Animal Cloning: A Risk Assessment

DRAFT Executive Summary

A. Overview

Cloning is the colloquial term used to describe the process of somatic
cell nuclear transfer (SCNT), and falls on a continuum of assisted
reproductive technologies (ARTs) currently used in agriculture. These
are summarized in the Technology Overview (Chapter II) of the Cloning
Risk Assessment. In the subsequent chapters and appendices of the 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 risks that may arise in species traditionally used for
food in which cloning has been accomplished (*e.g.,* cattle, swine,
sheep, and goats). Chapter III provides a general overview of the
process of risk assessment and how it has been applied to animal
cloning. Chapter IV addresses potential health risks to animals involved
in the process of cloning, while Chapter V addresses potential food
consumption risks that may result from edible products derived from
animal clones or their progeny. In order to make this process as
transparent as possible, all of the information that has been used is
publicly available. This limited our analysis to publications from
peer-reviewed journals and to one large data set on bovine clones made
available by a clone producer. The document concludes with appendices
containing background information, a complete bibliography, and a
glossary of terms.

B. Methodology

1\. Data Analysis

Both the animal health and food consumption risk assessments evaluated
information within a framework developed by CVM called the Critical
Biological Systems Approach (the Systems Approach), 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 includes puberty and
reproductive function throughout the reproductive life of clones. The
Post-Pubertal Maturation Node 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 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,
frankly deformed and diseased clones were excluded from the analysis
(because such animals would be condemned at slaughter, as currently
practiced with conventional animals), and emphasis was placed on
identifying subtle hazards that could have arisen as the result of the
SCNT process. Because of the assumption that hazards would be subtle,
data sets 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). Thus, although the data
sets considered by both risk assessments may have overlapped
considerably, the methods by which they were evaluated differed. Most
importantly, however, the conclusions of the two risk assessments may
differ with respect to the amount of risk present.

One of the conditions of this risk assessment is that all of the
information that CVM has used is also available to the public. Although
more information was received from clone producers than is available in
the Risk Assessment document, some has not been used in this iteration
of the risk assessment due to time constraints. By far the greatest
amount of information identified addressed bovine clones, followed by
swine clones. Although the data base on goat clones was quite small, it
is fairly comprehensive with respect to developmental nodes. Very little
information was available for sheep clones.

The risk assessment excludes clones derived from donor cells that are
transgenic (*i.e.,* cells that contain heritable DNA inserted by
molecular biology techniques) from the identification of adverse
outcomes. Transgenic clones are considered to occupy a different "risk
space" from "just clones" because the transgenic event is accompanied by
a series of construct-specific risks.

**2. Limitations on Conclusions from Qualitative Risk Assessments**

This is a qualitative, comparative risk assessment that does not attempt
to assign a quantitative value to estimates of risk or safety. The
strongest conclusions that can be drawn regarding positive outcomes in
risk assessments of this type are "likely to be as safe as" because
outcomes are weighed against comparators of known or inferred safety.
Safety is the condition of absence of adverse effects; as a result,
certainty of safety is approached, but never reached. Applied to the
health of animal clones, the finding of "as safe as" means that the
cloning process is likely to be as safe as other ARTs. Applied to the
safety of edible products derived from clones, the finding means that
food products derived from animal clones are likely to be as safe as
corresponding products from non-clones, or as safe as foods that we eat
every day. On the other hand, risks can be characterized in a
qualitative, comparative risk assessment because adverse outcomes can be
observed.

C. Risks to Animals Involved in Cloning

Although increased in frequency relative to other ARTs, none of the
adverse outcomes noted in animals involved in the cloning process are
qualitatively different from those observed in other ARTs. Risk appears
to be elevated in some species (*e.g.,* cattle and sheep) relative to
others (swine and goats). Adverse outcomes are noted with the highest
frequency in the earliest stages of a clone's development. Most embryo
clones fail to develop, and pregnancies terminate spontaneously due to
fetal abnormalities or difficulties with placentation. Many of these
anomalies tend to be related to fetal overgrowth, often referred to as
Large Offspring Syndrome (LOS), although other frank developmental
abnormalities have been noted.

**1. Pregnancy through the Perinatal Developmental Period**

Large Offspring Syndrome, or LOS, has been noted in many of the early
reports of cloning in cattle and sheep as well as other ARTs. LOS is
generally thought to consist of a nexus of clinical signs that may be
directly related to fetal oversize (at least 20% heavier than the
average birth weight for the breed), the size of the dam's uterus, and
dystocia (difficulty giving birth) during labor. LOS thus delineates
most of the risks to both animal clones and their surrogate dams. Other
signs, such as under-developed respiratory, cardiovascular and renal
(kidney) systems may not be due to intrauterine effects, but are often
considered part of the syndrome due to the frequency with which they
occur in combination with the overgrowth phenomena. Many animals born
with LOS have excess fluid in their placentae or organs (hydrops), which
may contribute to their large size. The causes of LOS are not known, but
may be related to a combination of incomplete reprogramming of the
somatic cell nucleus and the *in vitro* culture conditions that IVF and
SCNT embryos experience. Large Offspring Syndrome has not been reported
in swine or goats.

Cattle and sheep carrying clone pregnancies are at risk of developing
hydrops and dystocia. Neither of these complications has been reported
in swine or goats carrying clone pregnancies. Dystocia is an identified
hazard for any pregnancy, clone or not, that goes to term. A common
cause of dystocia is incompatibility between the size of the fetus and
the pelvic opening through which it must pass.

Other frank abnormalities that have been noted among stillborn and
neonatal clones include flexor tendon contracture, which may result from
crowding in the uterus, and respiratory failure. The latter, which is
one of the most commonly reported clinical signs in neonatal clones, may
result from numerous causes, including inadequate surfactant and failure
of the lungs to inflate. Many calf clones are born with large umbilici,
often with patent blood vessels, which can increase the risk of
bacterial infection. These umbilical problems are generally resolved
surgically.

**2. Juvenile Developmental Node**

Most prepubertal cattle, sheep, swine and goat clones appear to grow and
develop normally following the early neonatal period. None of the
reports evaluated in this risk assessment indicated any anomalies that
have not been observed in animals derived via other ARTs or natural
breeding. Healthy clones appear to be physiologically similar to their
conventionally bred counterparts or their genetic donor. Alterations in
physiological parameters (clinical chemistry and hematology measures)
that may have been noted early in the clone's life appear to resolve
within a month or two of birth, and the health of bovine clones, in
particular, is confirmed by veterinary records.

Some clones, although surviving the early neonatal period, died during
the juvenile period due to either congenital abnormalities that did not
resolve or failure to thrive. One dwarf calf that suffered skeletal
defects and gastro-intestinal problems did not recover and was
euthanized. Other calves in this group had no apparent health problems.

A few frank developmental defects have been noted. Cryptorchidism
(failure of testicular descent) was reported in three calves derived
from the same cell line. Hyperkeratosis, a thickening and crusting of
the skin that may predispose the animal to skin infections and observed
in naturally bred pigs, was observed in one porcine clone. Both
conditions may be related to genetics of the nuclear donor, although
hyperkeratosis in swine is more often related to nutrition. Neither
condition is necessarily attributed to cloning.

**3. Reproductive Function**

Because of the newness of the technology, very few animal clones have
lived to reproductive age, and there are very few publications that
specifically address reproductive function. In general, the cursory
mentions of bovine reproductive capacity indicate that most clones are
as fertile as their conventional counterparts, although there are
reports of individual heifer clones that may fail to become pregnant
after attempts at insemination. No specific studies of the reproductive
capacity of bull clones were identified, although there are reports of
"normal" fertility on individual bull clones in articles addressing
other portions of the life cycle of clones. No adverse outcomes (*e.g.,*
infertility, low sperm motility) have been reported for reproduction in
male goats or sheep, but only one study in each of these species has
been identified. No studies addressing the fertility of porcine clones
were identified.

**4. Post-Pubertal Maturation**

No studies specifically addressing the health of post-pubertal clones
were identified. Instead, cursory information from studies examining
other portions of the clones' life cycles was reviewed. No specific
reports of risks to clone health were found. This observation must be
tempered with the lack of clones that could populate this developmental
node, plus the lack of impetus to publish studies addressing the health
of clones of this developmental stage.

**5. Conclusions for Animal Safety Based on Available Information**

No reports of risks qualitatively different from those encountered by
animals involved in modern agricultural practices were detected,
although the frequency of the risks appears to be increased in some
species during the early portions of the life cycle of animal clones.

There is an increased risk of adverse outcomes for surrogate dams
bearing bovine and ovine clones relative to dams bearing non-clone
pregnancies. These risks are best characterized by dystocia, likely due
to LOS, and hydrops. The risk to swine and goat surrogates bearing clone
pregnancies does not appear to be increased relative to dams bearing
non-clone pregnancies.

Early reports of cloning in cattle and sheep indicated that most clone
pregnancies failed to result in live births. As the technology improves,
however, the proportion of live, normal births appears to be increasing.
Most of the increased risk for the bovine and ovine clones appears to be
related to LOS, although other frank developmental defects are observed.
These risks do not appear to apply to porcine and caprine clones.

As clones grow and develop, they appear to become as healthy as their
conventional counterparts. There do not appear to be any health risks to
apparently normal clones that survive beyond a few weeks of birth. Data
are very limited on the health and functionality of clones beyond the
juvenile developmental period. Information on reproductive function is
limited to a few studies, but appears to indicate that clones are not at
increased risk of reproductive failure relative to animals produced
using other ARTs or natural breeding. The information on
non-reproductive maturation in animal clones is essentially limited to
that provided by one clone producer. It indicates that clones in the
6-18 month cohort are virtually indistinguishable from their age- and
breed-matched comparators.

**D. Food Consumption Risks**

**1. Two-Pronged Approach to Identifying and Characterizing Food
Consumption Risks**

The food consumption risk assessment assumes that all clones and their
products would be subject to the same local, state, and federal
regulations as conventional food animals and their edible products.
These rules exclude frankly malformed, diseased, and otherwise unhealthy
animals from the human food supply. Therefore, this assessment focuses
on clones that appear to be healthy. Because scientific and regulatory
communities have traditionally assumed that it is highly unlikely that
"silent" pathways producing intrinsic toxicants exist in food animals,
the only hazards that could arise in animal clones would be from
incomplete or inappropriate reprogramming of the genetic information
from the donor somatic nucleus (*i.e.,* epigenetic effects). These would
allow a clone to develop with apparently normal appearance and
functions, but with sub-clinical physiological anomalies, and are
referred to as "subtle hazards."

In order to determine whether these subtle hazards pose food consumption
risks, CVM has developed a two-pronged approach. The previously
described first component, the *Critical Biological Systems Approach*,
is based on the hypothesis that a healthy animal is likely to produce
safe food products, and incorporates a systematic review of the health
of the animal clone or its progeny. 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 Critical Biological Systems Approach 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 food that is safe for
consumption; the latter implies that clones with anomalies are likely to
be considered unsuitable for food.

The second component, or 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 are as safe to consume as their conventional counterparts. 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, at least during these early stages of the development of the
technology, 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.

At this time, CVM anticipates that for economic reasons (*i.e.,* a cost
of tens of thousands of dollars per clone), "founder" animal clones are
not likely to be slaughtered for meat, and that most of the food
products from cloned lineages will enter the food chain as first
progeny[^1] of animal clones, or their subsequent offspring. If,
however, the technology would become sufficiently cost-effective, it is
possible that clones themselves could be consumed for food.

Animal clones could also be introduced into the food supply as meat if
conditions outside the producer's control forced herd culling (*e.g.,*
loss of funding), if clones suffered non-recoverable injuries, or if
older clones had reached the end of their functional utility (*e.g.,*
breeders). Milk from clones, however, could enter the food stream.
Information on the composition of clone meat or milk is extremely
limited. Very few of the bovine clones are old enough to have been bred,
given birth, and begun lactating. One study has been identified on the
composition of milk from clone cows; no studies on the composition of
meat from clones have been identified.

**2. Preliminary Conclusions Regarding Potential Food Consumption
Risks**

**a. Bovine Clones**

**i. Perinatal Bovine Clones**

The underlying biological assumption for this age cohort is that clones
may be fragile at birth possibly due to residual incomplete or
inappropriate reprogramming of the donor nucleus. Many of these animals
adjust to life outside the womb within a relatively short period of
time, either on their own, or with assistance from caregivers. The data
on the overall health of the clones are consistent with such a
hypothesis. Nonetheless, because of the consistently reported relatively
poor condition of clones at birth, and variability of their
physiological parameters, it is difficult to prove that these animals do
not differ materially from their age-matched conventional cohorts. Given
that live neonatal clones are unlikely to enter the food supply, they
pose an extremely limited risk for consumption as food. Moribund or
euthanized clones might enter the food supply via rendering; if they met
all of the conditions imposed for euthanized non-clone neonates, they
would not likely pose risks greater than the non-clones.

**ii. Juvenile Bovine Clones**

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. The results from the information supplied by the
clone producer and the peer-reviewed literature indicate that clones
show the appropriate physiological responses to developmental signals.
The data set from the clone producer indicates the overall health of
these animals is comparable to their age-matched comparators, with the
exception of the sequellae of umbilical problems and cryptorchidism.
Although these outcomes pose risk to the animals, if appropriately
managed, they do not appear to pose any food safety concerns. The
peer-reviewed literature indicates that although some animals at this
developmental node have alterations in physiological parameters such as
body temperature, some hormone and cytokine levels, these discrepancies
are resolved relatively rapidly (by approximately 50 days of life).
Thus, even for physiological parameters in which differences were
detected between clones and controls, most resolved soon after birth in
apparently healthy animals. Juvenile bovine clones are likely to be as
safe to eat as their contemporary conventional comparators.

**iii. Adult Bovine Clones**

The underlying biological assumption for this developmental cohort is
that there is no fundamental reason to suspect that animals derived via
SCNT would produce toxins, there are no introduced genes from other
sources, and any biological changes that are not immediately apparent
(*e.g.,* gross malformations) would, at most, tend to present subtle
changes that do not pose food consumption concerns. The weight of the
evidence is consistent with this biological prediction. Non-healthy
clones are immediately apparent, and culled from the herd. Data from the
clone producer indicate that healthy clones of the oldest (6-18 months)
cohort evaluated are virtually indistinguishable from their comparators
even at the level of clinical chemistry and hematology. The available
information on reproductive function in cows or bulls of this age cohort
is quite limited, but appears to indicate that clones have normal
reproductive function and give birth to healthy offspring. These
observations provide a high degree of confidence to the Center's
judgment regarding the health of bovine clones.

Edible products from healthy adult bovine clones are therefore likely to
be as safe to eat as those from non-clone adult cattle. The consistency
of the observations provide the Center with a high degree of confidence
in judgments regarding the health of (and likely food safety of edible
products derived from) bovine clones. The Center notes that although
these animals may be used for food, given the economic considerations
involved it is not likely that large numbers of clones will enter the
food supply. The most likely scenarios for these animals to be
introduced into the food supply are as the result of non-treatable
injury or old age.

There is one study presenting a compositional analysis of the milk from
cow clones. Although the sample size is small, and the differences in
animal diet and management between clones and comparator animals may
confound the interpretation of the results presented, this study
indicates that the milk of clones and the study comparators is very
similar, and that there are no apparent food safety concerns.

**b. Swine Clones**

The same underlying biological assumptions were employed in the
assessment of porcine clones as for bovine clones. The body of evidence
on the health of swine clones is considerably more limited than for
bovine clones. Although generating swine clones appears to pose more
technical difficulties than bovine clones, once piglets are born, they
appear to be healthy. The health status of perinatal animals is
generally presented as "normal" or "healthy" in peer-reviewed
publications. The most compelling argument for the normal health status
of swine clones has been in 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 conventionally-bred pigs. No significant differences were
observed in either behavior or physiological measurements, indicating
that these animals were not materially different from the comparators.
No information was available for review on the reproductive status of
swine clones or their non-reproductive maturation. Based on the
available information, edible products from swine clones are likely to
be as safe to eat as corresponding products from non-clone swine.
Confidence in this judgment is tempered by the relatively small data set
for these animals, although the consistent responses across species tend
to increase our overall confidence level.

**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 ovine clones to draw conclusions with respect to potential
risks that could be posed from the consumption of food products from
these clones. CVM was unable to find any publicly available reports on
the health status of ovine clones. There are several studies addressing
methodological issues for optimizing the generation of clones, but these
do not address post-natal health.

**d. Goat Clones**

This conclusion is based on the same underlying biological assumption
cited for the other livestock species, and a small but compelling data
set that indicates that goats appear to be particularly "cloning
friendly." Once embryo clones are transferred to surrogate dams and
pregnancies are confirmed, the "success rate" for live births is quite
high. The only anomaly noted was that approximately half of the cohort
of goats on which data are available appeared to have poor suckling
response immediately after birth, but by the second day were responding
normally and nursing from their surrogate dams. Clones appear to have
developed well through reproductive age, and the available data indicate
their physiological responses are appropriate for age and breed. Reports
on the reproductive development and function of male goat clones
indicate that they 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.

Edible products from goat clones are likely to be as safe to eat as
corresponding products from non-clone goats. The consistency of
reproductive function, even in a small cohort of animals, adds to the
confidence that can be placed in the judgment that these animals are as
normal and healthy as their conventional counterparts.

**e. Progeny of Clones**

The underlying biological assumption for progeny animals is that
generation of the cells that ultimately become ova and sperm naturally
resets epigenetic signals for gene expression. This process is thought
to effectively "clear" the genome of incomplete or inappropriate
signals. The data to confirm this underlying assumption are limited but
consistent across species. Cursory reports of normal reproductive
function of clone progeny add to the empirical demonstration that clone
progeny are as healthy and normal as their conventional counterparts.
The one instance in which milk from a daughter of a bull clone has been
evaluated seems to indicate that the milk of clone progeny is similar in
composition to non-clone contemporaries. Likewise, the one available
report of the reproductive development of a goat clone offspring
indicates that the animal functions appropriately for its breed.
Additional detailed data on the health status of clone progeny would
further increase the degree of confidence that could be applied to the
safety of edible products derived from healthy clone progeny. In the
absence of detailed information on the health of the progeny of
livestock clones, the mouse model has offered convincing evidence that
even anomalous phenotypes in clones such as prepubertal obesity are not
passed on to the offspring of the obese clones.

Edible products from the progeny of healthy clones are likely to be as
safe to eat as the corresponding products of progeny from conventional
animals. It is likely that clone progeny (from the first sexual breeding
of a clone through subsequent generations) will provide the overwhelming
majority of clone-derived cattle, swine, sheep, and goats in the US. The
weight of evidence evaluation for clone progeny is dominated by the
biological assumption of epigenetic resetting via the natural passage
through the germ line, and supported by the consistent lack of detection
of adverse outcomes in progeny of clones among and across species
(including the mouse model), and limited reports on the health of
individual clone progeny animals. Additional detailed data on the health
status of clone progeny would increase the empirical support for the
assumption underlying this conclusion. Nonetheless, we do not believe
that consumption of edible products from clone progeny would pose any
additional risk relative to consumption of similar products from
non-clone progeny.

**f. Summary of Risk Statements for Food Safety**

The current weight of evidence suggests that there are no biological
reasons, either based on underlying scientific assumptions or empirical
studies, to indicate that consumption of edible products from clones of
cattle, pigs, sheep or goats poses a greater risk than consumption of
those products from their non-clone counterparts. The level of certainty
is highest for bovine clones, followed in decreasing order of certainty,
by porcine, caprine, and ovine clones. Edible products from the progeny
of healthy clones are likely as safe to eat as similar products from the
progeny of non-clone animals, based on underlying biological
assumptions, compelling evidence from the mouse model system, and
limited data in the species evaluated. The one study of the composition
of milk from bovine clones does not indicate any food safety concerns.
The level of confidence that may be placed in these conclusions could be
increased by additional data, particularly with respect to composition
of edible products.

**E. Concluding Statements**

**Animal Health:** SCNT can pose 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. In
particular, the frequency of live normal births appears to be low,
although the situation appears to be improving as the technology
matures. In particular, cattle and sheep appear to be subject to a set
of syndromes 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. 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.

**Food Consumption Risks:** Edible products from normal, healthy clones
or their progeny do not appear to pose increased food consumption risks
relative to comparable products from conventional animals. Confidence in
this conclusion is relatively high due to empirical evidence from bovine
clones, and the consistency of empirical observations among the other
species. Progeny of clones are likely to be as safe to eat as their
non-clone counterparts based on underlying biological assumptions,
evidence from model systems, and limited, but consistent empirical
observations in the species evaluated. Additional data on the health
status of progeny, and composition of milk and meat from clones and
their progeny would serve to further increase the confidence in these
conclusions.

[^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 cloned animal as a parent (but could result from two
    cloned animals mating). Clones of clones would be considered as
    clones (*i.e.,* directly arising from an SCNT process).