Selected Excerpts

Animal Cloning: A Risk Assessment

Center for Veterinary Medicine
U.S. Food and Drug Administration
Monday, January 14, 2008; 9:46 PM

Conclusions Regarding Food Consumption Risks from Bovine Clones and their Progeny

As the first prong of our strategy to address the food consumption risks associated with clones, we have used the Critical Biological Systems Approach (CBSA) as a framework to search for subtle differences between clones and comparators that may pose food consumption risks. In general, these differences cannot be detected macroscopically but may be evident as differences in physiological parameters during the five developmental nodes. For bovine clones, our health-based assessment of food consumption risks is facilitated by a significant body of evidence from the peer-reviewed literature together with a large data set from Cyagra. Many bovine clones do not survive the neonatal period, and several abnormalities (e.g., those related to LOS [large offspring syndrome], prolonged recumbency, umbilical malformations) have been described during this developmental node. None of these abnormalities is unique to clones, and all have been observed in calves produced by other ARTS [assisted reproductive technologies] such as in vitro fertilization or following natural mating.

In clones that survive the neonatal period, some studies have identified differences in physiological measures between clones and comparators during the first few weeks of life. These findings support the notion that bovine clones are more physiologically unstable during the early juvenile period. There is evidence that the physiological transition from neonatal period to the juvenile period may take longer in calf clones (e.g., elevated body temperature during the first two months of life). Once their physiological status is stabilized, however, there is ample evidence to indicate that growth and development proceed normally in bovine clones. Similarly, several studies indicate that fertility in clones is normal, and there are no indications that the physiology or health of clones is compromised during the post-pubertal period.

In summary, we have searched for subtle differences between clones and their comparators to identify differences that may pose food consumption hazards. We have not found any such subtle differences, and based on this review of the health and physiology of bovine clones using the CBSA approach, we conclude that there is no reason to expect that food from bovine clones would pose additional food safety risks compared with the same products derived from conventionally-bred cattle.

Clone progeny are not expected to pose any increased food consumption risks compared with other sexually reproduced animals (NAS 2002b). Although the amount of data describing the health of progeny of clones is more limited than the amount describing the health of clones themselves, the results are consistent with the biological assumption. In the two studies that characterized the physiology of heifers produced by clones, growth, reproductive function, and telomere length were normal in clone progeny, and the incidence of general health problems was not increased in clone progeny compared with progeny of other sexually reproduced animals. Based on the CBSA approach, we therefore conclude that sexually reproduced progeny of clones are indistinguishable from other sexually reproduced animals, and pose no additional food consumption risks.

Conclusions Regarding Food Consumption Risks from Swine Clones and their Progeny

These studies and data evaluated indicate that there are no apparent anomalies present that would have a direct impact on the safety of food products derived from swine clones or their progeny. The measurements taken at 27 weeks of age are appropriate for the evaluation of food consumption risks because this is the approximate age at which pigs are sent to slaughter in the US. The identified abnormalities in the Archer et al. (2003a) (parakeratosis) and the ViaGen dataset (lung adhesion) are not unique to swine clones and do not pose a food consumption risk, as the affected tissues from the carcass would be condemned at the slaughterhouse and would not enter the food supply. The apparently normal status of the clinical measurements indicates that the clones in this study possess the same physiological functions and behaviors as their conventional counterparts, and thus do not contain subtle hazards that would pose food consumption risks compared with food from conventionally-bred swine.

Health information on the progeny of swine indicates that although occasional phenotypic abnormalities were observed in these progeny during the neonatal and perinatal periods (contracture of the flexor tendon, anal atresia, spraddle legs), none of these anomalies are unique to cloning and all occurred at frequencies similar to those observed in conventionally produced swine. Progeny of swine clones are healthy, grow at the normal rate, and do not appear any more susceptible to infection or disease that conventional pigs. Importantly, the studies reviewed indicate that physiological and phenotypic differences that might be observed in swine clones are not passed down to their progeny. These results provide further support for the hypothesis that epigenetic errors in clones are reset during gametogenesis, resulting in progeny that are healthy and physiologically normal. Therefore, based on the CBSA portion of this assessment, we conclude that progeny of swine clones, produced by normal sexual reproduction, do not contain any subtle hazards that would pose any increased food consumption risk compared with the offspring of any other sexually reproduced swine.

Conclusions Regarding Food Consumption Risks from Sheep Clones

Very few conclusions can be drawn about the health of sheep clones, due to the small database available for evaluation. Only one study provided detailed physiological data from sheep clones, and these data were limited to only a few metabolic and endocrine endpoints. Despite Dolly's high public visibility, there are very few other reports of non-transgenic sheep clones. Until additional specific information regarding the health of sheep clones becomes available, the only inferences that can be made would be drawn from interspecies extrapolation from other ruminant clones, i.e., cattle and goats.

Conclusions Regarding Food Consumption Risks from Goat Clones

Based on the data reviewed, there do not appear to be any anomalies present in the goat clones that would have an effect on the safety of food products derived these animals, and no subtle hazards were identified in these clones that could pose food consumption risks. Goats appear to be relatively "cloning friendly" with a high degree of successful live births following confirmation of pregnancy. All reports of health of the goat clones seem to indicate that they are normal and healthy. The available data on the physiological parameters of goat clones indicate that these animals respond as their conventional counterparts to internal signals for growth. The apparently normal status of the clinical measurements indicates that the clones in this study possess the same physiological functions and behaviors as their conventional counterparts. Further, unlike the other livestock clones, data on the reproductive behavior of male goat clones indicate that reproductive function is normal. Finally, although cursory in mention, it appears that male progeny of clone bucks also reach puberty at the appropriate time. Thus, although the number of animals that has been evaluated is not as large as in the case of bovine clones, goat clones appear to be healthy, and do not appear to be materially different from conventional goats.

Summary Statement on Composition of Milk from Clones

Several peer-reviewed studies describe the composition of milk from bovine clones. In addition to gross composition (percent solids, fat, protein, and lactose), some reports include a detailed analysis of fatty acids, vitamins, minerals, and amino acids, and in some cases, comparisons are made with previously published reference values for milk composition. These studies indicate that milk from cow clones is not significantly different in composition from milk from non-clones. Some minor differences have been identified in the composition of milk from clones compared to non-clones or reference values, but in each of these reports, the authors attribute the minor differences to diet, environmental conditions, small numbers of animals, and limited numbers of genotypes, rather than to cloning per se. None of these differences, however, indicate the presence of hazards that could pose food consumption risks, as they all fall within published historical values for milk. We therefore that milk derived from bovine clones does is not materially different from milk from milk from conventionally bred cattle.

Conclusions from Studies Evaluating the Composition of Meat and Milk from Clones and Their Progeny

The second prong of our Risk Assessment is based on the hypothesis that food products from healthy animal clones and their progeny that are not materially different from corresponding products from conventional animals are as safe to eat as their conventional counterparts. CVM has reviewed several peer-reviewed publications that have evaluated gross (e.g., milk yield, carcass characteristics) and fine (e.g., individual amino acid and fatty acid components) characteristics of meat and/milk from clones, and in two studies, their sexually-reproduced progeny. All but one of these studies indicate that none of the characteristics that we examined differed in any biologically significant way between the clone and comparator. The only exception is a preliminary study in bovine clones which provides evidence that lipid metabolism may be altered in clones, resulting in slight alterations in the fatty acid composition of milk and meat. However, without a comparison of these data to historical reference values, it is unclear whether these differences are representative of all bovine clones or are specific to the limited number of genotypes used in the study.

For swine clone progeny, a comprehensive, peer-reviewed analysis of meat from a large number of animals provides strong evidence that there are no compositional differences between meat from swine clones and meat from conventional swine, and that meat from clone progeny and their comparators is not materially different.

Therefore, in this prong of the Risk Assessment, CVM concludes that the weight of evidence indicates that meat and milk from clones and their progeny do not differ materially from meat and milk derived from their conventional counterparts, and therefore, based on compositional analysis, do not pose any additional food consumption risks compared with meat and milk from conventionally bred animals.

Conclusions from Allergenicity and Feeding Studies in Rodents

The second prong of this risk assessment is based on the hypothesis that edible products from healthy cloned animals and their progeny are as safe to eat as edible products from conventionally produced livestock. CVM reviewed three studies in which the rat was used as a surrogate animal model to investigate possible biological effects of eating meat or milk from cattle clones. One of these feeding studies was conducted over an extended period (14 weeks) and included standard toxicological endpoints as well as a functional observational (behavior) battery. None of these studies demonstrated any change in the physiology or pathology of the rat following consumption of meat or milk from clones. Moreover, no evidence has been found to indicate that the allergenic potential of meat or milk from cloned cattle is greater than that of meat or milk from non-cloned cattle. No behavioral changes were observed. These findings are consistent with our conclusions using the Compositional Analysis approach, i.e., that meat and milk from clones and their progeny are not materially different from meat and milk derived from conventional counterparts and thus do not pose any additional food consumption risks relative to food from conventional animals.

The risk of allergenicity is one that is often cited for foods that do not have a long history of consumption. Although there is no reason to suspect that cloning will cause the synthesis of new proteins in animals that appear healthy and normal, there are two possible pathways that might pose an increased allergenic risk from the edible products of animal clones. One is an increase in the relative amount of an individual protein component of milk or meat that may only be present in very low or trace amounts. Cows' milk has been associated with true allergies (Cows Milk Allergy or CMA) in approximately six percent of the US population (Bernstein 2003). Caseins, although the predominant proteins in milk, do not appear to be the key allergens associated with CMA. The other possible pathway is that processing of the proteins during their generation in the mammary gland or muscle cells somehow alters their antigenic presentation. The Center cautions that these are purely hypothetical pathways, and that there has been no demonstration that either of these actually occurs.

In theory, evaluating the relative concentrations of milk proteins in clone and comparator milk could provide information to determine if the first risk exists. The study by Tian et al. (2005) provides just such a comparison using SDS/page technology. In practice, however, even this study highlights the difficulty in establishing the appropriate comparator and minimizing variability. Milk from non-clone dairy animals may vary in relative composition due to the influences of breed, diet, number of lactations, where in the lactation cycle the milk is collected, etc. Further, the level of exposure (dose) required to elicit an allergenic response is not well understood, and has been the subject of much discussion in the scientific literature (Taylor 2002) and among international regulatory bodies (Codex Alimentarius 2003112). Nonetheless, the limited studies provided (Japan 2002) show that milk from both SCNT and BNT clone cattle showed similar digestibility characteristics both in vitro and in a rodent in vivo assay. In addition, a rodent bioassay for allergic response did not show any significant differences in response between clone and non-clone derived milk. Combined with the underlying biological assumptions, these data support the lack of a unique allergic response to milk derived from clone cattle.

Microbiological Effects

It has been suggested that epigenetic changes in animal clones could somehow alter the rumen and intestinal microflora of the ruminants (cattle, sheep, and goats), or the intestinal microflora of monogastric species (swine) (NAS 2002b). Such alterations in intestinal flora might be considered hazards because they could, in theory, result in increased levels of an existing zo?notic pathogen or the growth of a novel zo?notic pathogen. Shedding of these pathogens in fecal material could possibly result in a higher load of undesirable microbes on the carcass at slaughter, increasing the likelihood of contamination of the edible tissues.

The use of animal drugs has been postulated to alter the intestinal flora of treated food animals, resulting in an increased load of zo?notic pathogens in the food supply. The potential for animal drugs to induce this change was considered at length by the January 2002 CVM Veterinary Medical Advisory Committee on that topic113. Most of this independent scientific advisory committee found that animal drug use was unlikely to significantly impact pathogen load (or the prevalence of zo?notic pathogens), and that pathogen load has little or no impact on public health.

The Center is not aware of any studies that have characterized the intestinal flora of livestock clones, and the complexity of the intestinal microflora makes this an extremely difficult question to address directly. Although it is possible that epigenetic reprogramming in clones may have effects on the intestinal flora, this postulate can be challenged on the basis of animal health. The data reviewed in this risk assessment indicate that the vast majority of clones studied during the juvenile, reproductive and post-pubertal phases of life are as healthy as their sexually-produced counterparts. It therefore seems very unlikely that the milieu of intestinal microflora is abnormal in these animals, and that contamination of carcasses of clones due to bacterial shedding would pose a greater food consumption risk than that posed by conventional food animals.114 We further note that such alterations would not be unique to clones as all animals, regardless of their method of production, are subject to alterations in epigenetic programming.

Technology Changes

Significant changes in cloning technology, especially those accompanied by donor nucleus or o?cyte treatment regimens introducing new hazards into the overall process, would significantly increase the uncertainty associated with our judgments regarding the degree of risk that could accompany the resulting clones and clone food products. Without a careful evaluation of the animals arising from such methods, it would not be appropriate to speculate on the relative safety of the process from either an animal health or food safety perspective.

How Much (Information) Is Enough?

The question of determining when sufficient data have been collected in order to allow high confidence in risk-based decisions regarding edible products from animal clones is difficult to determine in the abstract. In practice, the answer is "it depends on what questions you ask, and how the data answer those questions."

Because the nature of the technology has generally precluded generating large datasets on clones with good statistical power, CVM constructed a systematic approach to frame the appropriate questions (hazard identification), evaluated the available data (hazard characterization), and attempted to characterize resulting risk (probability of harm given that exposure occurs). This weight of evidence approach allows for the evaluation of the data from the CBSA and Compositional Analysis prongs of the Risk Assessment as part of an overarching whole. The conclusions from this risk assessment represent the judgment of CVM veterinarians, animal scientists, toxicologists, and risk assessors. The underlying assumptions for clones and their progeny were that the animals needed to meet all relevant federal, state, and local laws and regulations for conventional animals, and the food products derived from clones or their progeny also had to meet relevant federal, state, and local laws and regulations.

When considered across the Developmental Node spectrum, the data on the health of livestock clones were remarkably consistent across species, despite initial anomalies that appear to be species-specific. For example, although LOS may be more prevalent in cattle and sheep, most surviving animals normalize initial anomalies and become "healthy and normal." This consistency has increased the value of even small datasets (e.g., goats), and has contributed significantly to the judgments regarding the health of these clones and their suitability as food sources. In addition, CVM evaluated a number of reports on the composition of meat and milk from clones and their progeny. No biologically important or safety-relevant differences were noted when compositions were compared to standard databases or contemporary comparator controls. If anything, these data confirm the rather wide variability in the composition of meat and milk eaten on a daily basis. In summary, no toxicological hazard of concern for the human consumer has been identified in any of the reported studies. Although additional data from other sets of animals, particularly in other species routinely used for food, could be useful in increasing the confidence that may be placed in overall judgments regarding food safety, the weight of the evidence at this time is sufficient for the agency to draw the conclusions it has made in this Risk Assessment with reasonable certainty.

Risk Management Plan for Clones and their Progeny

Surveillance for Changes in Cloning Technology and State of Knowledge that Could Affect Food Safety

1. Monitor and review additional animal health and food composition data on animal clones or their progeny as they become available.

- FDA will establish a close liaison with professional and scientific organizations such as the International Embryo Transfer Society (IETS), the Federation of Animal Science Societies, and the American Veterinary Medical Association to collect and access new animal health and production data as they become available, and will work with these organizations to collect and maintain an international, centrally-located database of animal clone and progeny health and production data, which would be made publicly available. In particular, the FDA is currently engaged in an ongoing project with the IETS to produce a publicly available international data base on the health of clones and the composition of food from them. This data base should become available in 2008.

2. Monitor and review changes in animal cloning techniques and technologies.

- FDA will routinely monitor the scientific literature and attend pertinent scientific conferences to stay abreast of animal cloning technologies. FDA will continue to maintain open and informal channels of communication with animal clone producers and researchers to remain up-to-date with these technologies.

3. Continue to consult with clone producers to review changes in the technology.

- FDA will continue to consult with clone producers to review changes in the technology. Clone producers with questions regarding whether their technology is different from that evaluated in the Risk Assessment are strongly encouraged to discuss their technology with FDA.

4. Monitor and maintain knowledge base on the biology of epigenetic mechanisms governing gene expression and their role in nuclear transfer.

- FDA will maintain an ongoing awareness of the scientific literature regarding the biology of animal clones and epigenetics, maintaining our scientific currency in accordance with our regulatory mission.

Risk to the health of animals involved in cloning

Increased risks of adverse health outcomes of the types previously observed in animals produced via other ARTs have been observed in surrogate dams and very young clones. Animal cloning, particularly in cattle and sheep, is associated with an increased risk of adverse health outcomes in the surrogate dams carrying late-term clone fetuses, as well as very young clones. Specific health issues of concern for the surrogate dams include the increased incidence of prenatal hydroallantois and/or hydrops in the surrogate dams carrying clone pregnancies to term. Health issues of concern for the clones themselves include perinatal symptoms related to LOS including, but not limited to, pulmonary and/or renal insufficiency, difficulty maintaining body temperature, and umbilical hernias.

In order to minimize the impact(s) of these animal health risks, we have been working with the International Embryo Transfer Society to prepare a publicly available manual on animal care standards for animals involved in the cloning process. This document is due to be released to the public on IETS' website in early 2008.

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