<<back
Chapter I: Executive Summary
Cloning is the colloquial term used to describe the process of somatic cell nuclear transfer (SCNT) that falls on a continuum of assisted reproductive technologies (ARTs) currently used in agriculture. In this Draft Risk Assessment, the Center for Veterinary Medicine (CVM or the Center) at the US Food and Drug Administration (FDA) presents a science-based review of the available information on cloning in species traditionally used for food (i.e., cattle, swine, sheep, and goats).
A. Overview
This Draft Risk Assessment addresses SCNT technology, its impact on the health of animals involved in that process, and food consumption hazards that may arise in animal clones and their progeny1 in the context of the use of ARTs in conventional animal agriculture. Chapter II is a summary of ARTs currently used in food animal breeding and a detailed explanation of SCNT. Chapter III describes the process of risk assessment, its application to animal cloning, and the nature of the hazards that may arise as the result of cloning. A synopsis of the processes involved in epigenetic reprogramming and their relevance to adverse outcomes noted in animals derived via SCNT and other ARTs is found in Chapter IV. Chapter V addresses potential health risks to animals involved in the process of cloning, including surrogate dams, clones, and their progeny. Chapter VI addresses potential food consumption risks that may result from edible products derived from animal clones or their progeny. Each chapter contains conclusions relevant to that subject; the Risk Assessment is summarized in Chapter VII, and our overall conclusions are presented there. In order to make this process as transparent as possible, all of our methodologies are presented in the text of the risk assessment; the information and data that CVM evaluated are publicly available, either in peer-reviewed publications, or in Appendices to this document. The process by which CVM drew its conclusions is presented in the Risk Assessment, along with explicit statements of potential bias and uncertainty. The document concludes with a complete bibliography, a glossary of terms, and appendices containing data and background information.
The Draft Risk Assessment is the result of a qualitative analysis that identifies and characterizes the nature of hazards that may be introduced into animals as a result of cloning, and puts them in the context of other assisted reproductive technologies currently practiced in the United States. The strongest conclusions that can be drawn regarding positive outcomes in risk assessments of this type are “no additional risk” because outcomes are weighed against known comparators. If a finding of “no additional risk” were to be applied to the health of animal clones, it would mean that the cloning process would not pose any greater risk to the health of the animals involved than other ARTs. Applied to the safety of edible products derived from clones, a finding of “no additional risk” would mean that food products derived from animal clones or their progeny would not pose any additional risk relative to corresponding products from conventional animals, or that they are as safe as foods that we eat every day. As with all risk assessments, some uncertainty is inherent either in the approach we have used or in the data themselves. Where uncertainties exist, CVM has attempted to identify the degree of uncertainty and the reasons for its existence.
1 For the purposes of this analysis, an animal clone is one arising directly from a somatic cell nuclear transfer event. A progeny animal is one derived from sexual reproduction that has at least one animal clone as a parent (but could also result from two animal clones mating). Clones of clones would be considered as clones (i.e., directly arising from an SCNT process).
B. Technology Overview (Chapter II)
Assisted reproductive technologies (ARTs) have been employed extensively in animal agriculture for over a century, and at least one (artificial insemination) has been practiced for several hundred years. These technologies form a continuum that ranges from the fairly minimal assistance provided to animals engaged in natural service through the more recent development of SCNT. ARTs have aided in the genetic improvement of domestic livestock species by the selection and propagation of desirable phenotypes, and accelerating the rate at which those characteristics have been incorporated into national herds. Artificial insemination, for example, permitted the propagation of valuable genomes without the sire being physically present, thereby allowing superior genetics to be spread beyond relatively small geographical areas.
Most commonly used ARTs rely on fertilization as a first step. This joining of egg and sperm is accompanied by the recombination of the genetic material from the sire and dam, and is often referred to as “shuffling the genetic deck.” From a breeder’s perspective, phenotypes resulting from sexual reproduction cannot be predicted—that is, the characteristics of the offspring from a mating may be estimated, but not predicted with certainty. Nuclear transfer, the most advanced of these technologies, does not require fertilization and allows for the propagation of known genotypes and phenotypes without the risk of genetic reshuffling. Thus, SCNT’s greatest immediate impact on animal breeding may be that it allows the propagation of genomes whose phenotypes are proven. It also allows the propagation of animals whose reproductive function may be impaired, or of very valuable animals that have died. SCNT, like the other newer forms of ARTs (e.g., in vitro fertilization, embryo splitting) results in some known adverse outcomes to the animals and possibly the dams bearing those pregnancies.
C. Risk Assessment Methodology (Chapter III)
Risk assessment is a science-based process used to identify hazards that may be present in predefined exposure scenarios, and to estimate the severity and chances of the outcome(s) occurring once that exposure occurs. Because many, if not all, of the individual steps that comprise a risk assessment contain various degrees of uncertainty, risk assessors should explicitly describe the sources of uncertainty and the effect(s) that the uncertainties may have on any judgment of risk. Risk assessment serves as the scientific underpinning from which risk managers may choose different options based on their understanding of, and responsibilities to, the broader contexts within which they operate.
Qualitatively, risk may be thought of as some function of the combination of exposure and the intrinsic properties of the substance or process under consideration by linking an exposure to the likelihood of an outcome. When performing a risk analysis, it is critically important to distinguish between a hazard and the potential risk(s) that may result from exposure. A hazard can be defined as an act or phenomenon that has the potential to produce an adverse outcome, injury, or some sort of loss or detriment. These are sometimes referred to as harms, and are often identified under laboratory conditions designed to maximize the opportunity to detect adverse outcomes. Thus, such observational summaries are often referred to as “hazard identification” or “hazard characterization.” Risk, then, is the conditional probability that estimates the probability of harm given that exposure has occurred. In a qualitative assessment such as this, however, risks can be discussed only within a qualitative context, and no quantitative interpretations should be made.
In order to address the hazards and risks to animals involved in cloning and the food products derived from them four issues must be addressed: identifying hazards and risks; determining the degree to which existing data address the question of risk; characterizing residual uncertainties; and selecting the most appropriate definition of risk for the risk assessment.
This Risk Assessment explicitly excludes transgenic clones from the identification of hazards or risks experienced by “just clones” because of the inability to determine whether the transgenic event or cloning was causally associated with an adverse outcome. In addition, the Risk Assessment has assumed that, at minimum, animal clones, their progeny, and food products derived from them would be subject to the same laws and regulations as conventional animals and their food products. Because no exogenous genes have been introduced into animals derived via SCNT, the underlying assumption regarding potential hazards that could arise is that anomalies observed in animal clones are due to incomplete or inappropriate reprogramming of the donor cell nucleus. Therefore, any remaining hazards leading to food consumption risks that would result from inappropriate or incomplete reprogramming would be subtle. These subtle hazards would allow an animal clone to develop with apparently normal functions, but with sub-clinical physiological anomalies. These could include alterations in the expression of key proteins affecting the nutritional content of food and possibly lead to dietary imbalances. Similar hazards arise in animals generated via other ARTs. The goal of this draft risk assessment is to determine whether any unique hazards arise that are not noted in comparators, or have not been identified in cattle, swine, sheep, or goats produced via other ARTs.
Both the animal health and food consumption risk assessments evaluated information within a framework developed by CVM called the Critical Biological Systems Approach (CBSA), which divides the life cycle of an animal clone into five functional developmental nodes. Developmental Node 1 incorporates the initial technical steps involved in SCNT, from cell fusion through fetal development. Developmental Node 2 encompasses the perinatal period, including late gestation, labor induction in the dam, delivery, and the critical few days after birth. The third developmental node, Juvenile Development and Function, covers the period of rapid growth between birth and the onset of puberty. The Reproductive Development and Function Node (Developmental Node 4) includes puberty and reproductive function throughout the reproductive life of clones. The Post-Pubertal Maturation Node (Developmental Node 5) consists of all non-reproductive functions of sexually maturing or mature clones, including growth, weight gain, disease frequency, aging, and, where available, lifespan.
The nature of each component of the risk assessment (i.e., animal health or food consumption) shaped the manner in which the available data were evaluated. For example, identification of adverse outcomes for animal health included both the animal clone and the surrogate dam carrying the pregnancy. Emphasis was placed on the clones’ development and probability of normal development, compared with other ARTs such as artificial insemination (AI), in vitro fertilization (IVF), and blastomere nuclear transfer (BNT). For food consumption risks, however, animal clones bearing gross anomalies were excluded from the analysis, and emphasis was placed on identifying unique subtle hazards that could have arisen as the result of the SCNT process. The rationale for this approach is found in Chapter IV, which provides the molecular evidence for the role of epigenetic reprogramming as the source of these subtle hazards. Because of the assumption that hazards would be subtle, datasets were evaluated on as fine a level of resolution as possible, including individual animals or even individual analytes per animal in order to have as sensitive a screen as possible for adverse outcomes (and thus potential food consumption risks). In this risk assessment, the most detailed level of resolution used for evaluating animal health has been physiological and biochemical measures of individual animals. It is likely, as technologies mature, that molecular techniques such as genomics, proteomics, and their integrated metabolomic measures will assist in such determinations, but to date, these methods have not been standardized or validated (NAS 2004).
D. The Implications of Epigenetic Reprogramming for Clones and their Progeny (Chapter IV)
Epigenetics has been defined as the study of stable alterations in gene expression potentials that arise during development and cell proliferation. In sexual reproduction, a new diploid genome is created by the fusion of two haploid genomes. The subsequent expression of that genome into a functional organism is governed by a “program.” There are several examples of epigenetic control of gene expression, of which DNA methylation is likely the best characterized.
Mammalian embryos experience major epigenetic reprogramming primarily at two times in their development, both of which have significant implications for cloning. One of these takes place soon after fertilization, and is referred to as preimplantation reprogramming; the other occurs during gametogenesis (the development of cells that ultimately become the sperm and egg). Because preimplantation reprogramming occurs after fertilization, and in the case of nuclear transfer, after fusion of the donor nucleus with the oöplast, it is the most immediately affected by the cloning process, and may be most directly implicated in the development of clones with defects. Gametogenic reprogramming may also be involved in the abnormalities noted in clones, but it likely has more far-reaching implications for progeny, because it generates the gametes used for the sexual reproduction of clones.
When cloning, the donor nucleus must be coaxed to direct embryonic development as if it were a fertilization-derived zygote. Most of the time this is not successful. Anomalous epigenetic reprogramming is observed at the global genomic and individual gene level in clone embryos and fetuses, and in similar developmental stages of animals produced using ARTs with significant in vitro culturing components. Many of these are lethal, as demonstrated by the low success rate of IVF and the even lower success rate of SCNT. In the small number of successful cases that ultimately result in normal-appearing and functioning animals, SCNT-derived embryos appear to be able to carry out reprogramming just about as well as fertilization-derived embryos. Live and apparently healthy clones may exhibit some level of epigenetic differences relative to fertilization-derived animals.
The Center assumes that if clones were to pose food consumption risks, the only mechanism by which those risks could arise would be from inappropriate epigenetic reprogramming, similar to those observed for other ARTs. It is important to note that the genes that are being dysregulated are the “normal,” naturally present genes that comprise the animal’s genome, and have not been introduced via recombinant DNA techniques from other sources (i.e., these are not transgenic or genetically engineered animals). Progeny of animal clones, on the other hand, are not anticipated to pose food safety concerns, as natural mating resulting from the production of new gametes by the clones is expected to reset even those residual epigenetic reprogramming errors that could persist in healthy, reproducing clones.
E. Risks to Animals Involved in Cloning (Chapter V)
This chapter compares SCNT with other ARTs with respect to effects on animal health and concludes that some animals involved in the cloning process (i.e., cattle and sheep surrogate dams, and some clones) are at increased risk of adverse health outcomes relative to conventional animals. None of these adverse outcomes, however, are unique to cloning.
Cows and ewes used as surrogate dams for SCNT-derived pregnancies appear to be at increased risk of late gestational complications such as hydrops, as well as dystocia at parturition, that occur at a lower frequency with other ARTs that have a significant in vitro culturing component. Surrogate swine and goat dams bearing clones do not appear to be at increased risk.
There is an increased risk of mortality and morbidity in perinatal calf and lamb clones compared with calves and lambs produced using other ARTs. In cattle and sheep, the increased risk appears to be related to large offspring syndrome. Survival of these clones appears to be a function of both the severity of the clinical signs and neonatal management. Morbidity and mortality do not appear to be increased in perinatal swine and goat clones.
After the perinatal developmental node, no new health risks have been identified in clones of any of the species considered in this risk assessment. Clones in the juvenile to prepubertal age cohort do not appear to be at an increased risk of morbidity or mortality compared to animals produced by natural service or ARTs. Most animals surviving the neonatal period appear to grow and develop normally. No increased risk of adverse health effects have been reported in clones approaching reproductive maturity. Finally, the available information indicates that there are no increased risks to the health of maturing clones relative to conventional animals. Currently, it is not possible to draw any conclusions regarding the longevity of livestock clones due to the relatively short time that the technology has existed.
Based on the biological assumptions and molecular data reviewed in Chapter IV, progeny of clones are expected to be normal. Consistent with these predictions, the data on the health status of clone progeny indicate that there is no increased risk of health problems in these animals compared with conventional animals.
F. Food Consumption Risks (Chapter VI)
1. Two-Pronged Approach to Identifying and Characterizing Food Consumption Risks
In order to determine whether epigenetically-caused subtle hazards pose food consumption risks, CVM has developed a two-pronged approach. The first component, the Critical Biological Systems Approach (CBSA), incorporates a systematic review of the health of the animal clone or its progeny. Its role in the evaluation of food consumption risk analysis is premised on the hypothesis that a healthy animal is likely to produce safe food products. It accepts that at this time, SCNT is a biologically imprecise and inefficient process, but recognizes that animals are capable of biological repair or adaptation. The cumulative nature of the CBSA allows for the incorporation of both favorable and unfavorable outcomes. The former, provided that all other measures appear to be normal, will result in the finding that the clone is likely to produce edible products that pose no food consumption risks; the latter implies that clones with anomalies are likely to be considered unsuitable for food. The second component, the Compositional Analysis Method, assumes that food products from healthy animal clones and their progeny that are not materially different from corresponding products from conventional animals pose no additional risks. It relies on the comparison of individual components of edible products, and the identification of the appropriate comparators.
Assessing the safety of food products from animal clones and their progeny is best accomplished by using both approaches: prospectively drawing on our knowledge of biological systems in development and maturation, and in retrograde, from an analysis of food products. Subtle hazards and potential risks that may be posed by animal clones must, however, be considered in the context of other mutations and epigenetic changes that occur in all food animal populations. No adverse outcomes have been noted in clones that have not also been observed in animals derived via other ARTs or natural mating that enter the food supply unimpeded.
Because the value of clones lies in their genetics, CVM anticipates that animal clones might enter the food supply as meat if removed from the herd due to injury or senescence, but these would likely be animals near the end of their reproductive lives. Milk from clones, however, might enter the food supply. Progeny of clones are more liked to be reared as animals intended primarily for food use.
2. Conclusions Regarding Potential Food Consumption Risks
Based on this review of the body of data on the health of animal clones, the composition of meat and milk from those animals and corresponding information on clone progeny, CVM has drawn the following conclusions:
a. Cattle Clones
Edible products from perinatal bovine clones may pose some very limited human food consumption risk.
The underlying biological assumption in place for this age cohort is that perinatal clones may be fragile at birth due to residual incomplete or inappropriate reprogramming of the donor nucleus. The data are consistent with that assumption; some perinatal clones do not survive for several reasons, including poor placentation, LOS, and in some cases, frank malformations. Although surviving clones can be fragile for a period of time, survivors tend to adjust to life outside the womb within a relatively short period, either on their own or with assistance from caregivers. A significant proportion of perinatal clones survives gestation and is born without significant health problems. Laboratory measures of key physiological functions do not indicate that surviving animals are very different from conventional newborns. It is therefore unlikely that food consumption risks have been introduced into these animals or that rendering these clones will pose risks in animal feed or to humans consuming animals fed material derived from the clones.
Edible products from juvenile bovine clones pose no additional food consumption risk(s) relative to corresponding products from contemporary conventional comparators.
The underlying biological assumption for this developmental node is that if any anomalies were to be found in the youngest clones and those animals were to survive to be healthy adults, the juvenile developmental node would be a period of equilibration and normalization. The data are consistent with such a hypothesis.
Juvenile bovine clones are largely healthy and normal. Although some clones in this developmental node are more physiologically unstable than their conventional counterparts, they are in the process of normalizing their physiological functions on the way to adulthood. This normalization has been observed consistently and is further demonstrated by the analysis of clinical chemistry and hematology data demonstrating that clones show the appropriate physiological responses to developmental signals. None of the physiological measures taken, including both clinical chemistry and hematology, indicate any food consumption hazards.
Edible products derived from adult bovine clones pose no additional risk(s) relative to corresponding products from contemporary conventional comparators.
This conclusion is based on application of both prongs (CBSA and Compositional Analysis) of the risk assessment approach. The body of data comprising the CBSA approach is consistent with the biological prediction that there are no underlying biological reasons to suspect that healthy animal clones pose more of a food safety concern than conventional animals of similar age and species.
The data show that healthy adult clones are virtually indistinguishable from their comparators even at the level of clinical chemistry and hematology. These data also confirm the observation that physiological instabilities noted earlier in the lives of the clones are resolved in the juvenile developmental node (see previous conclusions regarding other developmental nodes), and do not reappear as the clones age. There are some reports of early deaths of clones; as these animals would not enter the food supply, they do not pose a food consumption risk. Data on reproductive function in cows or bulls of this age cohort indicates that healthy bovine clones surviving to reproductive maturity function normally and produce healthy offspring. These data are consistent across studies. Given that reproduction is the most difficult “biological hurdle” placed on an organism, the observation of normal reproductive function provides an additional degree of confidence to the conclusion of the appropriate development of these animals.
All of the reports on the composition analysis of meat or milk from bovine clones show that there are no biologically significant differences in the composition of milk derived from clone and non-clone cattle. Additionally, data from one report show no difference in allergenic potential for meat or milk derived from clone cattle compared to meat or milk from non-clone comparators. Similarly, neither meat nor milk from clone or non-clone cattle induced mutations in a mutagenicity assay. Finally, none of the reports identified an endpoint that would pose a hazard for human consumption.
b. Swine Clones
Edible products from adult swine clones pose no additional risk(s) relative to corresponding products from contemporary conventional comparators.
This conclusion is based on the same underlying biological assumption as cited for adult bovine clones. Because the data are more heavily weighted towards adult, market sized animals, judgments regarding the safety of food products from swine clones are provided in one aggregate set of comments.
Once piglet clones are born, they appear to be healthy. The most compelling argument for the normal health status of swine clones results from the evaluation of the behavior and physiological status of a small cohort of relatively young (15 weeks), and approximately market age (27 weeks) swine clones relative to closely related conventional pigs. No significant differences were observed in either behavior, epigenetic, or physiological measurements, indicating that these animals were not materially different from the comparators. Another small dataset on swine clones reared in very unusual settings (i.e., deprivation of colostrums, initial husbandry in pathogen-free conditions, switching to commercial settings) is confounded with respect to outcome. Nonetheless, these clones were able to respond appropriately to this stress¸ and their carcass characteristics, reproductive performance, including semen quality, farrowing rates and litter sizes were within national averages. No biologically relevant differences were observed in the composition of meat from these clones or their comparators.
c. Sheep Clones
Except by relying on underlying biological assumptions, and by inference from other species, there is insufficient information on the health status of sheep clones to draw conclusions with respect to potential risks that could be posed from the consumption of food products.
With the exception of reports on Dolly, CVM was unable to find any publicly available reports on the health status of live sheep clones. There are several studies addressing methodological issues for optimizing the generation of clones, but these do not address post-natal health. There are reports of anomalies noted in fetal sheep clones that have died or been terminated, and reports on the pathology associated with animals that do not survive. Although these are instructive for understanding the molecular and developmental pathways that may be perturbed during the process of SCNT, these studies have limited relevance to addressing food safety because the deceased animals would not have been allowed to enter the food supply. CVM was not able to find any reports on the composition of milk or meat from sheep clones.
d. Goat Clones
Edible products from goat clones pose no additional food consumption risk(s) relative to corresponding products from contemporary conventional comparators.
This conclusion is based on the same underlying biological assumption cited for the other livestock species, and a relatively small but compelling dataset. Once clone embryos are transferred to surrogate dams and pregnancies are confirmed, the “success rate” for live births is quite high. The animals appear to have developed well through reproductive age, and the available data indicate their physiological responses are appropriate for age and breed. The reproductive development and function of male Nigerian Dwarf goat clones demonstrate that those animals functioned appropriately relative to age- and breed-matched comparators. One male progeny goat was derived from the buck clones; this animal also appeared to function in an age- and breed-appropriate manner. No meat or milk composition data were identified for goat clones.
e. Clone Progeny
Edible products derived from the progeny of clones pose no additional food consumption risk(s) relative to corresponding products from other animals.
Progeny of clones will likely provide the overwhelming majority of clone-derived food products (both meat and dairy) in the US. The underlying biological assumption for health of progeny animals is that passage through the process of creating the cells that ultimately become ova and sperm naturally resets epigenetic signals for gene expression, and effectively “clears” the genome of incomplete or inappropriate signals. The rationale for this assumption has been developed in Chapter IV, and dominates the conclusion that edible products from any clone progeny pose no additional food consumption risk(s) relative to those from any other sexually reproduced animals. It has been supported by detailed empirical2 evidence both in the mouse model system, which clearly indicates that phenotypic alterations noted in the parent clones are not passed to their sexually-derived progeny. Observations on the health and meat composition of progeny of livestock clones, with one extensive dataset on the progeny of swine clones in particular, provide direct data on the health of these animals and on the composition of their meat. The swine data support the underlying biological assumption that the progeny of clone animals are essentially indistinguishable from the comparable progeny of non-clone animals.
We therefore concur with the high degree of confidence that the outside scientific community (NAS 2002 a,b) places in the underlying biological assumption, and conclude that consumption of edible products from clone progeny would not pose any additional food consumption risk(s) relative to consumption of similar products from sexually-derived animals.
G. Concluding Statements (Chapter VII)
For Animal Health: SCNT results in an increased frequency of health risks to animals involved in the cloning process, but these do not differ qualitatively from those observed in other ARTs or natural breeding. The frequency of live normal births appears to be low, although the situation appears to be improving as the technology matures. Cattle and sheep exhibit a set of clinical signs collectively referred to as LOS that do not appear to be present in swine or goats. Surrogate dams are at risk of complications from birth if the fetus suffers from LOS, or from accumulation of fluid in the cavities of the placenta (hydrops). Clones exhibiting LOS may require additional supportive care at birth, but can recover and mature into normal, healthy animals. Most clones that survive the perinatal period are normal and healthy as determined by physiological measurements, behavior, and veterinary examinations. Progeny of animal clones also have been reported as normal and healthy.
For Food Consumption Risks: Extensive evaluation of the available data has not identified any food consumption risks or subtle hazards in healthy clones of cattle, swine, or goats. Thus, edible products from healthy clones that meet existing requirements for meat and milk in commerce pose no increased food consumption risk(s) relative to comparable products from sexually-derived animals. The uncertainties associated with this judgment are a function of the empirical observations and underlying biological processes contributing to the production of clones. There is less uncertainty about the health of clones as they age and have more time to exhibit the full range of functionality expected of breeding stock.
Edible products derived from the progeny of clones pose no additional food consumption risk(s) relative to corresponding products from other animals based on underlying biological assumptions, evidence from model systems, and consistent empirical observations.
The results of this comprehensive risk assessment agree with the preliminary findings of the NAS (2002a) conclusions that “The products of offspring of clone[s] … were regarded as posing no food safety concern because they are the result of natural matings,” and “In summary there is no current evidence that food products derived from adult somatic cell clones or their progeny present a food safety concern.”
2 Empirical refers to that which can be seen or observed alone, often without reliance on theory. In the context of this risk assessment, conclusions drawn on empirical evidence are those that are drawn strictly based on the data. These conclusions may later be put in the context of underlying biological assumptions.
|
||||||
 |
||||||
|
||||||