept any increase of the
exposure of consumers to oestradiol-17 β residues. As soon as the Eu ropean Communities accepts to
considers these residues resulting from these therap eutic and zootechnical use of oestradiol-17 β as
negligeable, it enters in a quantitative, or at least in a semi quantita tive, exposure assessment
procedure for these oestradiol-17 β residues and, starting from that, it has no good reason to object to
consider a wider exposure assessment covering all the residues resulting from the different sources of
oestradiol-17β. This comment has already been made in my reply to question 31.
437. My reply would not have been different at the time of adoption of the EC directive in
September 2003.
Dr. Boobis

Aggregate and cumulative exposure to hormones

438. As indicated above (in my reply to question 56), combined exposures to the same substance
from more than one source has been described as aggregate exposure whilst exposure to more than
compound acting by the same mechanism has been described as cumulative exposure. To my
knowledge no account is taken of hormone treatme nts of cattle for purposes other than growth
promotion, such as for therapeutic purposes, by the EC in its assessment of the aggregate or
cumulative effects of the hormones in meat from cattle treated for growth promotion. However, this
reply needs some clarification. Firstly, the issu e of aggregate and cumulative risk, as indicated above
(in my reply to question 56), depends upon assump tions about the nature of the dose-response
relationship. Second, though unlikely given the EC 's views on the growth promoting hormones, the
need for taking into account such other exposures would depend on how far exposure was considered

to be from any threshold, i.e. the margin of exposure. My reply would have been the same at the time
of adoption of the EC Directive in September 2003.
Dr. Guttenplan

439. The EC does not really take into account horm one treatments of cattle for purposes other than
growth promotion, such as for therapeutic or zo otechnical purposes in their risk assessments. Their
reasons are summarized in (EC rebut US sub. Para 114). "Exceptions to the ban on meat from
hormone-treated cattle were made only for the use of certain of these substances for zootechnical and
therapeutic purposes where no viable effective altern atives appeared to exist. This exception was
based on the assessment that owing to the nature and limited duration of the treatments, the limited
quantities administered and the strict conditions im posed to prevent misuse, that this use did not
constitute a hazard for public health." This is a reasonable response. My response would not have
been different in 2003.
58. Please comment on the EC statement in para. 94 of the EC Replies to Panel Questions
that "the only rationale that can be inferred from the available scientific data is that the
higher the exposure to residues from these ho rmones, the greater the risk is likely to
be", taking into account para. 105 of Canada Rebuttal Submission.

Dr. Boisseau

440. My reply to question 55 applies to this question as well.
Dr. Boobis

Dependency of risk on dose

441. Again, this depends critically upon the conclu sion regarding the nature of the dose-response
relationship. JECFA has concluded that there was a threshold fo r all of the potential adverse effects
of the hormones and that it was possible to estab lish ADIs. Hence, as indicated above, exposure
below the ADI is considered to be without appreciable risk. Estimated exposure from consumption of
meat from treated cattle would result in hormone intakes that were well below the respective ADI's, in
the case of oestradiol its intake would represent only 1.5% of the ADI (JECFA 2000a, b) . Hence,
within quite broad limits, higher exposure would not r esult in any increase in risk. This would be the
case until the ADI was exceeded. It should also be not ed that for the critical endpoints of concern in
the JECFA evaluation, including cancer, the risk would be significant only with prolonged exposure
(Coombs et al, 2005) . Hence, occasional exposure even above the ADI would be considered to pose
no appreciable risk.
Dr. Guttenplan

442. This is indeed a very weak statement by th e EC. However, the alternative would be to
suggest a risk that might be wildly inaccurate, due to the limitations imposed by the lack of solid data
on levels of hormones in meat. Perhaps a better approach would have been to suggest several
scenarios. These could be validated or disproved by subsequent studies.
59. Does the scientific evidence referred to by the European Communities identify any
adverse effects on the immune system from th e consumption of meat from cattle treated
with the growth promoting hormones at issue? Would your reply have been different at
the time of adoption of the Directive in September 2003? If so, why? [see para. 132 of
Canada Rebuttal Submission]

Dr. Boisseau

443. The scientific evidence referred to by the European Communities allows to identify adverse
effects of hormonally active substa nces on the immune system. Neve rtheless, as these data have not
been used by the European Communities to c onduct any quantitative risk assessment likely to
establish, for these effects associated with the hormonal properties of growth promoters, thresholds
and ADIs different from those proposed by JECFA, it is not possible to conclude that this scientific
evidence allows to identify any adverse effect s on the immune system associated with the
consumption of meat from cattle treated with the growth promoters at issue.
444. My reply would not have been different at the time of adoption of the EC directive in
September 2003.
Dr. Boobis

Effects on the immune system

445. The evidence on immune effects of hormones such as oestradiol referred to by the EC does
not identify any adverse effects on the immune system from consumption of meat from treated cattle.
In general, clear evidence for immune effects were observed only at high doses. There is no evidence
that doses such as those resulting from consumption of meat from treated animals have any effect on
the immune system (JECFA, 2000b; CVMP, 1999). It should also be noted, that in the case of
immune effects, exposure relative to endogenous levels is a critical issue. Given the large margin of
exposure on anticipated intake from residues in meat from treated animals, no effect on the immune
system is anticipated, as immune modulation is de pendent on dose and there are thresholds for such
effects (Barton and Clewell, 2000; Kroes et al, 2004).
446. My reply would not have been different at the tile of adoption of the EC Directive in
September 2003.
 Barton HA and Clewell HJ 3rd (2000). Eval uating noncancer effects of trichloroethylene:
dosimetry, mode of action, and risk assessment. Environ Health Perspect, 108 (Suppl 2):323-334

 Kroes R, Renwick AG, Cheeseman M, Klein er J, Mangelsdorf I, Piersma A, Schilter B,
Schlatter J, van Schothorst F, Vos JG and Wurtzen G; European branch of the International Life
Sciences Institute (2004). Structure-based thres holds of toxicological concern (TTC): guidance for
application to substances present at low levels in the diet. Food Chem Toxicol, 42:65-83

Dr. Guttenplan

447. The relationship between estrogen and auto immune diseases has received considerable
attention (Opinion SCVPH, April 30, 1999, section 2.4) . There is evidence that estrogens can be
involved in Lupus, rheumatoid arthritis, thyroiditis. In addition the development of allergies is
thought to be at least partially related to estrogens . The studies in experimental animals also did not
identify any immune-related effects, although it is no t certain the types of possible effects in humans
would be detected in experimental animals. No definitive studies have related intake of meat from
hormone-treated animals to the above disorders.
448. My reply would not have been different in 2003.
60. Does the scientific evidence referred to by the European Communities identify and
evaluate whether there is a difference in terms of potential adverse effects on human
health from the consumption of meat from cattle treated with hormones for growth

promotion purposes when these hormones are administered as feed additives (MGA) or
implanted? Are you aware of any differences?

Dr. Boisseau

449. I do not understand this question as MGA is only used as feed additive and the five other
hormones are not used as feed additive. Regardi ng the exposure assessment, the risk is potentially
higher with implanted growth promoters as there are more "options" in terms of misuses/abuses.
Nevertheless, to this respect, the scientific evid ence referred to by the European Communities does
not identify and evaluate wether there is a differe nce in terms of potential adverse effects on human
health from the consumption of meat from cattle treated with hormones for growth promotion
purposes when these hormones are administered as feed additives or implanted.
Dr. Boobis

Relevance of method of use of hormones

450. The scientific evidence referred to by the EC does not identify any difference in terms of
potential adverse effects on human health from the consumption of meat fr om cattle treated with
hormones for growth promotion purposes and when they are administered as feed additives or
implanted. I am not aware of any such differe nces. None would be expected, as in all cases
maximum intake would be well below the ADI. Ho wever, there is a situation which, at least
hypothetically, could give rise to a difference in eff ects. This would be the misuse or abuse of the
compounds when used as growth promoters, givi ng rise to increased intake because of a misplaced
implant or entry into the food chain of tissue contai ning the implant (i.e. cow's ear). However, whilst
this would lead to increased exposures, it is still unlikely this would exceed the ADI, and certainly not
for any period of time. It is also an unlikely o ccurrence in view of the way in which the hormones are
used and controlled.
Dr. Guttenplan

451. This question appears not to be specifically addressed, but MGA is the only hormone which
might be administered by both methods. The poten tial for excessive exposure to MGA exists by both
routes (oral and implantation), but it cannot be stated and I am not aware of which route is more likely
to contribute to high levels in meat (SCVPH, section 4.1.4).
61. In your view and in the light of information provided by the parties as well as the work
undertaken at JECFA and Codex, did th e the scientific evidence available to the
European Communities at the time it adopted its Directive (September 2003) allow it to
conduct an assessment (quantitatively or qua litatively) of the potential for adverse
effects on human health arising from the co nsumption of meat from cattle treated with
(a) progesterone; (b) testosterone; (c) trenbo lone; (d) zeranol; and (e) melengestrol
acetate? Would your response differ in light of the scientific evidence provided which is
subsequent to the adoption of the Directive?

Dr. Boisseau

452. It is difficult to reply to this question as I don't really know what were the data available to the
European Communities at the time it adopted its dire ctive (September 2003). On the other hand, it is
always possible to ask for more data in order to clarify more issues so that the willing to eliminate any
scientific uncertainty could result in an endless assessment process.

453. My reply would not have differed in light of the scientific evidence provided which is
subsequent to the adoption of the EC directive in September 2003.
Dr. Boobis

Availability of data for risk assessment prior to September 2003

454. In my view there was sufficient information av ailable to the EC to have enabled it to have
conducted an assessment of the risks to human heal th arising from consumption of meat from cattle
treated with any of the six hormones at issue (see comments above for details of the basis of this
response). My reply would not have been different at the time of adoption of the EC Directive in
September 2003.
Dr. Guttenplan

455. The evidence does indicate that potential adverse effects exist for all of the hormones.
However, the ability to make a risk assessmen t (qualitative or qualitative) does vary between
compounds.
456. Progesterone, testosterone have been extens ively investigated and the assessment seems
sound and is based on the no effect level and a saf ety factor. (JECFA meeting 52, report-WHA TRS
893).
457. There is more limited evidence available for Trenbolone and Zeranol and most of it is in vitro
(SCVPH 2002 Opinion) or not recent (e.g., JECFA meeting 34 th report, 1989 and 32 nd report, 1988).
However, both appear to be potentially significantly estrogenic. Experimental and analytical methods
have improved but it does not appear that accurate ADI's can be established at this point. Studies in
experimental animals and studies on levels in beef ar e still needed. However, from the data available
at the time of the Directive, the potential for adverse effects could not be ruled out.
458. Melengestrol acetate. The assessment for melengestrol acetate seems sound. Thorough
metabolic and estrogenic studies have been carried out . Actual levels in beef were not provided.
(JECFA 62 FNP 41/16).
459. My opinion would not have been different in 2003.
62. Does the scientific evidence relied upon by the European Communities support the EC
contention that the new scientific studies that have been initiated since 1997 have
identified new important gaps, insufficienci es and contradictions in the scientific
information and knowledge now available on these hormones such that more scientific
studies are necessary before the risk to human health from the consumption of meat
from cattle treated with these hormones for growth promotion purposes can be
assessed? Would your reply have been different at the time of adoption of the Directive
in September 2003? If so, why?

Dr. Boisseau

460. The scientific evidence relied upon by the Eu ropean Communities has certainly provided
new interesting data potentially useful for con ducting the risk assessment of growth promoter
residues. For all that, these new data do not de monstrate any important gaps, insufficiencies and
contradictions in the scientific information used by JECFA for conducting its risk assessments. In
order to decide wether or not more scientific st udies are necessary before the risk to human health
from the consumption of meat from cattle treated with these hormones for growth promotion purposes

can be assessed, it should be necessary to conduct a temporary risk assessment with these new data in
order to see to what extent the conclusions of this temporary risk assessment is significantly different
from those already performed by JECFA. If it would be the case, it would be necessary, at this time,
to identify the additional studies necessary to carry out in order to clarify all the outstanding issues
and to complete the risk assessment of the residues of hormones used as growth promoters.
461. My reply would not have been different at the time of adoption of the EC directive in
September 2003.
Dr. Boobis

Additional information provided by the studies initiated since 1997

Analytical techniques and bioassays for screening

462. The studies on developing improved analytical methods were considered by the SCVPH
(2002) to have been inconclusive.
463. "Despite a number of positive analytical results in this study, the low number of samples does
not allow a qualified validation of typical character istics such as sensitivity, specificity, accuracy and
reproducibility (study 1, study 8)." I agree with this conclusion.
464. "The obtained results suggest that the use of recombinant yeast and rainbow trout hepatocytes
to detect oestrogenic compounds is not justified in vi ew of their lack of sensitivity" (study 9). I agree
with this conclusion. It is of note that despite the use of similar strategies, the limit of sensitivity of
the yeast reporter assay used by Le Guevel and Pakde l (2001) in study 9 was substantial less that that
of Klein et al (1994).
Bovine metabolism of oestradiol-17 and oestrogenic potency of residues

465. One of the potentially relevant observations was the finding of oestradiol fatty acid esters
produced in cattle following treatment (study 3). Th e study of Hoogenboom et al (2001) showed that
the intrinsic oestrogenicity of these esters was much lower than that of oestradiol, by 25-200 fold.
Paris et al (2001), have shown that most likely due to kinetic differences, the oestrogenicity of the
fatty acid esters in vivo is up to 10-fold greater than that of oestradiol. However, it is apparent that
the difference in potency from the parent hormone is not very great or even apparent at low doses,
where effects were minimal. Given these findings and that the esters are not the major residue in meat
from treated animals, that in so me tissues such as muscle, levels are much lower than those of
oestradiol (Maume et al, 2001), and that total e xposure will be very low (J ECFA, 2000), particularly
when the balance of the diet is considered, these findings do not raise additional concerns regarding
the potential adverse health effects of the hormones wh en used to treat cattle. An additional point to
note is that in all of the studies cited above on the fatty acid esters of oestradiol, concentrations were
expressed in units of mass per litre or per kg. Howe ver, as only the oestradiol moiety is hormonally
active (Hoogenboom et al, 2001), this tends to overestimat e potency relative to that of oestradiol by a
factor of 2-fold (due to the difference in molecular weight relative to that of oestradiol).
 Maume D, Deceuninck Y, Pouponneau K, Pa ris A, Le Bizec B and Andre F (2001).
Assessment of estradiol and its metabolites in meat. APMIS, 109:32-38.

 Paris A, Goutal I, Richard J, Becret A and Gueraud F (2001). Uterotrophic effect of a
saturated fatty acid 17-ester of estradiol-17bet a administered orally to juvenile rats. APMIS,
109:365-375

Multiple implanting, multiple dosing

466. In study 5, the impact of misuse and multiple dosing on residual hormone levels in meat was
determined. Dosing at up to 10 times the approve d dose, resulted in an increase in the tissue
concentrations of some hormones in some tissu es to values above the MRL for those hormones for
which Codex has established an MRL. For those hormones for which an MRL has not been specified,
there were also increases in some tissues (Lange et el, 2001).
467. "Treatment with zeranol and testosterone propionate, even after multiple application, does not
cause any problems, as far as infringement of threshold levels is concerned."
468. "Exceeding of the MRL was found in the liver in one out of two animals after 3-fold and in
two out of two animals after 10-fold dose of the 200 mg trenbolone acetate-implant". No exceedances
were seen in muscle, kidney or fat, even at 10-fold the approved dose.
469. For oestradiol, the maximum increase observed in any tissue was not greater than
proportional to the dose applied. Hence, even at 10-fold the approved dose, intake would be well
below the ADI. This would be offset by the fact that not all tissues had such elevated levels, and the
probability of consuming such high r esidue levels of a regular basis is minimal. It should also be
noted that Codex did not specify an MRL for oestradiol, as it was considered unnecessary.
 Lange IG, Daxenberger A and Meyer HH (2001). Hormone contents in peripheral tissues
after correct and off-label use of growth promot ing hormones in cattle: effect of the implant
preparations Filaplix-H, Raglo, Synovex-H and Synovex Plus. APMIS, 109:53-65

470. In the study on misplaced implantation sites (D axenberger et al, 2000), substantial residual
hormone was sometimes found at the implantation site when this was not as recommended. However,
for these findings to have significance for the consumer a number of factors need to be considered.
These include the likelihood of off-label use of the hormones, the failure to detect the implantation
site, the use of the implantation site for food use, th e contribution of the contaminated meat to the diet
and the frequency of such contamination. No da ta have been presented on the prevalence of such
significant contamination as a consequence of the veterinary use of the hormones. Indeed, no
evidence is presented that such misuse does occur with the consequences suggested by the authors.
 Daxenberger A, Lange IG, Meyer K, Meyer HH, Daxenberger A, Lange IG, Meyer K and
Meyer HH (2000). Detection of anabolic residues in misplaced implantation sites in cattle. J AOAC
Int, 83:809-819

471. In studies on MGA (Daxenberger et al, 1999) tissue levels increased with dose, most
markedly in fat. Whilst in fat, there was a r oughly proportional increase with dose, in other tissues
(muscle, kidney, liver) the fold-increase was appreci ably less than the fold-i ncrease in dose. Using
the values obtained in the study of Daxenberger et al (1999) at 10 times the maximum approved dose,
consumption of all four tissues (liver, kidney, fat and muscle) at the JECFA levels (300 g muscle, 100
g liver, 50 g kidney and 50 g fat per day) would resu lt in a slight exceedance of the ADI (2.5 µg cf
1.8 µg). However, it should be noted that this would require all of the tissues to be from animals
treated with the high dose, and exposure would ha ve to be over a prolonged period of time. The
probability that this would occur is extremely low.
 Daxenberger A, Meyer K, Hageleit M and M eyer HH (1999). Detection of melengestrol
acetate residues in plasma and edible tissues of heifers. Vet Q, 21:154-158

472. In risk assessment amongst the objectives is to de termine whether it is possible to set health
based guidance values (e.g. ADI) and upper levels for exposure (reference values or MRLs). If

possible and necessary, such values should be estab lished. MRLs are established following use of
the drug according to Good Veterinary Practice (GVP ; also Good Practice in the Use of Veterinary
Drugs, GPVD). This is the policy of all agenci es and organisations involved in such activities (EEC,
1990; EC, 2005b; FAO, 2006). An additional question that may be asked is the consequence of abuse
or misuse. However, such inappropriate activity cannot be used as the basis for establishing MRLs.
This is because whilst use according to GVP can be foreseen and regulated, it is not possible or
appropriate to regulate any conceivable misuse or abuse, whether actual or hypothetical. Normally,
the risk management strategy to deal with this is to ensure adequate surveillance of residues and to put
in place a system of penalties for violation. This is the situation for veterinary drugs in all regions
where they are subject to market authorisation, in cluding the EU and the USA. In assessing the risks
from abuse or misuse, from a human health persp ective, the concern is whether the consequent
residues would result in exposure that exceeded th e ADI. Exceedance of the MRL or other nominal
tolerance level for residues his implications for detecting abuse or misuse in surveillance programmes,
but has no direct link with whether there is a health concern or not. For example, residue levels from
the use of zeranol according to GVP would result in TMDIs below the ADI. MRLs were established
accordingly, so that there is a ma rgin by which the residues can exceed the MRLs, yet intake will still
be no greater than the ADI (JECFA, 1988a, b).
473. In considering the consequences of abuse a nd misuse, the following aspects need to be
considered:
• The likelihood of violations or off-label use
• The residue levels occurring after such misuse or abuse
• The extent to which exposure to such residues will result in an exceedance of the ADI
• The likely frequency or period over which the ADI will be exceeded
• The acute consequences of exceeding the ADI
• The severity of the endpoint upon which the ADI is based
• The steepness of the dose-response curve for the endpoint upon which the ADI is
based

474. Taking account of all of these factors, the data generated by the EU research in question do
not provide any indication that it is not possible to conduct a risk assessment of the hormones used as
growth promoters. Nor do they provide any indicati on that even such misuse or abuse as investigated
gives rise to undue risk from the resultant residues, as intake would only very rarely exceed the ADI
and then only on a rare occasion.
Alteration of gene expression by oestrogenic compounds

475. The study referred to (study 17), reported in Leffers et al (2001) , showed that a number of
oestogenic compounds affected the e xpression of several genes in the ER α positive breast cancer cell
line, MCF7. The responsiveness of this cell line to o estrogens is well established. It was of interest
that all of the changes reported by Leffers et al (2001) were blocked by the selective ER α antagonist
ICI82.780. The relevance of effects observe d in a cultured cell line to the situation in vivo, where
kinetic and metabolic factors will influence the ma gnitude of the response is not known, nor is the
significance of changes in gene expression to the toxicity of the hormones known. Many of the
changes will reflect the proliferative response to an oestrogenic stimulus. However, in general
toxicogenomic data, in the absence on any info rmation on the functional consequences, is not
considered a sound basis for use in risk assessment (IPCS, 2003).
 IPCS (2003). Toxicogenomics and the Risk Asse ssment of Chemicals for the Protection of
Human Health (http://www.who.int/entity/ipcs/methods/en/toxicogenomicssummaryreport.pdf)

 Leffers H, Naesby M, Vendelbo B, Skakkeba ek NE and Jorgensen M (2001). Oestrogenic
potencies of Zeranol, oestradiol, diethylstilboestro l, Bisphenol-A and genistein: implications for
exposure assessment of potential endocrine disrupters. Hum Reprod, 16:1037-1045.

Recent findings on the mutagenicity and genotoxicity and of oestradiol-17β

476. The recent reports (study 3 and study 8) on mutagenicity confirm that oestradiol can produce
a genotoxic response in vitro in certain tests. Evidence was obtained that this was due, at least in part,
to the formation of reactive oxygen species. Much of the work was undertaken with relatively high
concentrations of metabolites a dded exogenously. For some of the genotoxic endpoints, oestradiol
and the metabolites tested were negative. In one of the cited studies ( Chakravarti et al, 2001) the
effects of the 3,4-quinone metabolite were inv estigated in vivo. A relatively high dose was
administered to mouse skin. Whilst mechanistic in formation of value can be obtained using a route
other than the one of concern (i.e. oral for dietary residues), in this case there is concern that kinetic
differences, particularly in the disposition of a qui none metabolite, make interpretation of the findings
difficult, In addition, the mechanism for genotox icity observed was not established, and the authors
acknowledge that this could have b een due to redox cycling. As indicated above, such a mechanism
normally exhibits a threshold (see reply to question 19 above). It is known that compounds such as
quinones can show marked route-dependent differen ces in their genotoxic effects. Indeed, several
authorities, including the EC have accepted a threshold for the genotoxicity of some of these
compounds in vivo following oral administration (European Chemicals Bureau, 2006) . It was of
interest that the mutations observed in vivo by Chakravarti et al (2001) in mouse skin involved
adenine and not guanine. This is significant as it is the N7-guanine adducts th at were "considered to
play a crucial role in the initiation of oestrogen-dependent tumours" (SCVPH, 2002).
 Chakravarti D, Mailander PC, Li KM, Higginbotham S, Zhang HL, Gross ML, Meza JL,
Cavalieri EL and Rogan EG (2001). Evidence that a burst of DNA depurination in SENCAR mouse
skin induces error-prone repair and forms mutations in the H-ras gene. Oncogene, 20:7945-7953

477. The genotoxicity and mutagenicity of oestradiol and, more particularly, its metabolites in
vitro was already well established. No new evidence has been provided on the genotoxic potential of
oestradiol in vivo. The study on the quinone metabolite on mouse skin does not further the risk
assessment of the compound. No evidence was pr ovided that oestradiol, or indeed any of its
metabolites, is genotoxic in vivo following oral administration.
Recent findings on the biological effects of testosterone and progesterone

478. The SVCPH (2002) concluded in their report that on the basis of the most recently published
papers "there is no evidence that progesterone or testosterone have genotoxic potential".
Recent findings on the biological effects of trenbolone and zeranol

Biotransformation

479. The metabolism of zeranol and trenbolone had been further investigated (study 4). These data
do not appear to have been published in the peer reviewed literature to date.
480. The data on trenbolone show that the alpha enantiomer in liver slices from bovine is
extensively conjugated and hence inactivated. Ther e is some conversion of the alpha to the active
beta isomer by human liver microsomes, but the kine tics of the reaction and the extent of conjugation
have not been determined. No data were presen ted on levels of the alpha enantiomer in meat from
treated cattle. However, these data do not affect th e risk assessment of trenbolone acetate. This is
because a) the toxicological studies were conducted in animals that would have been exposed to the

metabolites of concern, b) JECFA considered residu es of both the alpha and the beta enantiomers in
recommending MRLs for trenbolone acetate.
481. The study on zeranol suggested that some of the metabolites might undergo autooxidation.
However, the extent of any such reaction in int act cells was not investigated, nor was the likely
detoxication of the products formed. Perhaps more im portantly, it is the toxicology of zeranol and its
residues in meat that is at issue, and the evidence is such that it was possible to identify a threshold
dose for all of the effects of concern.
Binding to sex hormone binding globulin

482. In this study (study 10), which does not appear to have been published in the peer reviewed
literature, the interaction of growth promoting horm ones with the binding of testosterone to plasma
proteins was investigated. Some of the hormones, but not zeranol, were able to displace testosterone
partially and only at concentrations very much hi gher then would ever be achieved from ingestion of
meat from cattle treated even with high doses of th e hormones for growth promotion. Hence, these
findings have no significance for the risk assessm ent of the hormones. Although zeranol did not
appear to bind to plasma proteins with high a ffinity, the ADI was established on the basis of the
exogenous dose that had no effect. The fraction of protein binding should be the same for the ADI
and the TMDI. Hence, the risk assessment of this hormone will not be affected by whether zeranol
binds to plasma proteins or not.
Mutagenicity and genotoxicity

483. Study 4 reports recent observations on the ge notoxicity and mutageni city of zeranol and
trenbolone. Both compounds were negative for tests of mutagenicity, i.e. induction of lacI mutations
in E coli and induction of hprt mutations inV79 cells. Zeranol did not produce DNA adducts in rat
hepatocytes whilst a low level of DNA adducts w as observed with trenbolone. Both were very
weakly positive in a micronucleus test, at high (pot entially cytotoxic) concentrations. As indicated
above (see my reply to question 21), micronuclei can arise via a non-genotoxic mechanism,
particularly at concentrations that may have caused some toxicity. In addition, the 32P-post-labelling
assay is not specific, and data cited above suggest that DNA adduction can arise by mechanisms other
than direct interaction with DNA. These data are insufficient, given the number of well conducted
studies in which the compounds were negative, to alter the conclusion that neither zeranol nor
trenbolone acetate has genotoxic potential in vivo. Indeed, the SVCPH (2002) concluded that "both
compounds exhibited only very weak effects" in th ose in vitro tests in which positive effects were
observed.
Recent findings on the biological effects of MGA

Biotransformation

484. In study 4, unpublished preliminary findings on the in vitro metabolism of MGA were
reported. This study provided some evidence fo r the formation of multiple metabolites of MGA by
liver from human, rat and bovine. However, these fi ndings do not affect the risk assessment of MGA
because a) the toxicological studies were conducted in animals that would have been exposed to all of
the metabolites of concern, b) JECFA assumed that all of the residues in meat from animals treated
with MGA were as hormonally active as MGA when it proposed MRLs in 2002 (JECFA, 2002b). It
was subsequently shown that this was a conservative decision, as not all of the residues were as active
as MGA itself (JECFA, 2006c).
 JECFA (2002b). Residues of some veterinary drugs in animals and foods. FAO Food and
Nutrition Paper 41/14, Rome, Italy

 JECFA (2006c). Residues of some veterinary drugs in animals and foods. FAO, Rome, Italy
(in press)

MGA binding to sex hormone binding globulin

485. In study 10, preliminary and relatively poorly reported data on the interaction of MGA with
the binding of testosterone to plasma proteins were presented. MGA, at concentrations very much
higher than those that would arise from consumption of meat from animals treated with the hormone
had some displacement activity against testoste rone. The SCVPH (2002) commented on the absence
of data on reproducibility in these studies and that in some experiments no concentration curves were
developed. In conclusion, these findings do not alter the risk assessment of MGA.
Mutagenicity and genotoxicity of MGA

486. In study 4, MGA was negative in studies of the induction of hprt mutations in V79 cells, the
induction of micronuclei in V79 cells and the induction of lacI mutations in E coli. Pure MGA had no
effect on apoptosis, which could potentially confo und interpretation of studies using V79 cells.
Preliminary studies with rat liver slices, reported in an abstract but not yet published in the peer
reviewed literature, suggested that MGA could produce unidentified adducts with DNA. As indicated
above, there are mechanisms of adduct formation that do not involve direct interaction of the inducing
compound with DNA. Overall, a report of puta tive covalent binding to DNA observed using 32P-
post-labelling is not sufficient to over-ride the consistently negative results of MGA in a range of tests
for mutagenicity. Hence, on the basis of the findings in study 5, there is no reasons to change the risk
assessment or MGA.
Recent data on endocrine and developmental effects of the hormones

Experimental studies in rabbits

487. The EC commissioned a study on the effects of in utero exposure of rabbits to the three
exogenous hormones, MGA, trenbolone acetate and zeranol (study 11). To date, only information on
metabolism and disposition have been published (Lange et al, 2002). Given the time that has elapsed
since this paper was published (submitted Septem ber 2001), it is somewhat surprising the data from
the remainder of the study have not been published.
488. The Lange et paper (2002) demonstrates transplacental transfer of the three hormones. This is
not surprising given the physicochemical propertie s of the compounds (lipid solubility, non-polar,
molecular size) (Syme et al, 2004) . In addition, endogenous hormones are known to cross the
placenta. It is notable that in the study of Lange et al, fetal concentrations of the hormones and their
metabolites were similar to or less than, someti mes much less than, those in corresponding maternal
tissues, suggesting that there was no net accumulation of the compounds in fetal tissues. It is also
noted that the number of animals studied was very small, a point commented on by the authors
themselves.
 Lange IG, Daxenberger A, Meyer HH, Ra jpert-De Meyts E, Skakkebaek NE and
Veeramachaneni DN (2002). Quantitative assessment of foetal exposure to trenbolone acetate,
zeranol and melengestrol acetate, following maternal dosing in rabbits. Xenobiotica, 32:641-651

 Syme MR, Paxton JW and Keelan JA (2004). Drug transfer and metabolism by the human
placenta. Clin Pharmacokinet, 43:487-514

489. The unpublished component of this study was an investigation of the potential health
consequences of in utero exposure of rabbits to th e three hormones. From the information provided,
low dose exposure in utero caused modest changes in some parameters, but was not associated with
either cancer or adverse effects on reproductive capacity. There were no changes in sperm number. It
is not clear whether the changes observed were consistent and hence compound-related as a only a
single dose was used for each compound. Nor is it ap parent whether the magnitude of all of changes
discussed reached statistical significance (often the changes were described as slight and no measure
of variance is provided). The doses used in this study would have provided much higher levels of
exposure than those predicted to arise from residue s in meat. In the case of trenbolone acetate and
zeranol exposure was via the subcutaneous rout e, thus bypassing presystemic metabolism in the
intestine and/or the liver. In th e case of MGA the oral dose was ov er 16,500 times the ADI. Hence,
even if the effects observed were of toxicological significance the ADI would provide a more than
adequate margin of protection.
490. Overall, this study cannot be said to confirm a risk to human health from consumption of
meat from animals treated with these hormones.
In utero exposure and breast cancer: a study in opposite sexed twins

491. This study (Kaijser et al, 2001 ; study 13) showed an association between birth weight and
risk of breast cancer. This is consistent with in ut ero exposure to oestrogens as a risk factor in breast
cancer. However, it does not establish such a relationship. The authors note that "Although
statistically significant (P=0.03), these estimates were based on small numbers in the extreme
categories. However, when the data were categor ized in more equally sized groups, the associations
were similar and retained statistical significance, albeit with lower point estimates." There is no
specific consideration of exposure to hormones present in meat from treated animals. The risk of such
exposure, with respect to the hypothesis proposed by Kaisjer et al (2001) would depend on the mode
of action and the dose-response relationship. A recent study (de Assis et al, 2006) suggests that higher
birth weight per se can increase the risk of breast ca ncer. However, the significance of this study in
experimental animals to humans has yet to be de termined. Given that exposure to oestradiol from
meat of treated animals would be extremely low, particularly relative to endogenous hormone levels,
which increase during pregnancy, (e.g. see Weiss, 2000 ) the findings of the Kaisjer et al study
provide no evidence for risk from exposure to oestradiol residues in meat from treated animals.
 De Assis S, Khan G and Hilakivi-Clarke L (2006). High birth weight increases mammary
tumorigenesis in rats. Int J Cancer (in press)

 Kaijser M, Lichtenstein P, Granath F, Erlandss on G, Cnattingius S and Ekbom A (2001). In
utero exposures and breast cancer: a study of opposite-sexed twins. J Natl Cancer Inst, 93:60-62

 Weiss G (2000). Endocrinology of parturition. J Clin Endocrinol Metab, 85:4421-4425

492. Overall, this study cannot be said to confirm a risk to human health form consumption of
meat from animals treated with these hormones.
Retrospective study on long-term effects in child ren of following suspected exposure to oestrogen-
contaminated meat

493. This paper (Chiumello et al, 2001; study 12) reviews the increased incidence of gynocomastia
in male children observed over a certain interval in a school in Milan, Italy. As oestradiol is known to
cause gynocomastia, the authors speculated on the possi ble role of oestrogens, perhaps from meat of
treated animals, as causative agents in these effects. However, this is entirely speculative, as no data
were obtained to suggest that there had been any such exposure. Further, it was not possible to

establish whether oestrogens were involved at all, as there are a number of other risk factors for
gynocomastia. Indeed, the SCVPH (2002) concluded that "As the reason for this incident remains
unknown, the relevance of these data remains unclear".
 Chiumello G, Guarneri MP, Russo G, Stroppa L and Sgaramella P (2001). Accidental
gynocomastia in children. APMIS 109 (Suppl 103): S203-S209

494. Overall, this study cannot be said to confirm a risk to human health form consumption of
meat from animals treated with these hormones.
Conclusion on the evidence from the studies initiated since 1997

495. There is little information in the scientific studies initiated by the EC since 1997 that support
the contention that they have identified important ne w gaps, insufficiencies an d contradictions in the
scientific information and knowledge on the horm ones, and that additional studies are necessary
before the risks to health of consumption of meat from treated animals can be assessed. Whilst
additional information has been obtained on a number of aspects of the hormones in question, this was
often not definitive, sometimes it was not relevant , in some instances it confirmed or expanded on
previous knowledge. The evidence obtained did not indicate any additional concern regarding the risk
from exposure to residues of the hormones in meat from cattle treated for growth promotion.
496. My reply would have been the same at the time of adoption of the Directive by the EC in
September, 2003.
Dr. Guttenplan

497. Yes, I see several important gaps. For instance:
498. It is not known if eating beef from hormone-tr eated animals substantially elevates the blood
levels of estrogen/estrone in prepubescent children. This can probably be investigated.
499. It is not known if eating beef from hormone-treated animals substantially elevates the level of
estrogenic activity in blood.
• Experiments on the identification, quantifi cation, bioavailability and accumulation of
lipoidal esters of estrogen in humans and experimental animals should be conducted.
• Epidemiological studies comparing adverse effects in matched populations of
children eating beef from hormone-treated and untreated animals have not been
reported.

[Please see the following references for the two questions above:
– paras. 58-94 and 125-129 of US First Submission, paras. 28-32 of US Rebuttal
Submission
– paras. 116-124 of Canada First Submission, paras. 74, 130-135 of Canada Rebuttal
Submission (Exhibit CDA-23)
– paras. 108, 147, 162-169 of EC Replies to Panel Questions, paras. 143-174 of EC
Rebuttal Submission (US case), and paras. 148-166 of EC Rebuttal Submission (Canada
case)
– Exhibit CDA-32 provides a detailed table outlining the chronology of JECFA's
assessment of these hormones and the resulting documentation]

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