- 1.1 Introduction
- 1.1.1 Recent Historical Perspective
- 1.1.2 The Role and Remit of the VPC 2002 Working Group
- 1.2 Biological Effects of Hormones and Endpoints of Health Concern
- 1.2.1 General Properties of Hormones
- 1.2.2 Health Endpoints of Concern
- 1.2.3 Endocrine Disruption
- 1.2.4 The Use of Oestradiol in Cattle
- 1.3 The Scientific Evidence Available to the VPC Working Group
- 1.3.1 Exposure to Hormonally Active Substances
- 1.3.2 Bioavailability of Hormonally Active Substances
- 1.3.3 Cancer Risks of Oestrogenic Substances
- 1.3.4 Altered Gene Expression by Oestrogenic Substances
- 1.3.5 Genotoxic and Mutagenic Effects of Oestrogenic Substances
- 1.3.6 Developmental and Reproductive Effects of Hormonally Active Substances
- 1.3.7 Environmental Impact of Hormonally Active Substances
- 1.3.8 Other Considerations of the WG
- 1.4 Recent Opinions and the Future
- 1.5 Conclusions
Chapter 1: The Use of Hormomally Active Substances in Veterinary and Zootechnical Uses – The Continuing Scientific and Regulatory Challenges
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Published:26 Nov 2009
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L. S. Levy, in Analyses for Hormonal Substances in Food-producing Animals, ed. J. F. Kay, P. S. Belton, and G. Downey, The Royal Society of Chemistry, 2009, ch. 1, pp. 1-47.
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1.1 Introduction
“Those who cannot learn from history are doomed to repeat it.” (George Santayana, 1863–1952)
“What experience and history teach is this – that people and governments never have learned anything from history, or acted on principles.” (George Wilhelm Hegel, 1770–1831)
The scientific, regulatory and political debate surrounding the use or banning of hormones or hormone-like substances in the production of meat and meat products and for veterinary use is, for me, the quintessential example of the limitations of all three of these facets of risk assessment/management inputs in producing unequivocal answers. It is thus a salutary lesson to scientists, regulators and policy-makers, politicians and risk assessors to understand all strands of this tangled issue. Whether or not it helps us to make better decisions in the future, for this and other continuing debates in risk assessment, will depend on which of the two above apparently contradictory quotations regarding the utility in understanding the past you subscribe to.
1.1.1 Recent Historical Perspective
A range of hormonally active substances, such as diethylstilboestrol (DES), had been used for growth promotion in cattle and sheep since the early 1950s. This latter use is termed zootechnical as opposed to veterinary or therapeutic use. Concerns about a possible risk of cancer from residues of such substances had been expressed in the early 1970s and eventually the European Community (EC) introduced a ban on the use of DES in 1987 and, in addition, banned the use of all hormonally active substances as growth promoters in food-producing animals in 1988. A similar condition was placed on all countries, so-called “Third Countries”, wishing to export meat from such animals to the EC.
The United States and Canada objected to this ban to the World Trade Organization (WTO). As a result, in 1997, the WTO Expert Panel found that the ban was not based on science – for example, on a risk assessment or on relevant international standards.1
The European Commission appealed against this ruling. In February 1998, the Appellate Body upheld the WTO Expert Panel's view, in that they found that the ban had been imposed without credible evidence to indicate that there were health risks posed by eating hormone-treated meat. As a result, the European Commission was given 15 months to remove the ban or produce a risk assessment.
As a response to this ruling, in early 1998, the European Commission (EC) sponsored 17 research studies to help clarify the findings in the Appellate Body report. These covered toxicological and carcinogenicity aspects, residue analysis, potential abuse and control problems and environmental effects of hormone use.
At the end of 1998, the EC's Scientific Committee on Veterinary Measures relating to Public Health (SCVPH) was asked to carry out an assessment of the risk to human health from the use of the six hormonally active substances, particularly from residues from bovine animals where such substances were administered for growth promotion. These substances were: 17β-oestradiol, testosterone, zeranol, progesterone, trenbolone acetate and melengestrol acetate. In April 1999, the SCVPH produced its first Opinion on the subject.1
The SCVPH concluded that the risks from hormone-treated meat were “higher than previously thought”. Further, it proposed that there was a significant body of scientific evidence suggesting that 17β-oestradiol should be considered a complete carcinogen. It also concluded, with different standards of evidence, that there were risks to consumers from the other five hormones examined.
Most importantly, the SCVPH concluded that no threshold concentrations could be defined for the hormones – this precluded the setting of Acceptable Daily Intakes (ADIs) or Maximum Residue Limits (MRLs). However, they were unable to estimate the extent of any risk.
In the UK, the then Minister of Agriculture, Fisheries and Food asked the Veterinary Products Committee (VPC) to assess the evidence in the SCVPH Opinion. The VPC is an independent scientific committee that has the remit to give scientific and veterinary advice on veterinary medicines and other products used for animal production and husbandry to the Veterinary Medicines Directorate (VMD). The VMD has statutory duties in relation to veterinary medicines and products in the UK. The VPC set up a Sub-Group to do this, which reported in October 1999. At the same time, the Safety Working Group of the Committee for Veterinary Medicinal Products (CVMP) – the European Commission's own organisation with responsibility for advising on the safety of veterinary medicines – also examined the SCVPH Opinion.
Following detailed deliberations, the VPC Sub-Group was unable to support the SCVPH's conclusion that the risks associated with eating hormone-treated meat “might be higher than previously thought”. The Sub-Group also found that it had sufficient concerns about the scientific reasoning in a number of key areas, to throw serious doubt on the conclusions of the SCVPH. However, the Group identified a number of areas where additional expert evidence should be sought to add to the data and help prevent selective scientific conclusions being drawn in the future.
The CVMP also produced a report in 1999 in response to the SCVPH Opinion (EMEA/CVMP/885/99). The CVMP was unconvinced by the SCVPH data and arguments, and concluded that its (the CVMP's) previous recommendations with regard to the ADIs and MRLs of the five hormones examined were still applicable (17β-oestradiol, altrenogest, progesterone, flugesterone acetate and norgestomet). The CVMP also noted that its conclusions were practically the same as the FAO/WHO Joint Expert Committee on Contaminants and Food Additives.2
The UK Government accepted the view of the VPC-that they were unable to support the conclusion of the SCVPH of a “higher risk than previously thought” from eating hormone-treated meat. The UK has, however, always fulfilled its obligations to enforce the EU ban and continues to do so.
In May 2000, the SCVPH produced a review3 of its Opinion after having examined the reports of both the VPC and CVMP. The SCVPH noted that these two independent evaluation reports showed a high degree of consensus on the possible risks. However, it did not seek to answer the questions raised in the reports, but concluded that they did not provide convincing data and arguments that demanded revision of the SCVPH's previous conclusions. The SCVPH review acknowledged that there were obvious gaps in the present understanding on the hormones in relation to animal metabolism and residue deposition but it anticipated that the EC's research programmes the EC had instigated would provide additional data on these topics.
Following the completion of these 17 studies sponsored by the EC, the SCVPH was asked to review their previous Opinions of 1999 and 2000, the data from the 17 studies and other recent scientific literature from any source. In April 2002, the SCVPH released yet another Opinion.4 In this, it reconfirmed the views in the previous SCVPH Opinion and concluded that no amendments to these were justified.
In September 2003, the European Parliament and Council of Ministers passed Directive 2003/74/EC.2 This Directive put further restrictions on the use of veterinary medicinal products containing oestradiol or its ester-like derivatives. Originally, the intention was for all uses of 17β-oestradiol to be banned and restrictions tightened on other hormones; however, the UK and other Member States expressed concerns about the potential loss of a number of valuable veterinary therapeutic products.
This Directive required that oestradiol and its derivatives should not be used for oestrus induction/synchronisation in cattle, horses, sheep or goats after October 2006. These substances were still allowed to be authorised for the treatment of foetus maceration or mummification and the treatment of pyometra in cattle, or oestrus induction in cattle, horses, sheep or goats. However, the Directive required the European Commission to present a report by October 2005 on the possible alternatives to oestradiol for these therapeutic uses. A new Directive 2008/97 now bans the use of oestradiol in cattle, etc. The key conclusions of the SCVPH Opinion are reproduced below:
“The review of the 17 studies launched by the European Commission and a recent scientific literature allows the following conclusions:
Ultra-sensitive methods to detect residues of hormones in animal tissues have become available, but need further validation.
Studies on the metabolism of 17β-oestradiol in bovine species indicate the formation of lipoidal esters, disposed particularly in body fat. These lipoidal esters show a high oral bioavailability in rodent experiments. Thus, the consequence of their consumption needs to be considered in a risk assessment.
Experiments with heifers, one of the major target animal groups for the use of hormones, indicated a dose-dependent increase in residue levels of all hormones, particularly at the implantation sites. Misplaced implants and repeated implanting, which seem to occur frequently, represent a considerable risk that highly contaminated meats could enter the food chain.
There is also a dose-dependent increase in residue levels following the oral administration of melengestrol acetate at doses exceeding approved levels, with a corresponding increased risk that contaminated meats could enter the food chain.
Convincing data have been published confirming the mutagenic and genotoxic potential of 17β-oestradiol as a consequence of metabolic activation to reactive quinones. In vitro experiments indicated that oestrogenic compounds might alter the expression of an array of genes. Considering that endogenous oestrogens also exert these effects, the data highlight the diverse biological effects of this class of hormones.
No new data regarding testosterone and progesterone relevant to bovine meat or meat products are available. However, it should be emphasised that these natural hormones are used, only in combination with 17β-oestradiol or other oestrogenic compounds in commercial preparations.
Experiments with zeranol and trenbolone suggested a more complex oxidative metabolism than previously assumed. These data need further clarification as they might influence a risk assessment related to tissue residues of these compounds.
Zeranol and trenbolone have been tested for their mutagenic and genotoxic potential in various systems with different endpoints. Both compounds exhibited only very weak effects.
Data on the genotoxicity of melengestrol acetate indicate only weak effects. However, pro-apoptotic effects were noted in some cell-based assays, which were attributed to the impurities in commercial formulation. Further experiments should clarify the toxicological significance of these impurities.
Model experiments with rabbits treated with zeranol, trenbolone or melengestrol acetate, mirroring their use in bovines, were designed to study the consequences of pre- and perinatal exposure to exogenous hormones. All compounds crossed the placental barrier easily and influenced to varying degrees the development of the foetus, at the doses used in the experiments.
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 increased adult breast cancer risk.
Several studies were devoted to the potential impact of the extensive use of hormones on the environment. Convincing data were presented indicating the high stability of trenbolone and melengestrol acetate in the environment, whereas preliminary data were provided on the potential detrimental effects of hormonal compounds in surface water.
In conclusion, after re-appraisal of the data from the 17 studies and recent scientific literature, the SCVPH confirms the validity of its previous Opinions (in 1999 and 2000) on the Assessment of Potential Risks to Human Health from Hormone Residues in Bovine Meat and Meat Products, and that no amendments to those Opinions are justified.”4
Due to this new SCVPH Opinion, in 2002 the VPC was again asked to examine the scientific evidence for a ban on the use of hormones in food-producing animals and to advise on whether therapeutic uses posed any risk to consumers. A new VPC Working Group was formed with the following Terms of Reference:
“to evaluate the latest Opinion of the Scientific Committee on Veterinary measures relating to Public Health (SCVPH) dated April 2002 and advise on its conclusions and;
to advise on whether the latest Opinion of the SCVPH, and the research studies on which it is based, addresses the conclusions reached in the report by the VPC Sub-Group published in October 1999.”
1.1.2 The Role and Remit of the VPC 2002 Working Group
The Working Group (WG) was set up in November 2002 and consisted of members of the VPC plus a number of additional invited experts required covering the specialisation required to address all the relevant scientific and veterinary areas. It was agreed by the WG that, in addition to a critical evaluation of the SCVPH Opinion and the 17 EC-funded studies, other recent relevant scientific publications would be sought, evaluated and included, based on the knowledge and expertise of WG members. In some cases, only the study reports of some of the 17 studies submitted to the EC were available. In other cases, peer-reviewed publications were available, based on results within some of the study reports.
The WG also agreed that, apart from a scientific evaluation of existing data, it was important to highlight significant gaps in knowledge and areas where uncertainty existed. This was felt to be particularly relevant as the WG was aware that it had no mandate to make, or even propose, on policy in this area; thus, it was crucial for the report to express uncertainties where they existed in the science base which would inform policy makers and other stakeholders.
Another issue of concern within the WG was to ensure that the reader understood the various uses to which hormonal substances had been, or could be, used in bovine meat production and ensuing meat products. Their use as growth promoters may be regarded as zootechnical. This was the original use for which the EU ban was intended. Oestrus induction in cattle and horses is also a zootechnical use whereas other veterinary uses such as the treatment of pyometra are regarded as therapeutic.
It was noted by the WG that, although the toxicological evidence of the substances under discussion would be based on studies on specific laboratory animals, the actual risk assessment to humans would be dependent on the dose used, the time of administration relative to slaughter, the pharmacological formulation and route of application to these meat-producing animals, and information on human consumption; questions of whether risks, however minimal, should be permitted – depending on whether the use is zootechnical, hence commercial or therapeutic, and so to the benefit of the health and welfare of the animal – were beyond the mandate of the WG. In other words, such an expert group can only provide a scientific opinion, not make policy.
The WG noted that illegal or improper use of growth-promoting substances, in the form of implants and/or in feed, might present an added exposure to humans who consumed meat or meat products from animals so treated. However, they also noted that this would be no different, in terms of risk management, from the illegal or inappropriate use of any other veterinary products and, as such, would be beyond the remit of the WG.
1.2 Biological Effects of Hormones and Endpoints of Health Concern
1.2.1 General Properties of Hormones
Hormones are vital in normal development, maturation and physiological functioning of many vital organs and processes in the body. However, like any other chemicals of natural or synthetic origin, hormones can be toxic to living organisms under certain circumstances. The toxicity may be due to an excess of its normal (“physiological”) action. This may be the result of excessive exposure to the substance, for example following absorption of a large dose, or because the physicochemical nature of the substance gives it greater (more “potent”) or more prolonged activity of the same type, or because the hormonal action (endocrine effect) occurs at an abnormal time during development or adult life, or is an action on an organism of the inappropriate sex. Hormones, like other chemicals, may also exert direct toxic actions not related to their endocrine (“physiological”) effects.
Hormones are very “active” substances, whereby relatively small doses may have profound effects. However, this does not mean that they do not follow typical dose-response relationships, or that all their actions are necessarily permanent in the current or future generations. The relationship between the dose applied to an organism and the effect produced may be linear or it may follow a more complex relationship because it will depend on the extent of absorption of the substance, how it is transported, metabolised and excreted from the body (“pharmacokinetics”) and its access across internal barriers to its sites of action. The severity and duration of hormone-induced toxicity will therefore be related to the local concentration at the target site in the body, at least above any possible minimum threshold for an effect and below the concentration at which the mechanism causing the effect becomes saturated and the action reaches its maximum. Many toxic actions are reversible once exposure ceases because normal physiological processes and repair mechanisms return the affected cells and organism to normality. However, irreversible damage may result if there is extensive damage to a tissue.
1.2.2 Health Endpoints of Concern
Endogenously produced sex steroids exert a wide range of biological effects on the body with most tissues/organs affected to a greater or lesser degree (not just the reproductive organs). These effects vary according to age and gender. Therefore, exposure to exogenous steroidogenically active compounds at certain levels has the potential to affect many organs.
Overwhelming evidence suggests that sex steroids exert effects that are dose-dependent and that a threshold dose exists, below which no biological effect will occur. This threshold may vary according to age, gender and tissue/organ.
Production, bioavailability/metabolism and action of endogenous sex steroids are closely controlled. Exogenous exposure to synthetic sex steroids may therefore be compensated for such that no biological effect ensues.
During development, sex steroids play an organisational role that involves programming of various tissues/organs in a gender-specific way. These effects are largely irreversible (e.g. sexual differentiation of the external genitalia).
Differences in the degree of exposure to sex steroids during development between individuals can occur naturally (e.g. higher exposure in twin pregnancies) and this may alter predisposition to future disease such as breast or testicular cancer.
Importantly, for the six hormonal substances considered by the WG, effects of concern would have thresholds, although they may be difficult to define. As noted above, because of “feedback” systems within the body, the introduction into the body of an exogenous source of a sex steroid hormone may be compensated for, so that no biological effect is produced. However, this may not apply to the foetus, postmenopausal women or pre-pubertal children – thus making these groups potentially more vulnerable.
The major areas of concern expressed in the literature and in the SCVPH Opinions were to health effects of hormonal substances in bovine meat and meat products related to cancer, mutagenicity and reproductive effects, in particular endocrine disruption. Generally, cancer and mutagenicity are well described, reasonably well understood by most readers and need little general description. However, endocrine disruption has become, in recent years, an area where there has been concern about potential harmful outcomes for a wide range of chemicals hitherto unsuspected of causing such effects. Many of these are less well described and are thus described below.
1.2.3 Endocrine Disruption
Hormones such as androgens and oestrogens play important roles in the day-to-day functioning of the body. Effects are not restricted to the reproductive system but are pervasive, affecting most tissues in the body, including the brain, bone, muscle, liver, fat, cardiovascular and immune systems.5 During development, androgens and oestrogens also have important organising effects in which the function of certain tissues may be permanently altered. The role of androgens in masculinising the male (reproductive system, genitalia, brain and rest of the body) during foetal life is the most dramatic example of this. It is well established that when there is inappropriate production or inhibition of normal androgen/oestrogen production/action, whether in foetal, childhood or adult life, then important health disorders are likely to occur.5
Against this background, it is understandable that there has been widespread concern about the potential health consequences that might result from human exposure to environmental chemicals that possess intrinsic oestrogenic, androgenic or anti-androgenic activity. In addition to xenobiotics, such effects are also attributed to phytoestrogens.3 These chemicals have been loosely termed “endocrine disruptors”, based on their potential to alter normal hormone action.6,7
Whether or not this potential is realised in the body of the recipient is dependent on many factors, one of which is the potency of the chemical in question, i.e. what dose of compound will exert a detectable effect? The oestrogenic potency of any ingested compound is determined by a combination of factors. These include:
absorption, metabolism, entero-hepatic recirculation,
binding to plasma proteins such as sex hormone-binding globulin (SHBG),4 and
affinity for binding to either oestrogen receptor-α (ERα) or oestrogen receptor-β (ERβ).
Other factors such as rates of breakdown of the compound and local availability in particular tissues may also be important. It is not easy to predict from simple measurements of any one of these parameters what the overall oestrogenic potency of a compound will be in the whole animal. Most studies on the oestrogenic potency of compounds, to which there is human exposure, have utilised in vitro cell transfection systems that assess the ability and affinity of the compound to compete for binding to oestrogen receptors, ERα or ERβ. Compounds that have a high affinity for these receptors are likely to be potent oestrogens and may, therefore, exert biological effects on oestrogen target tissues. However, as other factors mentioned above can influence potency, studies involving in vivo administration of such compounds provide the most accurate guide as to whether or not they may have target tissue effects. The immature rat uterotrophic assay is the most widely used endpoint of oestrogen bioactivity in vivo. Though this provides perhaps the most useable measure of oestrogenic potency in vivo, activity in this assay does not necessarily mean that there would be similar biological activity at other oestrogen target sites, such as in the breast or in bone.10
Most endocrine disruptors have only very weak intrinsic hormonal activity when compared with the natural (endogenous) hormones (testosterone, oestradiol) that are made within the body.5 It is also commonly overlooked that all hormonal systems in the body are tightly controlled and this usually involves a feedback “balancing system” or systems that constantly check the extent of action of the hormone in question and adjust its concentration up or down accordingly.5 Therefore, in theory, exposure to exogenous endocrine disruptors at a concentration sufficient to cause an effect should be compensated for by altered production of the endogenous hormone in question.
An important exception to this principle is exposure in foetal/perinatal life when hormones are exerting organisational effects and these feedback systems may not be operative.5 Also in postmenopausal women, no ovarian oestrogen synthesis occurs and the residual oestrogen production, which occurs predominantly in subcutaneous fat, is not subject to significant feedback control. Thus in these circumstances, ingestion of exogenous hormone will lead to additive increments of exposure. This is something that was given particular attention by the WG in their deliberations.
Some compounds may possess no intrinsic hormonal or anti-hormonal activity, yet still be capable of exerting hormonal effects. This can occur if the compound affects the production, bioavailability or metabolism of endogenous (potent) hormones. Such compounds potentially pose a more serious health threat, as alterations in endogenous hormones will cause clinical disorders; there is a growing number of examples of such compounds.11 Evaluation of potential effects of residues of hormone growth promoters in meat ingested by humans therefore needs to be considered against the background outlined above. In contrast to most endocrine disruptors, several of the growth promoters are intrinsically potent hormones (e.g. oestradiol). This means that effects are more likely if there is significant human exposure. However, human exposure is most likely to residues of the growth-promoting agent present in muscle/fat, which will normally be at very low concentrations (pg to μg kg−1 day−1). This may rule out possible effects.12–14 Moreover, as human exposure will be via food, the absorption and metabolism of the compound in the gut becomes very important. Oral absorption of oestradiol is good, however the quantity reaching the systemic circulation is greatly reduced by extensive first-pass metabolism in the intestines and liver, and oestradiol is generally considered to be inactive when administered orally.15
1.2.4 The Use of Oestradiol in Cattle
Cattle are the main food-producing species in which oestradiol products are used for therapy or growth promotion. In order to put the contribution to the food chain from therapeutic and zootechnical use of oestradiol in context, the endogenous production of oestrogens arising at various stages of the reproductive cycle is described below.
1.2.4.1 The Reproductive Cycle of the Cow
The reproductive cycle of the “average” dairy cow, calving approximately once a year, involves four to five oestrous cycles followed by pregnancy. On average, she spends approximately 75% of the year being pregnant and produces milk for all but the last 40–60 days of pregnancy.
Endogenous production of oestradiol and oestrogens varies throughout the reproduction cycle. In the “cycling” cow there are two or three small peaks of oestradiol during the 21-day oestrous cycle, which accompany waves of follicular development, and one major peak at oestrus. Reported concentrations of oestradiol in plasma and milk vary according to the assay method used but are typically four to five times higher during oestrus than in the remainder of the cycle. During pregnancy oestradiol concentrations in plasma and milk rise dramatically and are typically ten-fold higher than in the cycling animal. Therefore most milk comes from pregnant animals and thus contains higher concentrations of natural endogenous oestrogens.
In addition, a proportion of cows/heifers entering the food chain are pregnant. Meat from these individuals can also contain higher concentrations of oestrogen produced by the foeto-placental unit. The WG noted that when the removal of the ban on the inclusion of meat from cattle over 30 months into the food chain occurs, approximately 25% of cull cows entering the food chain are likely to be pregnant.16 Meat from these animals will add significantly to the oestrogen concentrations currently entering the food chain from this source. However it should be noted that removal of the restriction would only return the oestrogen load to pre-ban concentrations.
1.2.4.2 Therapeutic Use of Oestradiol in Cattle
The uses and indications for oestradiol salts have been recognised for some time and are clearly defined. Oestradiol benzoate was authorised for the treatment of pyometra and endometritis in cattle. Therapy may also be beneficial to enhance oestrous behaviour in suboestrous or anoestrous animals in the induction of lactation as well as in the dilation of the cervix in cases of abortion. Various oestradiol salts were also luteolytic and are incorporated into oestrous synchronisation devices (PRIDs). Equally, the administration of 1 mg of oestradiol by injection in conjunction with the intravaginal progesterone-releasing device (Cidr) increases synchrony and may enhance the expression of oestrous behaviour. However, since the ban noted above, no oestradiol-based medicines are available in the UK except mescalin for use in dogs.
1.2.4.3 Intra-uterine Infections after Calving
Post-partum endometritis occurs mainly in dairy cows. Various reports estimate that prevalence in these animals ranges between 3 and 8%. This condition occurs during early lactation when discharges need to be eliminated to aid hygienic conditions for milk production.
A proportion of the cases occur in cows that have already experienced a post-calving ovulation and have functioning corpora lutea. A proportion of these will respond to a luteolytic injection by producing endogenous oestrogens and resolving the condition by “self-cure”. However, a significant proportion simultaneously experience post-partum anoestrus due to ovarian inactivity. This subgroup is not suitable for luteolytic therapy. For this group there are two alternative strategies. The first uses an intramuscular injection of oestradiol benzoate to mimic the effects of normal ovarian follicular cyclicity. The result is relaxation of the cervix, improved muscle tone and increased supply of leukocytes to the uterus. These induced changes result in evacuation of the uterine contents and elimination of infection. Following the injection, the blood concentration of oestradiol does not rise above the normal physiological range. Indeed, as the remnants of the foeto-placental unit are a source of oestrogen, early evacuation may result in a more rapid fall in milk oestrogen concentrations in these cows. The second accepted therapy involves intra-uterine infusion(s) of an antimicrobial (often an antibiotic) solution. This approach aims to reduce the intra-uterine infection and thus promote a return to normal ovarian cyclicity.
An overall ban on the therapeutic use of oestradiol and/or its esters would thus prevent the former therapy and greater use of antibiotics would be necessary. As these cows are producing milk that may enter the food chain, minimal use of antibiotics is required. Also a proportion of these infections may not respond to antibiotic therapy. Therefore, the result of a ban would be an increased risk of antibiotic resistance and reduced standard of welfare for a proportion of cows.
1.2.4.4 Veterinary Medicinal Products Containing Oestradiol
At the time of the WG's activity in 2002, and before the current ban, there were four veterinary medicinal products containing oestradiol available in the UK as shown below in Table 1.1. These products were formulated to release their oestradiol content in one burst of short duration and are therefore not suitable for growth promotion. One of these products was licensed exclusively for use in the bitch whilst the other three are licensed for zootechnical and therapeutic uses in the cow. Crestar devices were used purely for oestrous synchronisation in dairy and beef heifers and beef cows, and are not used in lactating dairy cows. PRIDs were used both for treatment of suboestrous and anoestrous as well as for oestrous synchronisation in both beef and dairy animals.
Example of oestradiol containing veterinary medicinal products
Product . | Active ingredient . | Concn (mg ml−1) . | Route of administration . | Indications . | Dose . | Withdrawal . |
---|---|---|---|---|---|---|
Mesalin | Oestradiol benzoate | 0.2 | Subcutaneous or intramuscular | Mesalliance in the bitch | 0.01 mg kg−1 3 and 5 days post mating | N/A |
Oestradiol benzoate | Oestradiol benzoate | 5 | Subcutaneous (bitch) | Mesalliance in the bitch | 0.3 mg g−1 1–4 days post mating | N/A |
Intramuscular (cow) | Pyometra and endometritis in the cow | 3 mg 500kg−1 | Milk – 0 d | |||
Meat – 15 d | ||||||
Crestar | Oestradiol valerate | 2.5 | Intramuscular | Oestrous synchronisation in combination with implant | 5 mg | Not for use in milking cows |
Meat – 14 d post implant removal (23–24 d) | ||||||
PRID | Oestradiol benzoate | N/A | Intravaginal | Oestrous synchronisation and stimulation of ovarian activity in anovulatory and suboestrous cows | 10 mg | Milk – 0 d |
Meat – 24 hrs |
Product . | Active ingredient . | Concn (mg ml−1) . | Route of administration . | Indications . | Dose . | Withdrawal . |
---|---|---|---|---|---|---|
Mesalin | Oestradiol benzoate | 0.2 | Subcutaneous or intramuscular | Mesalliance in the bitch | 0.01 mg kg−1 3 and 5 days post mating | N/A |
Oestradiol benzoate | Oestradiol benzoate | 5 | Subcutaneous (bitch) | Mesalliance in the bitch | 0.3 mg g−1 1–4 days post mating | N/A |
Intramuscular (cow) | Pyometra and endometritis in the cow | 3 mg 500kg−1 | Milk – 0 d | |||
Meat – 15 d | ||||||
Crestar | Oestradiol valerate | 2.5 | Intramuscular | Oestrous synchronisation in combination with implant | 5 mg | Not for use in milking cows |
Meat – 14 d post implant removal (23–24 d) | ||||||
PRID | Oestradiol benzoate | N/A | Intravaginal | Oestrous synchronisation and stimulation of ovarian activity in anovulatory and suboestrous cows | 10 mg | Milk – 0 d |
Meat – 24 hrs |
1.2.4.5 Food Safety Considerations Following Application of Oestrus Control Products
Crestar was not licensed for use in lactating dairy cattle and, despite the longer half-life of oestradiol valerate, the withdrawal period should ensure that no residues should reach the food chain via meat. In contrast PRID and oestradiol benzoate were licensed for use in both dairy and beef animals. However, as long as withdrawal periods are observed there were considered to be no residue implications associated with these products. The only area of concern noted would be the intramuscular injection site where significant residues may be present if the withdrawal periods were not observed. (It is, however, worth noting that therapeutic doses of oestradiol result in pg concentrations that have a half-life of 8 hours and do not exceed normal endogenous blood concentrations.)
The rationale for continued use was that, used for therapeutic or zootechnical reasons, these products do not cause the concentration of plasma or milk oestradiol to rise outside the physiological range. The use of oestradiol benzoate will cause an elevation in plasma and milk oestradiol concentrations; however these elevated concentrations are still well below those of naturally circulating oestradiol in pregnant animals.
As an illustration, the WG reported that if one considers the total number of treatments with oestradiol benzoate and PRID in 2002 and assumes that they were all delivered to lactating dairy cattle, and take the worse-case scenario, this use elevates oestradiol concentrations to the equivalent of a pregnant animal for 2 days. In 2002 there were approximately 94,500 treatments sold (oestradiol benzoate 29,870 doses; PRID 64,448 doses); assuming these were all used in the 2.25 million dairy cows in the UK, this equates to 0.042 doses per animal. The worse-case impact of this use could therefore be said to be the equivalent of extending pregnancy by 2 hours per cow in the national herd. To put this into context, a 1.2% increase in the proportion of dairy cows in calf to continental beef bulls would result in a similar increase in the duration of pregnancy (by virtue of the longer gestation period of these breeds).
Interestingly, the WG Report noted that the last 30 years have seen an increase in the use of continental sires from virtually zero to approximately 30% of dairy cows services; this management change alone had resulted in an additional 5.25 million days of pregnancy or 2.33 extra days per cow – equivalent to some 2.65 million oestradiol treatments per year.
1.2.4.6 Growth Promotion – Multiple Implantations into Cattle
Implanting hormonal growth promoters was and is currently widespread in the beef cattle industry of many non-EU countries for the better performance in growth and improvement of feed efficiency. In 1999, more than 96% of all US cattle in feedlots were implanted at least once (NAHMS, 2000, cited in 17). These hormonal implants may enhance growth during suckling, growing and finishing stages of production.18,19 Implant residues entering the food chain as a result of the implants administered during the suckling and growth phases of production will be lower than those arising from implants during the finishing stages. The weight gains are significant. A combination of trenbolone acetate and oestradiol improved average daily gain and feed efficiency during finishing stages by up to 20% and 13.5% respectively.20 The magnitude of the response to these anabolic implants in the performance of beef cattle varies depending on the type of implants, quantity of growth promoter, duration of exposure, age of animals and combination of implants. Improved performance in steers originating from the dairy herd has also been noted.
In general, anabolic implants have minimal or negative effects on meat quality including lower marbling, high shear force and advanced carcass maturity resulting in lower quality grades. Repeated (five sequential implants) implanting has been claimed to have detrimental effects detectable by consumer taste panel scores.19 However, consumers failed to detect these differences in meat after 7 and 14 days aging when more moderate (two sequential implants) implant regimes were used.21 Therefore, there is no organoleptic characteristic by which consumers can be expected to detect meat originating from implanted animals. To date, the WG noted that there was no validated technique to detect and assign the low residual concentrations of oestradiol in the finished edible products to natural sources or to implant residue. This is an area where research was noted to be urgently needed.
As noted above, these implants are no longer allowed in the European Union (EU), which also prohibits the importation of beef and its products derived from hormone-treated cattle.
1.2.4.7 Alternatives to Oestradiol-containing Products
In the absence of oestradiol-containing products, alternatives would need to be employed. For oestrous synchronisation regimes prostaglandin or the progesterone-releasing device (Cidr) could be employed. Alternatives for the treatment of pyometra and endometritis could include the use of prostaglandins for a combination of their direct ecbolic and luteolytic effects. However, it would not be possible to substitute for the current “off-label” use for enhancement of oestrous behaviour.
1.2.4.8 Zootechnical versus Therapeutic Use
The WG was of the view that the growth-promotion activity should be seen separately from the other zootechnical uses and the therapeutic uses of 17β-oestradiol and other hormonally active substances. One strongly expressed view was that, if the current EU position on the ban of 17β-oestradiol for growth-promotion purposes were to be maintained and extended, it would be most unfortunate to lose its use for other zootechnical or therapeutic purposes. There was also agreement that the therapeutic uses of 17β-oestradiol were more important than other zootechnical uses. It was noted that many of the alternatives to 17β-oestradiol would also result in a comparable rise in endogenous oestradiol. As an example, the use of prostaglandins, if used as an alternative, would raise endogenous oestradiol concentrations, so having a similar outcome to the administration of 17β-oestradiol in the first place. For this reason alone, it seemed sensible to continue with the use of 17β-oestradiol. It is well established that prostaglandins can exert both respiratory and reproductive effects following accidental exposure; for this reason it was felt that the operator risks associated with the use of prostaglandin products should not be overlooked.
1.2.4.9 Implications of Removal of Oestradiol-containing Products
Finally, the WG noted that it was important to consider the implications of removal of the use of oestradiol-containing products in food-producing species. Some of the possible implications of the removal of oestradiol products are:
an increase in the use of prostaglandins, which have health and safety implications for the operator as well as increasing endogenous oestrogens,
an increase in the use of antibiotics for the treatment of endometritis,
the development of microbial resistance due to increased use of antibiotics,
welfare implications through sub-optimal treatment of affected cattle,
“off-label” use of oestradiol-containing products licensed for use in companion animals is likely to occur,
unregulated use of oestradiol formulated on an ad-hoc basis from chemical suppliers may occur.
1.3 The Scientific Evidence Available to the VPC Working Group
The following sections contain a précis of the scientific evidence used by the WG in its deliberations and presented in their report. It consisted of discussion of the papers and reports emanating from the 17 EC-funded studies as well as other relevant studies identified by members of the WG. It also contains the conclusions of the WG within each of the specific areas.
1.3.1 Exposure to Hormonally Active Substances
1.3.1.1 Analytical Techniques
The SCVPH 2002 Opinion discussed four of the 17 EC-sponsored studies which concerned analytical techniques for the detection of trace hormones in meat and one study which developed screening bioassays for known oestrogenic and androgenic compounds in yeast, trout hepatocytes and human endometrial cancer cells (Presence of oestrogen in meat) would have been of particular relevance, but the WG were informed that no publication would be forthcoming.5 Two studies, both entitled Analysis of 500 samples for the presence of growth promoters would appear to represent key research involving new methods for the detection of trace hormones in meat, based on GC/MS. However, the report of one comprised only a one-page abstract of a lecture, the text of which includes a number of anecdotes but no new study data. The other study was supported by two publications.22,23 The derivation of new laboratory methodology was adequately described. But other than the description of steroids in four samples of residue-positive meat and liver, there are no data on samples that reflect the concentrations of the compounds under consideration in a representative set of samples.
On the basis of the results from these last four studies, the SCVPH report concluded, appropriately, that “the low number of samples does not allow a qualified validation of typical characteristics such as sensitivity, specificity, accuracy and reproducibility”.
1.3.1.2 Bioassays for Screening
The SCVPH 2002 Opinion discussed one study that developed a screening bioassay to detect known oestrogenic and androgenic compounds in yeast, trout hepatocytes and human endometrial cancer (Ishikawa) cells.24 The study revealed a highly variable sensitivity between the tests for oestradiol, and a variable differential response in in vitro potency tests that may in part be explained by the metabolism of some of the compounds by trout hepatocytes and Ishikawa cells. No data were derived by application of these techniques to meat; if they were to be so applied, exhaustive chromatography to isolate individual steroids would be required in order for the tests to provide useful data. The SCVPH report concluded that, in view of their lack of specificity and sensitivity, the assays performed in recombinant yeast and trout hepatocytes are not justified. The SCVPH Opinion of 2002 on the unsuitability of the yeast assay seemed reasonable to the WG. The WG also noted that this is a profoundly different conclusion from the SCVPH Opinion of 1999, when it was the availability of this highly sensitive (as it was then regarded) new bioassay that led them to consider that previous data on low oestrogen concentrations might be flawed.
1.3.1.3 WG Conclusions and Recommendations
The Working Group concluded that a number of new analytical methods had been developed that might helpfully be applied to the analysis of residues in the meat of cattle, but no substantial data had been presented from their application, nor had they been fully evaluated. These new techniques should be applied to meat in sufficient sample sets to provide reliable estimates of the relevant residues in untreated and implanted animals in the form that they enter the human food chain.
The suitability of three complementary bioassays for screening tissues for oestrogenic and androgenic compounds had not been demonstrated. Unless rigorous chromatographic separation techniques are developed, these bioassays should not be used for assessing residues in meat.
1.3.2 Bioavailability of Hormonally Active Substances
The SCVPH 2002 Opinion discussed two EC-funded studies relating to the bioavailability of hormonally active substances. One study14 involved the development of a new assay procedure for quantification of oestradiol concentrations in edible tissues and subsequent measurements of oestradiol concentrations in tissue samples from cattle following oestrogen implantation. The new assay included the analysis of lipoidal esters of oestradiol. Validation of the analysis of free oestrogens was complete but was only partial for the analysis of lipoidal esters. Nonetheless, the conclusion that lipoidal esters account for approximately 50% of the total oestradiol concentration in control or single-implanted steers appears sufficiently sound, as is the conclusion that this fraction should be taken into account when assessing the overall intake of oestrogens from treated cattle. Another study25,26 investigated the metabolism of 17β-oestradiol by bovine hepatocytes and human intestinal and breast cells and tested their oestrogenic properties in the rat uterotrophic bioassay. These studies showed that 17α-oestradiol as well as lipoidal esters of 17β-oestradiol may be formed in vivo in animals implanted with 17β-oestradiol as a growth promoter. 17α-oestradiol had only about 10% of the in vivo oestrogenic potency of 17β-oestradiol, whereas the lipoidal oestrogens had ten-fold higher potency than 17β-oestradiol when tested in vivo in the rat uterotrophic assay.
Based on these above studies, the SCVPH 2002 Opinion concluded that metabolism of 17β-oestradiol in bovine species results in the formation of lipoidal esters, and that these esters are largely disposed of in body fat and may contribute significantly to an additional oestrogen exposure via meats. Lipoidal oestrogens may have higher potency in the breast due to their postulated transport via the lymphatic circulation and might potentially bioaccumulate in edible fat or meat. However, their oral bioavailability in humans following dietary exposure via contaminated meat products is unknown.
According to the WG, these studies on 17β-oestradiol metabolism and evaluation of oestrogenic potency in vivo appear to have been well conducted. The demonstration25 that certain residues may have potency in the uterotrophic assay is suggestive of bioavailability in vivo at this particular oestrogen target site. But it remains unclear whether similar actions would occur at other sites and whether any biological or “adverse” effect would result. Since the 1999 SCVPH Opinion, more recent data14 have shown that in steers implanted with one (normal practice) or with two or four implants inserted simultaneously (misuse), dose-dependent increases in concentrations of lipoidal oestrogens are found in fat, ranging from 30–40 ng kg−1 (one implant) up to 100–140 ng kg−1 (four implants). Similar or slightly higher concentrations of 17β-oestradiol were detected and much lower concentrations of 17α-oestradiol. In muscle, concentrations of all three compounds were generally <100 ng kg−1, whereas relatively high concentrations of 17α-oestradiol were detected in liver and kidney samples (200–800 ng kg−1). Based on the rat uterotrophic studies reported,25 no significant effects were detected in vivo for any of these three oestrogens at doses of 25 nmol kg−1 day−1 (∼7000 ng kg−1) over a 6-day period.
Assuming similar absorption and metabolism profiles in the human and rat, these findings would suggest that consumption of meat/fat from 17β-oestradiol-implanted cattle is unlikely to provide biologically significant oestrogenic exposure, even if unusually large amounts, from animals bearing four times the recommended number of implants, were eaten regularly. However, this conclusion makes numerous assumptions relating to absorption, metabolism and bioavailability and takes no account of the (theoretical) possibility that lipoidal oestrogens might bioaccumulate over time in fatty tissue, such as in the breast. The Maume et al. (2001) paper14 also considered the concentrations of oestrogens in animals with multiple implants in relation to the maximum human daily dietary intakes. For adults their estimates indicate an intake of <5% of ADI from a standard 500 g meat intake, but for pre-pubertal boys the ADI might be exceeded.
The WG felt that the SCVPH conclusion on the formation of lipoidal esters based on in vitro oestrogenic activity expressed in T47 D breast cancer cells26 (Hoogenboom et al., 2001) was reasonable, although it is not clear to what extent hydrolysis of lipoidal esters occurred before binding to ER in T47 D cells, and thus did not reflect a direct effect of the esters themselves. The degree to which any such hydrolysis would occur in humans is unknown.
1.3.2.1 WG Conclusions and Recommendations
Theoretically, if considerable amounts of 17β-oestradiol or lipoidal oestrogens are present as residues or contaminants in hormone-treated meat samples, they could exert significant effects on important oestrogen target tissues such as the breast. From the information available it appears that such exposures are unlikely to occur, even in situations in which misuse (i.e. over-implantation) of implants has taken place. However, the WG added that this conclusion should be re-assessed when, and if, new data become available to show that bioaccumulation of lipoidal oestrogens in fatty tissue occurs in vivo after oral administration. The data from Maume et al. (2001)14 in relation to pre-pubertal boys should be confirmed by others and if confirmed may be a cause for concern in this group.
1.3.3 Cancer Risks of Oestrogenic Substances
1.3.3.1 Breast Cancer Risk
Over the last few years there have been a number of publications that have had a substantial impact on our thinking on the effects of endogenous and exogenous oestrogens on the incidence of breast cancer. The data are directly relevant only to postmenopausal women.6
The Endogenous Hormones Breast Cancer Collaborative Group (2002) collated and analysed data from the nine published studies on the relationship between the plasma concentration of steroid hormones in postmenopausal women and the risk of subsequent development of breast cancer. Statistically significant relationships were found for several hormones. The strongest associations were for 17β-oestradiol (stronger still when only the protein-free fraction was considered) and testosterone. The relationship with testosterone was markedly weakened after adjustment for 17β-oestradiol; this is consistent with the relationship being determined by conversion of testosterone to 17β-oestradiol by the action of the aromatase enzyme. The Collaborative Group estimated that the relative risk for breast cancer was 1.25 for each doubling of plasma 17β-oestradiol concentrations. It is, however, near certain that this underestimates the true risk,7 since only a single blood sample from each subject was analysed in each of the studies. Additionally, the studies used a wide variety of analytical techniques, some of which were inaccurate and/or inappropriate for use in postmenopausal women.
The WG noted that recent publications from two very large studies have confirmed that use of hormone replacement therapy (HRT) by postmenopausal women for several years significantly increases their risk of breast cancer.28,29 The Women's Health Initiative (WHI) conducted a randomised, placebo-controlled trial of combination HRT (oestrogen plus progestin) versus no HRT in 16,608 North American women and found that breast cancer incidence was increased with a hazard ratio of 1.24.29 Notably, this study also reported that the breast cancers presented at a significantly later stage in the HRT users. The Million Women Study (MWS) recorded HRT usage in around one million postmenopausal women in the UK.28 Consistent with the WHI study, a higher incidence of breast cancer was found in combination HRT users. Importantly, in the context of the possible impact of ingested exogenous oestrogenic residues, MWS also reported a higher incidence in women taking oestrogen-only HRT (relative risk of 1.30 versus never users). These two studies confirmed that incidence of breast cancer in postmenopausal women is enhanced by the regular ingestion of oestrogens, mainly in the form of oral conjugated equine oestrogens, in quantities sufficient to reduce menopausal symptoms and preserve bone integrity. However, it is not possible from these studies to ascertain a concentration of 17β-oestradiol that does not enhance the risk of breast cancer (i.e. an NOAEL cannot be established).
Yen et al (1975)30 had described the effects of ingested oestradiol on plasma oestradiol, oestrone and gonadotrophin concentrations in postmenopausal women. Their data showed that 2 mg micronised oestradiol led to a maximum plasma concentration of 110 pg ml−1 5 hours after ingestion and this was a 437% increase i.e. from a starting concentration of 20 pg ml−1, falling to baseline by 24 hours. Thus, the increment in oestradiol from ingesting 2 mg was 90 pg ml−1=320 pmol l−1. Over 24 hours the mean increment would be no more than 150 pmol l−1.
The highest concentration of oestradiol detected in meat was 56 ng kg−1 in kidney.31 For a postmenopausal woman eating a kilogram of such kidney from a treated animal, the theoretical increment would therefore be 0.004 pmol l−1 (based on the finding that the 97.5th percentile of consumers eat 40 g of kidney per day). This increment is approximately three orders of magnitude below the most sensitive assays available and below any concerns related to breast cancer risk. Assuming that all of the oestradiol in meat is bioavailable and unaffected by food preparation, this would be expected to lead to mean concentrations increasing from approximately 40 pmol l−1 to 40.004 pmol l−1, an increase of only 0.01%.
1.3.3.2 Reduced Breast Cancer Risk in Future
The WG noted that in the UK in 2002, there were over 100,000 women receiving tamoxifen for treatment of breast cancer, of whom about 75% were postmenopausal. Modern aromatase inhibitors (e.g. anastrozole, letrozole, exemestane) have shown themselves to be superior in efficacy to tamoxifen,32 and it was widely expected that in the next few years this population will instead be treated with aromatase inhibitors. The efficacy of these compounds is determined by their suppression of plasma oestradiol concentrations from 25 pmol l−1 to below the detection limit of available assays (<3 pmol l–1). However, their efficacy could be compromised by the ingestion of oestradiol in doses that achieved increments of plasma oestradiol in single figures of pmol l−1.
1.3.3.3 In utero Exposure and Breast Cancer Risks
Evidence to support a role of intra-uterine factors such as 17β-oestradiol concentrations and development of breast cancer in the female is well established.33,34 One of the approaches used to explore the potential involvement of hormones in affecting predisposition to cancer is to compare twin versus singleton pregnancies, as oestrogen concentrations in twin pregnancies are invariably higher than in singleton pregnancies.35 The SCVPH report discussed one EC-commissioned study based on the Swedish Twin Registry that sought to evaluate whether risk of breast cancer was higher in twins.36
1.3.3.4 Oestrogen and the Human Gut
Ingestion of meat from animals treated with hormonally active substances is likely to result in highest levels of exposure in the gut. Therefore, potential effects of oestrogenic and/or androgenic compounds on the gastrointestinal tract need to be considered. There are clear gender-related differences in gastric acid production (40% higher in males37 ) and in the incidence of gastro-duodenal ulcers (higher in men38 ), Crohn's disease (higher in females39 ) and colorectal cancer (higher in males than in pre-menopausal women40 ).
There is reasonably convincing evidence that the gender difference in gastric acid production and colorectal cancer stems from differences in oestrogen production/action in males versus females, as oestrogen treatment reduces gastric acid production,41 and oestrogen exposure, whether endogenous or via hormone-replacement therapy, is protective against colorectal cancer.40 The precise involvement of oestrogens in progression of Crohn's disease is less clear.42 Based on these observations, the WG felt that, on balance, it would be concluded that any additional exposure of the gut to oestrogenic compounds present in meat from growth-promoted animals would have a health-beneficial, rather than -detrimental, effect.
1.3.3.5 WG Conclusions
The WG noted that recent studies have confirmed hormone replacement therapy increases the risk of breast cancer in postmenopausal women. However, it also noted that the maximum increase in oestradiol concentrations which might occur following consumption of oestradiol-treated meat by a postmenopausal woman is most likely to be below any concerns related to cancer risk. Oestrogen therapy appears to be protective against colorectal cancer and, therefore, arguably, any additional exposure following ingestion of oestradiol-treated meat would, if anything, have a health-beneficial effect for this commonly occurring cancer.
1.3.4 Altered Gene Expression by Oestrogenic Substances
The SCVPH 2002 Opinion discussed one EC-funded study that measured changes in gene expression of a number of hormone sensitive genes in a breast cancer (MCF7) cell line.43 The study found the expression of the different hormone responsive genes varied for the different oestrogens (zeranol and five related compounds, 17 β-oestradiol and three other oestrogenic substances). The SCVPH's only comment on this study was to note the down-regulation of GSTμ3, a Phase II enzyme that is involved in protection against DNA damage by free oxygen radicals.
The results showed that zeranol was of similar potency to 17β-oestradiol in this test system, although there were gene-specific differences in the extent of expression following treatment with the oestrogenic substances. Zeranol was much more potent than the naturally occurring fungal contaminant, zearalenone. It was also noted that zeranol, as a mycotoxin, may arise from fungal growth in cereals. Although interconversion of these substances can occur, this occurs at a low rate, suggesting zeranol may pose a greater hazard than the widely occurring zearalenone. Zeranol was the most potent inhibitor of the expression of MRG1/p35srj, which is involved with a nuclear transcription activation factor.
The WG noted that a number of authors have demonstrated that zeranol shows no or only limited binding to cellular binding proteins in contrast to 17β-oestradiol. This indicates that zeranol may be more potent in hormonal activity than 17β-oestradiol, due to higher bioavailability.44–47 Currently, its bioavailability by the oral route following consumption of meat products containing zeranol is not known. However, the higher oestrogenic potential seen in this in vitro test system is not consistent with reports of lower oestrogenic potential in a variety of in vivo assays e.g. vaginal cornification, uterotrophic assay and depression of serum gonadotrophin concentrations in castrated monkeys (reviewed in Lindsay, 1985). Furthermore, zearalenone and zeranol were shown to have similar physiological effects in a variety of in vivo assays. This suggests that this gene expression test system may not be a good indicator of in vivo oestrogenic potency.
Leffers et al. (2001)43 also showed 17β-oestradiol down-regulated GST μ3 at extremely low concentrations and suggested that this response might be a result of the altered redox status within the cell, rather than due to regulation by the oestrogen receptor. Together with the down-regulation of other phase II genes, they suggested that this could reduce protection against DNA damage and that changes in the relative balance of gene expression of Phase I and Phase II metabolism may be important in the proposed production of genotoxic catechol metabolites of 17β-oestradiol.
1.3.4.1 WG Conclusions and Recommendations
The evidence that oestradiol gives rise to genotoxic metabolites is considered further later. The low binding activity of zeranol and its ability to alter gene expression of important hormone responsive genes makes it important to determine the bioavailability and biological significance of zeranol residues in meat.48 Initially this would require studies of serum concentrations following consumption of meat from zeranol-treated animals.
1.3.5 Genotoxic and Mutagenic Effects of Oestrogenic Substances
In its 1999 Opinion, the SCVPH concluded there was sufficient evidence that 17β-oestradiol was genotoxic. This Opinion was based on positive responses in a variety of in vitro indicator assays. The VPC Sub-Group report (1999) pointed out that standard mutagenicity tests on 17β-oestradiol (bacterial mutation, mammalian gene mutation, in vitro micronuclei, the bone marrow micronuclei test and germ cell cytogenetic assay) were all negative. Furthermore, the studies on which the SCVPH based the opinion were all non-standard studies (methotrexate resistance, microsatellite formation), or were unconvincing due to the absence of a dose-response. The SCVPH concluded, however, that there was evidence for induction of oxidative damage, DNA adducts and aneuploidy.
1.3.5.1 17β-Oestradiol
The 2002 SCVPH Opinion states there is now conclusive evidence that 17β-oestradiol is genotoxic since it induces mutations in mammalian cells, oestradiol metabolites induce mutations in mouse skin in vivo and catechol oestrogen quinones form DNA adducts in cultured cells and mouse skin. This SCVPH Opinion is based on a study commissioned by the EC25,26 and other published papers. The study by Hoogenboom and colleagues showed that 17β-oestradiol and several of its metabolites were negative in a series of apparently well-conducted bacterial mutagenicity assays using a variety of strains and metabolic activation conditions employed in order to improve the potential sensitivity of the test. Furthermore, they also reported negative responses in the Comet assay using human intestinal cells (CaCo-2). The other published papers considered by the SCVPH are discussed below.
A number of recent papers strengthen the evidence that 17β-oestradiol can be activated to produce genotoxic metabolites by its conversion into catechol oestrogens which may be oxidised to form semiquinones and quinones (e.g. 49–51 ). These quinones can form DNA adducts leading to depurination. The metabolites may also generate potentially mutagenic oxygen radicals by redox cycling. Inactivation via O-methylation, glucuronidation or sulfation also occurs.
The mutagenic potential of oligodeoxyribonucleotides adducted with hydroxyoestrogen moieties was studied.52 A series of synthetic oligonucleotides was produced, each containing a modified nucleotide. These were used to create vectors, which were then used to transfect COS-7 monkey kidney cells. They were shown to induce G to T transversions in this model system. This study demonstrates that oestrogen metabolite adducts introduced into naked DNA are pre-mutagenic. The system bypasses normal cellular controls (activation/inactivation pathways and DNA repair) of the intact animal.
A metabolite of 17β-oestradiol, 2-methoxyestradiol (2-MeE2), was claimed to induce transformation in the SHE assay, chromosome aberrations and mutations at the HPRT and Na+/K+ ATPase loci.53 The study is poorly reported. 2-MeE2 induced mutations at only one of the doses tested, which was a mid-point dose in the case of the Na+/K+ ATPase assay. Furthermore, it is not clear whether cytotoxicity has been assessed appropriately and the assays lacked statistical power due to the low control frequency and low numbers of cells analysed. At best the evidence is marginal. The induction of chromosome aberrations is even less scientifically convincing. Concurrent cytotoxicity data are not given, but assuming the concentrations are similar to those measured in the mutation assay, then an increase in aberrations was only seen at toxicity concentrations in excess of the internationally acceptable limits. Nearly all the damage was due to “chromosome pulverisation”, an effect attributed by the authors to asynchronous division within multinucleate cells, and not therefore due to clastogenicity. Aneuploidy and polyploidy were also induced. While there is some evidence that the metabolite was able to induce cell transformation, the significance of this finding is less clear as the SHE assay detects both genotoxic and non-genotoxic substances. A wider range of metabolites was tested in the same systems.54 Despite significant methodological and reporting inadequacies, the WG agreed that there does appear to be some evidence that some of the metabolites can induce chromosome aberrations, mutations and cell transformation in SHE cells. 4-hydroxyestrone and 2-methoxyestrone, but not 17β-oestradiol, oestrone, 2-hydroxyoestradiol or 4-hydroxyoestradiol, induced mutations at the HPRT locus. Estrone, 4 hydroxyestrone and 4-hydroxyoestradiol, but not oestradiol or the other hydroxyl metabolites tested, gave some evidence of weak induction of chromosome aberrations.
The evidence that 17β-oestradiol is a point mutagen is derived from the publication of Kong et al (2000),55 based on the induction of mutations at the HPRT loci in V79 cells. However, this study is not acceptable by generally recognised standards of quality or accuracy. In the study report there is insufficient information to evaluate whether an adequate number of cells was treated, there is no dose-response (significant increases were seen in cultures treated with 10−11, 10−10, 10−7 and 10−6 M 17β-oestradiol but not with 10−8 and 10−9 M) and there is no evidence that the protocol ensured the independence of the individual mutant colonies picked for assessing the mutation spectra. The mutation induction data appears to be based on separate experiments, combined into a single results table, making it impossible to determine the data obtained in each separate experiment and thus to see how the reported increases relate to control (or spontaneous) values. Some of the DNA base changes in the “induced” mutants are incorrectly assigned. A key observation is the occurrence of two “hotspots” of mutation. These specific changes are rarely found and furthermore may have arisen due to failure to ensure the independence of mutants selected.8 Certainly, the postulated mechanism of action of oestradiol due to free-radical induced DNA damage would not be predicted to produce such a unique profile of DNA base changes. Our current understanding of spontaneous mutation indicates that a major fraction of mutations originates from oxidative damage. Thus, if the proposed genotoxicity of oestradiol is due to oxidative damage, then one might predict a mutant profile similar to those produced spontaneously. Therefore, this study cannot be considered sufficient evidence of mutation potential and the claim that 17β-oestradiol does not act via a receptor because mutation is not reduced in the presence of an anti-oestrogen is also not substantiated.
The study of Chakravarti et al. (2001)56 is cited as evidence that oestradiol-3,4-quinone induces mutations in mouse skin in vivo. Dorsal skin of SENCAR mice was treated with this metabolite and the mice were sacrificed after one hour to measure DNA adducts and at intervals thereafter for measuring mutations in the H-ras gene. The dose used was 200 nmol oestradiol-3,4-quinone; the treated surface area was not defined. The mutations induced were sequenced. N3-adenine adducts (rapidly depurinating) and N7 guanine adducts (slowly depurinating) were seen. It appears that those arising at N3, but not at N7 guanine, gave rise to mutations. Whilst this study provides evidence that a metabolite of 17β-oestradiol can give rise to a genotoxic effect in vivo, the mutation frequencies obtained (2.2 × 10−5 mutations per base pair) are extremely high and there is no concurrent measure of toxicity. The relevance of this dose level to concentrations to which humans are exposed from eating meat from treated animals would require further investigation.
In a study not considered in the SCVPH Opinion, Yared and colleagues reported on the genotoxic effects of oestrogens in breast cells using the micronucleus and Comet assays.57 17β-Oestradiol, oestrone and oestriol were tested for their ability to induce micronuclei in an assay using cytocholasin B and DNA damage detected by the Comet assay in a human mammary cell line (MCF-7) and primary human mammary epithelial cells, both of which have the oestrogen receptor. Oestradiol induced an increase of micronuclei at 10−9 M. Higher concentrations also showed an increase above controls, but in an inverse dose-response. Oestrone induced a dose-related response in micronuclei. No increase was observed for oestriol. A dose-related induction of proliferation was also observed for all compounds. Positive responses in the Comet assay were seen for β-oestradiol and oestrone and to a lesser extent for oestriol in both cell types.
1.3.5.2 Testosterone and Progesterone
Testosterone had previously been reported to be negative in the L5178Y gene mutation assay and in in vivo somatic and germ cell assays for chromosome aberrations.58 No data were available on progesterone. The SCVPH considered further the JECFA/WHO evaluation of these hormones (WHO Food Additives Series 43; WHO, 20002) and considered there is no evidence that progesterone or testosterone have genotoxic potential. No other publications have been published to add to this.
1.3.5.3 Zeranol and Trenbolone
The 1999 SCVPH Opinion concluded that trenbolone was not genotoxic on the weight of evidence from numerous studies. Isolated positive responses were reported for micronucleus induction and cell transformation in SHE cells but not in C3H10T1/2 cells. There were, however, no standard assay results available on zeranol. On the basis of further work commissioned by the EC,59 the SCVPH 2002 Opinion concluded that these substances exhibit only very weak effects.
The mutagenicity of these substances was also investigated.59 β-Trenbolone was negative in a cell mutation assay (V79/hprt) and at the lacI loci in E. coli. Marginal positive results were claimed for micronuclei induction in V79 cells and for DNA adducts in hepatocytes. Zeranol did not induce DNA adducts in rat hepatocytes, mutations at the lacI locus in E. coli or mutations in mammalian cells (V79/hprt). A borderline response was seen for induction of micronuclei in vitro. The positive micronucleus responses for both compounds were only obtained at near-cytotoxic concentrations. The authors conclude that further work is required to evaluate the genotoxicity of these substances and their metabolites, and that non-standard systems may be required to detect weak effects.
The WG considered there to be a number of methodological flaws with Metzler and Pfeiffer's gene mutation study – insufficient cells were treated and maintained through the expression period and assessed for mutations; a single dose only was assessed and the cytotoxicity values are not presented. Similarly the micronucleus results were obtained at a single concentration only and the measures of cytotoxicity were not presented, although it would appear that near cytotoxic concentrations were used. The method did not use the cytocholasin B method and its sensitivity was consequently affected by the inhibition of cell proliferation by trenbolone.
A further study considered by the SCVPH involved the interaction of hormonal substances and their metabolites with sex hormone-binding globulin (SHBG) or the analogous sex hormone-binding protein (SBP) from trout plasma. Zeranol and its metabolites were included in this study, and were found to have low binding affinity to these proteins; this would result in high bioavailability when present in plasma, but also fairly rapid metabolism. This unpublished study is discussed further.
1.3.5.4 Melengestrol Acetate
At the time of the SCVPH 1999 Opinion, the information available on melengestrol acetate (MGA) was sparse. In its 2002 Opinion, the SCVPH considered the recent JECFA evaluation (WHO Food Additives Series 45; WHO, 20002), but noted that most of the references were to unpublished reports. An EC-funded study addressed this issue59 and showed that MGA was negative in a cell mutation assay (V79/hprt), in a micronucleus test in V79 cells and in a gene mutation assay for LacI mutations in E. coli.59 SCVPH (2002)4 concluded from this study that MGA showed only weak effects. However, the WG considered the published study provided insufficient information for evaluation and thus no conclusion can be made on the mutagenicity of MGA.
1.3.5.5 WG Conclusions and Recommendations
1.3.5.5.1 17β-Oestradiol
Most of the “new” information referred to in the 2002 SCVPH Opinion has been generated using non-standard methods that produce information of questionable relevance to effects that may occur in the intact animal. A number of the studies discussed in the report are of poor quality. However, there is now additional evidence that metabolites of 17β-oestradiol can form DNA adducts in vitro49,60 and in vivo.49 While the catechol metabolites of 17β-oestradiol induce DNA adducts in SHE cells, 17β-oestradiol itself does not do so.60 There is some evidence for the induction by oestradiol of DNA damage (single strand breaks) and micronuclei formation in cells with the oestrogen receptor.57 It is not known whether the micronuclei are due to clastogenicity or to aneuploidy.
Evidence for the induction of mutations by 17β-oestradiol itself has only been obtained in non-standard assays, including those without normal cellular controls (e.g. 52 ). There is still no evidence that 17β-oestradiol itself is a gene mutagen. The key study of Kong et al. (2000),55 purporting to show that 17β-oestradiol is a gene mutagen, suffers methodological and interpretation flaws. The mutagenicity seen in the in vivo study involving skin painting56 may have been associated with extreme doses. There is, however, some evidence for a clastogenic potential for 17β-oestradiol (reviewed by 49 ), although some studies have failed to differentiate between aneuploidy and structural damage. There is further evidence that oestradiol is an aneugen and an inducer of other genotoxic effects (e.g. DNA amplification, microsatellite formation). The significance of these latter endpoints for hazard and risk evaluation is still not clear.
Overall, the weight of evidence from many genotoxicity studies, both standard and non-standard, indicates there may be a genotoxic potential for metabolites of 17β-oestradiol, but this direct evidence is by no means substantial. However, there is further indirect evidence for genotoxicity. A plausible hypothesis has been advanced49,51 that 17β-oestradiol is carcinogenic in humans and animal models by a combination of effects on cell proliferation and by genotoxicity. The hypothesis is primarily based on reasonable evidence that 17β-oestradiol is not carcinogenic solely due to epigenetic phenomena such as induction of cell proliferation.
Although there is evidence that oestrogen metabolites may be directly genotoxic in vitro, in vivo their formation is affected by opposing activation and inactivation metabolic pathways, the presence of anti-oxidants and DNA repair capacity and thus it is likely this genotoxicity will have a thresholded response. The importance of anti-oxidant defence systems is demonstrated by the reduction in transformation and formation of DNA adducts by oestrogen metabolites in the presence of ascorbic acid.60
There were no standard tests conducted in vivo, even on 17β-oestradiol metabolites, which indicated a mutagenic potential for 17β-oestradiol in vivo. Since DNA repair pathways, anti-oxidant defence and Phase II inactivation pathways can be overwhelmed at high doses, it is necessary to obtain evidence of genotoxicity in well-conducted assays, employing realistic doses.
It is important to determine whether the 17β-oestradiol metabolites can be produced in vivo in sufficient quantities to result in genotoxicity. Thus, it is recommended well-conducted in vivo studies are performed to determine whether 17β-oestradiol is able to induce genotoxic damage in vivo under realistic exposure conditions.
1.3.5.5.2 Testosterone and Progesterone
On the basis of the limited information available to the WG, there was not further evidence of genotoxicity of these substances and no recommendation for further work.
1.3.5.5.3 Zeranol and Trenbolone
The WG concluded that there were insufficient data to indicate zeranol or trenbolone are genotoxic. This conclusion is the same as that reached in the (previous) 1999 SCVPH Opinion. Further studies would be required to evaluate this fully.
1.3.5.5.4 Melengestrol Acetate
The WG considered there to be insufficient data available to evaluate the genotoxicity of MGA.
1.3.6 Developmental and Reproductive Effects of Hormonally Active Substances
The potential for chemicals with intrinsic endocrine activity to perturb development and function of the reproductive system, especially in the male, has been a driving force for concern about the issue of environmental endocrine-active chemicals. Results of studies in experimental laboratory animals have been equivocal, and there are as yet no data to show that such effects occur in humans due to any environmental chemical exposure.
1.3.6.1 Recent Data
The 2002 SCVPH opinion considered three EC-funded studies that addressed reproductive and developmental sequelae of exposure to hormonally active compounds. One was an animal study, involving gestational and lactational exposure of rabbits to zeranol, trenbolone acetate (TBA) and MGA.61 The other two were human studies: a retrospective case-control follow-up of young men and women suspected of having been exposed to meat from hormone-treated animals when they were children in 197762 and a study based on data from the Swedish Twin Registry, looking at breast cancer risks in twins.36
Studies to directly assess the effects of zeranol, TBA and MGA on the development of the testis and reproductive system in rabbits were investigated. These studies involved gestational and lactational exposure to these compounds at moderate or high doses, ranging from 0.25 mg kg−1month−1 by implant for zeranol to 0.5 mg kg−1day−1 for MGA. Exposure to TBA or MGA was also investigated during adulthood. The SCVPH quote the authors’ conclusions as indicating “that prenatal exposure to low doses of MGA, TBA or zeranol may affect the function of the reproductive tract in rabbits, although the effects are not as severe as those observed after exposure to the high doses. The effects are most pronounced if the exposure occurred early in life. All three compounds readily cross the placental barrier and accumulate to a variable degree in fetal tissues. The effects of zeranol and TBA are more severe than the effects of MGA in animals exposed during development; however, MGA has marked effects on spermatogenesis when administered in adults”. As only a superficial description of the results of this study was given in the SCVPH 2002 opinion, the WG found it difficult to draw any conclusions with confidence. The only mention of a significant change was an increase in concentrations of oestrone after exposure to either MGA or to zeranol during early adolescence, a change unlikely to be of biological significance.
The final report on the above study provided a more conclusive view on this investigation. Pilot studies used relatively high doses of the test compounds and this led to various problems that resulted in use of lower degrees of exposure for the main (reported) study. In the pilot study, treatment with zeranol (dose unspecified, but an implant that delivered >0.25 mg kg−1month−1) resulted in major reproductive abnormalities, including cryptorchidism and gross suppression of spermatogenesis. However, no details of this pilot study are given (other than a description of testicular histology) and the use of a lower dose for the main study suggests that it was considered that the pilot study was compromised in some way. Similarly, prenatal exposure to TBA in the pilot studies was confounded by major perinatal mortality of the offspring and only one dam gave birth to offspring that survived after exposure to the lowest dose of TBA (0.5 mg kg−1week−1).
In the main study, only minimal effects were observed in animals exposed to doses of the three test compounds during different life phases (for zeranol, a monthly subcutaneous implant of 0.25 mg kg−1 to the dam; for TBA a weekly subcutaneous injection of 0.5 mg kg−1; for MGA 0.5 mg kg−1 orally daily). No consistent significant treatment-related effects were observed, though four cases of unilateral cryptorchidism were observed, two after adolescent exposure to TBA and one after adolescent exposure to zeranol; one animal with unilateral cryptorchidism was observed after gestational exposure to TBA. No other gross changes of the reproductive system were observed. Abnormal spermatogenesis, as evaluated by a non-standard, but published, method, was evident in the cryptorchid testes as expected. But there was no evidence for abnormal changes in scrotal testes (though this is based on deductions from the limited tabulated data provided), with the possible exception of animals exposed in adulthood to MGA. The latter (small) effect most likely occurs as a consequence of the progestational activity of MGA. Even assuming that MGA does have effects on spermatogenesis when administered to adult rabbits, the dose used (0.5 mg kg−1day−1) is presumed to have no relevance to humans exposed via residues in meat, unless there is ingestion of part of an implant.
Based on cellular morphology, the report refers to the abnormal persistence of “single” “foetal-like” germ cells in the testes of treated animals, although it is not specified in which treatment groups these cells were noted. Such cells are of interest because testicular (germ cell) cancer in humans probably arises from transformed foetal germ cells that have persisted in the testis since foetal life. However, the study was unable to confirm the possible foetal nature of these cells using a battery of specific markers as none of the available antibodies worked on rabbit tissues. Evidence for effects of TBA and zeranol on gonocyte development in the foetal testis was provided by increased numbers of these cells being immunopositive for PG-2, but as the role of this antigen is unknown, it is not possible to evaluate the significance of this observation (which was based on only two animals per group).
Sporadic changes in reproductive hormone concentrations were reported at certain ages in certain treatment groups, but no consistent, easily interpretable pattern was observed; no evidence of major dysfunction of the testis or of the hypothalamic-pituitary-testicular axis emerged from this study. Similarly, no evidence for any change in semen quality was found in any of the treatment groups. From limited studies on maternal and foetal samples from control and treated animals, it was shown that residues of MGA and zeranol and metabolites of TBA were clearly identifiable in various tissues of relevant treated animals, but were not detectable in controls.
This study experienced confounding problems due to “side-effects” of the administered compounds during pregnancy. This is not unusual as pregnancy is susceptible to hormonal disruption, as it is a hormone-dependent process.63 This may indicate that the doses of the test compounds being used were too high, although this was not a specified conclusion of the report. The use of generally lower doses for the main study largely avoided these confounding problems and provided only sketchy evidence for any significant impact on reproductive development and function as a result of in utero or postnatal exposures to MGA, TBA or zeranol. Perhaps the only lingering concern was the sporadic occurrence of cryptorchidism, which was confined to treated animals, though this was restricted to animals exposed during adolescence; this may indicate either that the cryptorchidism was treatment-unrelated (i.e. the problem was present prior to initiation of treatment, as cryptorchidism is not uncommon in rabbits) or that the final stage of inguinal testicular descent had been compromised. The latter is well established to be an androgen-dependent process, but the very limited data available for testosterone concentrations show no indications of suppression.
Other than the occurrence of cryptorchidism, none of the other findings in treated animals were suggestive of consistent, abnormal changes in development or function of the male reproductive system. Moreover, they occurred in animals in which exposure to the growth-promoting hormones was far in excess of that likely to occur in humans as the result of ingestion of meat/fat from growth promoter-treated cattle. This provides reassurance that adverse effects on the developing human male reproductive system are unlikely to occur.
The SCVPH 2002 Opinion concluded that in utero or pre- and peri-pubertal exposure to hormones (including animal evidence on synthetic products) may affect pubertal development and that epidemiological studies with opposite-sexed twins indicated prenatal exposure to hormones may be linked to adult cancer risk. These conclusions derived from two EC-funded studies.
The study of Chiumello et al. (2001)62 was thought by the WG extremely difficult to interpret and has several shortcomings. It followed an outbreak of gynaecomastia in school children in Italy in 1977, when it was presumed that accidental exposure to an oestrogenic compound of some sort was involved. The source and nature of the compound were never identified. In this situation the presumed exposure mimics what would happen normally during natural puberty when endogenous oestrogen concentrations would rise and stimulate breast development (in the female). If such exposure were continued over a period of time, effects on final height and other parameters might occur that could have significant impact for the individual. Remarkably, in the follow-up study height was not measured (or is not listed on the questionnaire or in the final report). Instead the focus was solely on reproductive issues and only minor changes were found. The most significant finding was an increased incidence of small (atrophic) testes in men who had attended the affected school in 1977. However, even this finding is suspect. First, it is well established that individuals who believe they may have a reproductive problem are more likely to volunteer/participate in studies that involve clinical examinations and blood tests related to reproduction (they get a free check-up); evidence of this was apparent from the report. Second, it is completely unknown whether or not the boys with atrophic testes were “exposed” to the contaminated meat. Third, as this contamination was not proven, nor the nature of any hormonal contaminant identified, it is not possible to draw conclusions from this study.
Even if it accepted that the children in question had been exposed to an oestrogen such as DES, used for meat growth promotion, the WG felt that there was no evidence to suggest that such outbreaks are other than very isolated and rare phenomena. This suggests that exposure of children to oestrogenic compounds in meat is not sufficient to induce precocious breast development to the point where it is clinically significant. The most frequent occurrence of precocious puberty in girls arises in individuals who have been adopted at an early age from a developing country and then reared in a Western country.64,65 What underlies the extraordinarily high incidence (20–25%) of precocious puberty in such individuals is still unclear but may involve precocious activation of the hypothalamic-pituitary axis.
The study of Kaijser et al. (2001)36 was based on the Swedish Twin Registry and sought to evaluate whether subsequent risk of breast cancer was higher in twins. This study showed that with increasing female birthweight, the risk of developing breast cancer in pre- or post-menopausal life was increased step-wise. Though comparison of twins versus singletons can reveal a relationship between oestrogen concentrations in pregnancy and the risk of reproductive cancer in the offspring,33,34 there are several difficulties in making such associations. First it is unclear what the relationship is between oestrogen concentrations measured in blood of the pregnant mother and those in the foetus, in particular the concentrations in oestrogen target tissues. Second, in twin pregnancies there is normally lower birth weight and this and other factors that affect growth of the foetus in utero can be significant risk factors for development of reproductive cancers in both sexes. This is illustrated in Study 13, in which risk of breast cancer in a female twin was considerably increased when there was a male twin present, and this appeared to be related partly to an increase in birth weight of the female twin. The latter effect might be related to increased androgen exposure from the male foetus, as the female foetus makes negligible amounts of sex steroids.
1.3.6.2 WG Conclusions
While the WG felt it reasonable to conclude that the hormone environment in utero is a factor in determining subsequent risks of some reproductive cancers, this is a complex area to interpret. It is certainly not straightforward to conclude on the basis of these findings that pre-natal exposure of the foetus to exogenous hormones, in particular hormones used as growth promoters in livestock, will be capable of inducing comparable effects. Issues such as potency, bioavailability, pharmacokinetics and transfer to the foetus all have to be taken into account.
By reference to offspring from women who were treated with extremely high doses (>0.1 mg kg−1day−1) of the potent oestrogen diethylstilboestrol during pregnancy, only a very modest increase in testicular cancer risk occurred in the male offspring66 and only rare cases of vaginal cancer occurred in the female offspring.67 It would therefore seem unlikely that exposure to the less potent growth-promoting compounds, at what would be very much lower concentrations,12–14 would pose a significant risk with regard to the development of reproductive cancers. Moreover, experimental studies in rodents that involve administration of test compounds to pregnant animals and consequent exposure of the foetus may be poor models for the human, because of major differences in endogenous hormone concentrations, timing/duration of foetal development,63 etc. Again, the dramatic changes in diet, BMI, later age at first pregnancy, rates of smoking, etc. in women in Western countries over the past 50 years have established effects on foetal growth and development.11 Against this changing background, discerning potential contributory effects from low-level exposure to growth-promoting hormones or their metabolites is probably an impossible task.
1.3.7 Environmental Impact of Hormonally Active Substances
1.3.7.1 Recent Data
Although the SCVPH Opinion (2002) concentrated on risks to human health, Section 6 (p. 21) and Annex 1 (pp. 24–27) also considered environmental effects. Three studies are mentioned by the SCVPH in relation to environmental effects:
Endocrine disrupting activity of anabolic steroids used in cattle. The paper by Schiffer et al. (2001)68 contains results from this study that are of relevance to environmental risk assessment.
Screening water samples for estrogenic and androgenic anabolic chemicals. The results from this study had not been fully published, but a discussion is published in a brief paper by Jégou et al. (2001).69
Endocrine disrupting effects of cattle farm effluent on environmental sentinel species. The results from this study have been published in brief form in a review by Orlando and Guillette (2001).70
The WG evaluated the SCVPH's conclusions and compared these with the published evidence from the three cited studies. Additional information was not sought, and it is possible that further publications have emerged from the three SCVPH studies.
Section 6 of the SCVPH Opinion simply stated that previous SCVPH Opinions have not addressed environmental concerns, but that relevant results from the 17 Studies are presented in Annex 1. This section also draws attention to the existence of a report by the Scientific Committee on Toxicity, Ecotoxicity and the Environment.71 This report is an overview of the evidence for endocrine disruption, particularly in wildlife. It does not specifically address risks from hormone residues in beef, but does briefly consider human and wildlife studies that examine the effects of oestradiol and 17α-ethinyloestradiol. The main focus for wildlife studies is on chlorinated organic compounds (e.g. PCBs and DDT) and TBT, rather than hormones. Several recommendations are made to improve the predictive and monitoring tools for detecting endocrine disrupting chemicals that might occur in the environment. However, the direct relevance of this CSTEE report to the environmental risk assessment of hormones used in beef is rather limited.
The SCVPH Opinion Annex 1 reviews the three studies with relevance for the environment, and drew five principal conclusions:
Aquatic animals are most sensitive to endocrine disruptors due to a greater potential for tissue accumulation.
The environmental impact of anabolic steroids is potentially great.
Further studies to determine the biological and chemical stability of such steroids in soil and water are warranted.
Little information is available on the endocrine disrupting potential of the metabolites of MGA.
Surface water downstream from a cattle feedlot was contaminated with oestrogenic and androgenic compounds, but the identity of these could not be established. Fish morphology near to cattle feedlots showed signs of endocrine disruption but, once again, the specific cause of this could not be identified.
The WG considered these five conclusions in turn, as follows:
Aquatic animals are most sensitive to endocrine disruptors due to a greater potential for tissue accumulation. Two issues were thought to be confused here: the inherent sensitivity of an organism to a toxicant and the extent to which organisms in different environmental compartments (e.g. freshwater, seawater, land or air) may be exposed to these contaminants. This conclusion requires further work in two areas before it can be accepted. There needs to be further ecotoxicological testing of the relative sensitivity of different terrestrial and aquatic organisms to growth-promoting hormones used in beef production. There also needs to be further environmental chemistry to determine the pathways taken by these hormones once they are released into the environment, to examine whether it is likely that they will reach aquatic systems.
The environmental impact of anabolic steroids is potentially great. This conclusion was based upon the findings from Schiffer et al. (2001)68 on the degradation kinetics of excreted trenbolone acetate (TBA) and melengestrol acetate (MGA) under different manure storage conditions. The study showed that both hormones are excreted in faeces and can be detected in soil for up to several months when contaminated dung that has been stored for 4.5 to 5.5 months is applied. There was evidence that both trenbolone and MGA adsorb strongly to soil. The authors speculated that various physical or biological processes could eventually remove these hormones from soil, but no work was done to determine which, if any, of these removal processes is most likely. This was a well-performed study, but it did not demonstrate, or seek to demonstrate, that either of these hormones had an adverse impact on the environment. It simply demonstrates that there is a pathway for these hormones from beef cattle, through dung and into soil. A pathway from soil into either terrestrial or aquatic organisms and subsequent biological effects in these organisms would need to be demonstrated before one could state that there is an environmental impact.
Further studies to determine the biological and chemical stability of such steroids in soil and water are warranted. This conclusion is also based on Schiffer et al. (2001)68 and agrees with Schiffer et al.'s conclusions. This is an appropriate conclusion to reach; clearly the conclusion stated in (ii) above cannot be supported until these stability studies are done.
Little information is available on the endocrine disrupting potential of the metabolites of MGA. This is also based on Schiffer et al. (2001).68
Surface water downstream from a cattle feedlot was contaminated with oestrogenic and androgenic compounds, but the identity of these could not be established. Fish morphology near to cattle feedlots showed signs of endocrine disruption but, once again, the specific cause of this could not be identified. These views were thought to be based on a study which has apparently not been published in the peer-reviewed literature, except for a brief summary in Jégou et al. (2001).69 The conclusions are also based on a study, which has only been published briefly as part of a review.70 These published papers do not provide sufficient information to judge the quality of the work, although the researchers involved are acknowledged leaders in the field.
1.3.7.2 WG Conclusions and Recommendations
The three environmental studies cited by the SCVPH Opinion were important initial efforts to understand the environmental risks posed by use of growth-promoting hormones in beef production. However, it is clear, as acknowledged by SCVPH, that these studies do not provide strong evidence that growth-promoting hormones used in beef production are the cause of oestrogenic and androgenic activity in water below feedlots, or of de-masculinisation of fish. Application of Hill's criteria or Koch's postulates to these results suggests that much more work needs to be done before uncertainties are reduced to a degree at which an evidence-based decision can be made. In particular, the identity of the substances responsible for such effects needs to be established, probably through Toxicity Identification Evaluation procedures, and a more extensive set of sites should be investigated so that problems of pseudo-replication are avoided.
One study had been reported fully in the peer-reviewed literature and showed that at least two of the hormones, or their metabolites, may be found in soil after dung spreading. However, pathways from soil (where these compounds may be strongly bound and therefore unavailable) to sensitive biological receptors, have not been established. In contrast to this study, the rather limited published results from the other two investigations showed that endocrine disrupting substances seem to be present, and may be exerting biological effects, at river sites near to cattle feedlots. However, the substances responsible for these effects have not been identified, so a pathway from effects on environmental receptors to application of hormones in beef remains un-established.
The WG believed results from these studies are insufficient to demonstrate cause and effect. Research to establish a source–pathway–receptor linkage is required. Importantly, the WG assumed that if the re-introduction of growth-promoting substances for use within the EU were considered in the future, then a full environmental risk assessment would need to be conducted according to good current scientific practice.
1.3.8 Other Considerations of the WG
1.3.8.1 Formal Risk Assessment of Hormonally Active Substances
The WG discussed whether it would be possible to undertake a formal risk assessment on hormonally active compounds from the available data on residue concentrations and consumption data. The individual substances of concern all have ADIs and thus, in theory, it would have been possible to compare any estimated dose with the current ADIs. To this end, the UK Food Standards Agency is able to provide reliable data on food consumption for toddlers and adults that include dairy, meat and aquaculture products. The information on residue concentrations for the growth-promoting substances (whether natural or synthetic) in bovine meat and meat products, together with the data on UK food consumption, would in the future enable a total body dose to be calculated should this be required. This would require a better understanding of residue concentrations from both proper and improper use of the hormonal substances and, in the light of the newer scientific information, whether or not new ADIs might be required.
It is arguably also important to distinguish the natural hormones, which, if present in food, simply supplement those already circulating in the body, from the synthetic analogues which may have subtle differences in receptor binding, metabolism, etc. For natural hormones it could be argued that the traditional approach of deriving ADIs by applying safety factors to no observed adverse effect levels (NOAELs) is excessively conservative. The risks following ingestion of natural hormones in food which simply supplement those already circulating in the body can only truly be assessed from human data and should be put into context with the intake from other sources, the dose relative to normal circulating concentrations and the human evidence of the adverse effects of elevated circulating hormone concentrations. If the evidence suggests that any chronic increase in oestrogen concentrations will tend to increase the risk of breast cancer, as seems to be the case, we need to derive a view of an acceptable degree of increase in that risk, and prioritise the sources of exposure for control against that value. Essentially this might mean advice to avoid any contribution of oestrogens from the diet for anyone already taking supplementary oestrogens.
The WG therefore believed that there is a need to gain a much better understanding of the impact of very small increases in oestrogen concentrations in human populations. If it is demonstrated that the contribution from meat from oestrogen-treated animals is not increased above that from untreated animals then the actual treatment is an irrelevance in the debate.
Synthetic hormones (and lipoidal esters) cannot be treated in the same way, since we do not have enough information about their interaction with natural hormones in humans, and probably the only way to arrive at an estimate of a safe human dose is to obtain more data in experimental animals. It would also be important to attempt to model the interaction of synthetic hormones with natural oestrogens in order to explore the possibility of their increasing or decreasing the incidence of tumours in humans.
1.3.8.2 Ban on Over-thirty-month Cattle
The WG considered the effect of the end of the ban in the UK in 2005 on cattle over 30 months of age entering the food chain. It noted that this would lead to a massive increase in the endogenous concentrations of oestradiol in meat reaching the consumer; although it was also noted that this increase would be to pre-ban concentrations of nine years earlier. This in itself would likely dwarf any increase in hormone concentrations reaching the consumer as a result of the use of growth-promoters, were they to be re-introduced. However, set against this consideration was the argument that, if 17β-oestradiol increases the risk of breast cancer as previously discussed, then any avoidable increase, however small, would have to be viewed as undesirable.
The view was also expressed by the WG that other factors beyond our control are likely to (and probably already do) have a bigger effect on hormone concentrations in food than the use of growth promoters, e.g. a change in the type of sire used in the national dairy herd. The point was also raised that, if we were to continue to exclude growth promoters on the grounds that it would raise the exposure of hormones to the consumer, then logically one should consider excluding meat from pregnant animals and possibly those in oestrus.
JECFA15 concluded in their evaluation of the numerous studies using authorised doses of the three natural steroids either alone or in combination that the hormone concentrations in edible tissues and blood were sometimes statistically significantly higher than the corresponding values found in concurrent controls but were always within the physiological range of these substances in bovine animals. The highest concentrations of progesterone and 17β-oestradiol are found in lactating and pregnant cows whereas extremely high concentrations of testosterone are found in bulls; the concentrations in treated calves and steers are significantly less than these natural concentrations.
The excess contribution of the residues to the ADIs set by JECFA is <4% for oestrogens, approximately 0.003% for progesterone and 0.2% for testosterone. Bearing in mind that post-pubertal humans produce very much larger quantities of these hormones, the margin of safety for adults consuming meat from treated animals is very high. The pre-pubertal child produces fewer natural steroids but will always consume fewer than the respective ADIs.
1.3.8.3 Conclusions and Recommendations of the WG
As can be seen from the above information and discussions, the WG gave very careful consideration to both the views of the SCVPH Opinion as well as the published papers and reports coming out of the 17 EC-funded studies. In addition, the WG included in their deliberations other papers and discussions which helped to put the use of these hormonally active substances into a broader context so as to assist risk assessors, policy-makers and regulators. The conclusions of the WG are reproduced below in their entirety.
1.3.8.4 Current Scientific Evidence For or Against Adverse Effects
The previous sections have discussed the current new evidence relating to mutagenicity, carcinogenicity and endocrine disrupting effects of the hormonally active substances and for humans who may be consuming meat from treated animals. Most of the evidence relates to 17β-oestradiol and the following key features are considered relevant to this and any future hazard or risk assessment:
17β-estradiol can be activated to catechol oestrogens and then oxidised to form semiquinones. The metabolites may also generate potentially mutagenic oxygen radicals by redox cycling.
There is good evidence for the formation of DNA adducts from metabolites of 17β-oestradiol in vitro and in vivo.
Synthesised oestrogen metabolite adducts are pre-mutagenic in sub-cellular test systems.
There is some evidence for mutagenic potential (induction of chromosome damage and mutations) for some metabolites of oestradiol in mammalian cells in vitro.
The evidence for induction of chromosome aberrations and gene mutations in vivo is poor and is derived from non-standard studies.
There are, however, reasonable arguments against the carcinogenicity of 17β-oestradiol being due solely to epigenetic processes.
It would be prudent to consider oestradiol and its metabolites as a complete carcinogen whilst more substantial evidence for its mode of action is obtained.
Despite its possible genotoxicity, it is reasonable to consider that 17β-oestradiol may have a threshold for carcinogenicity due to the presence of homeostatic feedback mechanisms, the requirement for activation pathways to exceed inactivation pathways and the presence of antioxidants in vivo.
When it came to the current evidence base for 17β-oestradiol, however, in spite of certain data gaps, the view of most of the Working Group was that there is ample information to show that zootechnical and therapeutic uses of 17β-oestradiol do not pose any risk to humans unless an active implant site is ingested.
In relation to the other hormones considered, a number of points emerged in the Working Group discussions. One view was that in regard to the five other hormones (testosterone, progesterone, trenbolone, zeranol and MGA), one could agree with the SCVPH assessment, as expressed in Directive 2003/74/EC of the European Parliament and the Council of 22 September 2003, “that the current state of knowledge does not make it possible to give a quantitative estimate of the risk to consumers”. However, the majority of the Working Group felt that, in spite of the acknowledged data gaps and uncertainties, the available evidence on genotoxicity, tumorigenicity, hormonal activity and endocrine disrupting effects was supportive of the view that eating meat from animals treated with these five hormones was unlikely to be harmful to human health.
As a rider to these statements, it should be noted that they are based on the assumption that the consumer is exposed to no greater concentrations of residues than those arising from “correct” or “recommended” use of hormones. The likely misuse of growth-promoting substances is noted elsewhere in this report.
A number of additional points were made by members of the Working Group:
In spite of the 17 additional studies funded by the Commission, little progress has been made to determine the safety of hormone growth promoters.
One member felt strongly in support of the findings of the SCVPH – that much more work needs to be done before the safety of the six substances under consideration can be assured and approval given for their growth-promotional purposes.
At the time, the 1999 VPC Sub-Group reported, they were unable to support the conclusions reached by the SCVPH “that risks associated with the consumption of meat may be greater than previously thought”. However, whilst the last 1999 VPC Sub-Group reported that “none of the publications reviewed provide any substantive evidence that oestradiol was mutagenic/genotoxic”, the more recent evidence does indicate that:
metabolites of oestradiol do have the potential to be genotoxic, in vitro and in vivo; and
steroid metabolites previously considered to be nothing more that inactivation products may have patho-physiological actions themselves.
1.3.8.5 Overall Conclusions and Recommendations
The Working Group were of the view that human exposure to residues of hormonally active substances, including growth promoters in meat, could exert biological effects if exposure is at a sufficiently high level. Therefore, the two key issues are:
determination of the dose-response induction of biological effects by the hormonally active substances in test animals and, ideally, humans in order to identify a Lowest Observable Effect Level (LOEL), and
determination of the level (and range) of the additional human exposure and uptake from eating meat from treated animals.
These determinations should be made in adults and in developing (foetal/neonatal) animals and humans to identify the most sensitive index of effect. These effects would be in addition to those occurring naturally due to endogenous hormones.
The research so far has provided some, but not all, the basic, but essential, information outlined above. Without it, no definitive conclusions can be drawn; although the weight of available evidence suggests that likely levels of human exposure to hormonally active substances in meat from treated animals would not be sufficient to induce any measurable biological effect.
Specifically, it is very unlikely that the presence of 17β-oestradiol and its metabolites in meat from treated animals would significantly increase the risk of adverse effects in consumers. This is due to their low concentrations in comparison to those arising from endogenous production and from other dietary sources. Any increase would be likely to be small in the context of the whole food basket.
In reaching these conclusions, the Working Group expressed a number of qualifications and reservations based on the current lack of evidence of a risk to humans. These included:
all scientific judgements made by the Working Group were based on the assumption that the consumer is exposed to residues at no greater concentrations than those that would be caused by the “correct” or “recommended” use of the exogenous hormones, be it for growth promotion or other zootechnical uses or therapeutic purposes;
the Working Group understands that misuse of hormonally active substances for growth promotion is more likely than misuse for oestrus synchronisation or therapeutic uses; and
substances with hormonal action may be used in combination, both legally and illegally, while the toxicological and safety factors available (e.g. MRLs and ADIs) only relate to single substances.
the Working Group had to decide what to do in the absence of information or where there was uncertainty of interpretation of information. One member expressed the view that, for the substances under consideration, there was a large element of uncertainty, so the precautionary principle must assume the primary consideration. The many uncertainties associated with the current lack of knowledge could be addressed by further research where this was both feasible and affordable. The Working Group was unanimous that all uncertainties must be made clear, especially those that were considered crucial in the risk assessment process.
As has been noted in this report, and acknowledged in the SCVPH Opinion, there are important gaps in the evidence base that preclude producing definitive risk assessments for 17β-oestradiol or the other five hormonally active substances. Not all data gaps are equally important for the purposes of risk assessment and the Working Group highlighted a number that could improve future risk assessments. As an example, it would be helpful if the CVMP and JECFA could make available data on pharmacokinetics and metabolism of assessed compounds that were supplied in manufacturers’ dossiers. This openness and transparency would allow greater public scrutiny of the facts and confidence in the hazard and risk assessments produced.
The Working Group felt that none of the basic issues could be addressed without a structured approach. There is a need to establish precisely the:
relationships between the potential use of growth promoters (including over-use) and concentrations of residues in meat;
levels of exposure in consumers (i.e. taking account of intake, absorption, bioavailability and metabolism); and
dose-response relationships for the effects of the hormonally active substances (and their metabolites) in experimental animals or in humans.
Further data on lipoidal oestrogens, possible bioaccumulation and possible synergistic effects of cocktails of hormonal substances would also be desirable.
The Working Group noted specific needs:
To establish in humans a detailed dose-response curve that relates exposure to specific hormonally active substances to the amount of meat consumed from treated animals.
To establish in experimental animals the relationship between intake of hormonally active substances, or their metabolites, and target-organ effects (selecting the likely most sensitive target organ depending on the nature of the activity of the compound). This study to be conducted for adults and then foetal and/or neonatal exposure to be considered.
To consider lipoidal esters of oestrogen in future studies of the possible passage of oestrogen in implants through cattle to humans. The bioavailability and metabolism of lipoidal esters following ingestion should be investigated to allow the biological significance of the oestrogens to be assessed.
To carry out studies to confirm whether the ADI for pre-pubertal boys could be exceeded if they consumed a standard 500 g portion of meat from an animal that had been treated with a number of hormonal implants. If confirmed this would be of concern.
To establish an independent laboratory test to confirm that meat has not been derived from animals produced with the aid of growth-promoting hormone implants.
1.4 Recent Opinions and the Future
Since the publication of the WG 2005 report, there have been a number of publications relevant to all of the scientific areas discussed above. Of particular importance is a new Opinion of the Scientific Panel of Contaminants in the Food Chain,72 which reviewed all relevant papers published between 2002 and 2007 in relation to testosterone, progesterone, trenbolone acetate, zeranol and melengestrol acetate used as growth promoters in meat production. The request for the Opinion came from the European Commission to the European Food Standards Authority (EFSA) to which the SPCFC belong. The terms of reference were somewhat similar to that of the VPC WG 2005 but a little more limited in that they were only required to summarise to new data. The conclusions that they reached are reproduced below.
1.5 Conclusions
New data published since 2002 confirm and extend the current understanding of the effects of steroid hormones and hormone-like substances used as growth-promoting hormones (GPHs), which are not only via interactions with their specific receptors.
In in vitro systems, the potencies of zeranol, trenbolone and melengestrol acetate in terms of oestrogen, androgen and progesterone receptor affinities and modulation of gene expression, as well as cell proliferation and apoptosis, may be equal to, or exceed, those of the most active natural hormones. There is a lack of information with respect to the in vivo significance of these effects at exposure levels associated with residues in meat.
Sensitive analytical methods have become available permitting the identification and quantification of the growth-promoting hormones (all five compounds under consideration) and their currently known major metabolites. Regarding the natural hormones, testosterone and progesterone, these methods also allow discrimination between endogenous and exogenous hormone residues. These advanced methods have as yet only been used in a very limited number of experimental studies, and await to be applied on a broader scale.
In the absence of data from surveillance studies, the exposure to residues of the hormones used as growth-promoting agents cannot be quantified. In particular, the available data on the metabolism of trenbolone, zeranol or melengestrol acetate in cattle, and the amount and nature of residues in animal tissues following routine use of these compounds in beef cattle operations, are too incomplete to be assessable.
An increasing number of publications presenting epidemiological data indicate a correlation between red-meat consumption and hormone-dependent cancers of the breast and prostate. Due to the high number of confounding factors, the contribution of residues of hormones in meat cannot be quantified in these studies.
Large-scale cattle production and the use of growth-promoting hormones in cattle operations in Third Countries has been associated with undesirable effects in sentinel aquatic species in contact with cattle farm effluents.
The CONTAM Panel concluded that the new data that are publicly available do not provide quantitative information that would be informative for risk characterisation, and therefore do not call for a revision of the previous assessments of the Scientific Committee on Veterinary Measures relating to Public Health (SCVPH).
As can be seen, there is nothing fundamentally new in these conclusions that contradicts those of the VPC WG 2005 in relation to the specific substances. Of note however, the final paragraph seems to support the risk assessment of the previous SCVPH 1999, 2002 Opinions that were used to underpin and maintain the EU ban.
Of scientific importance is that the better analytical methods have been developed which are able to distinguish between endogenous and exogenous sources for the compounds of concern in tissue residues. These are said to be of only limited utilisation and would need further development for routine residue surveillance schemes.
Critically, all experts groups seem to agree that the available scientific and epidemiological data is insufficient to come to firm conclusions regarding risk assessments for the compounds of concern, for humans consuming meat, for any of the health endpoints.
Clearly, this story is not over. The research will continue and new Opinions will be provided by expert committees at the requests of regulatory bodies. This is not surprising as the health consequences are serious (cancer and reproductive perturbations) and the major proportion of the human population will be exposed to meat and meat products where use of these substances as growth promoters is permitted.
The WTO Panel report numbers – WT/DS26/R/USA and WT/DS48R/CAN.
http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003L0074:EN:HTML, accessed 29 March 2009.
Phytoestrogens are a group of chemicals produced naturally by certain edible plants. The commonest are the isoflavones (e.g. genistein, found in soybeans and legumes), coumestans (found in young sprouting legumes), lignans (found in linseed, many cereals, fruits and vegetables) and prenylated flavonoids (found in hops and beer). Human dietary exposure can therefore be substantial, although very variable depending on the composition of the diet. A recent in-depth review of the evidence for both adverse and beneficial effects of dietary phytoestrogens concluded that many of the available data are equivocal and fail to distinguish between effects of the compounds themselves and effects of other dietary constituents.8 (COT 2003). However, it was also emphasised that the nature and extent of any potential effects of phytoestrogens in the diet will depend critically on both the degree of exposure and the age at exposure.
In humans, sex steroids do not circulate in the bloodstream in a readily bioavailable form. The majority (>95%) are bound to SHBG or other plasma proteins, such as albumin. An equilibrium exists between the amount of protein-bound and free sex steroid in plasma.9 (Hammond 2002). Although endogenously produced sex steroids bind to SHBG, many synthetic steroidal compounds do not, e.g. the potent oestrogens diethylstilboestrol (DES) and ethinyl oestradiol. The possibility that ingested compounds may bind to SHBG and displace already bound endogenous sex steroid, thus making the latter bioavailable, must also be considered. Theoretically, a compound with minimal or no intrinsic oestrogenicity itself could induce oestrogenic effects if it was able to bind with higher affinity than oestradiol to SHBG and thus displace it.
Confirmed in a letter from Dr Belingieri, 17 July 2003.
In postmenopausal women, no ovarian oestrogen synthesis occurs and the residual oestrogen production occurs predominantly in sub-cutaneous fat and is not subject to significant feedback control; in these circumstances, ingestion of exogenous hormone leads to additive increments of exposure.
It has been estimated by the evaluation of multiple samples that the true relative-risk from plasma oestradiol is double that estimated by single-sample studies.27 (Hankinson et al, 1995). Thus, the relative risk of breast cancer from a doubling of oestradiol concentrations is likely to be approximately 1.50.
Mammalian Gene Mutation Database, available at: http://lisntweb.swan.ac.uk/cmgt/index.htm, accessed 29 March 2009.