Safety assessment of food products from r-DNA animals

Safety assessment of food productsfrom r-DNA animals§

Martin A. Lema a,b,*, Moises Burachik a,c

a Biotechnology Office, Secretariat of Agriculture, Livestock, Fisheries and Food,

Av. Paseo Colon 922, Piso 2, of. 246, Buenos Aires city C1063ACW, Argentinab National University of Quilmes, Roque Saenz Pena 352, Bernal B1876BXD, Bs. As., Argentinac University of Buenos Aires, Facultad de Ciencias Exactas, Pabellon II, Ciudad Universitaria,

Buenos Aires city 1428, Argentina

Abstract

Recombinant-DNA (transgenic) animals intended for food production are approaching themarket. Among them, recombinant-DNA fishes constitute the most advanced case. As a result,intergovernmental organizations are working on guidelines which would eventually become inter-national standards for national food safety assessments of these products.

This article reviews the emerging elements for the food safety assessment of productsderived from recombinant-DNA animals. These elements will become highly relevantboth for researchers and regulators interested in developing or analyzing recombinant-DNAanimals intended to be used in the commercial elaboration of food products. It also providesreferences to science-based tools that can be used to support food safety assessments. Finally, it

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Comparative Immunology, Microbiologyand Infectious Diseases 32 (2009) 163–189

§ Disclaimer: The information and views contained in this article are the sole responsibility of the authors,and they should not be ascribed to and do not necessarily represent the opinions or policies of anyorganization, institution or government. This article is only intended for academic purposes, and does notsubstitute for ad hoc expert advice and case-by-case risk analysis. The authors disclaim all responsibility andliability arising from the use of this information, and strongly suggest referring to the current state of the art,as well as to applicable regulations and regulatory bodies, when preparing to actually obtain or assess the foodsafety of an r-DNA animal.* Corresponding author at: Biotechnology Office, Secretariat of Agriculture, Livestock, Fisheries and Food, Av.

Paseo Colón 922, Piso 2, of. 246, Buenos Aires city C1063ACW, Argentina. Tel.: +54 11 4349 2070;fax: +54 11 4349 2178.

E-mail address: [email protected] (M.A. Lema).

0147-9571/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.cimid.2007.11.007

mailto:[email protected]

http://dx.doi.org/10.1016/j.cimid.2007.11.007

proposes recommendations for the further development of biosafety assessment methodologies inthis area.# 2007 Elsevier Ltd. All rights reserved.

Keywords: Food safety assessment; Transgenic animals; Recombinant-DNA animals; Non-heritable applica-tions; Codex Alimentarius

Résumé

Les animaux à ADN recombiné (transgéniques) destinés à l’alimentation humaine se rapprochentde la mise sur le marché. Parmi eux, les poissons transgéniques constituent le cas le plus avancé. Cecia amené des organisations intergouvernementales à proposer des lignes de conduites qui pourraientdevenir des standards internationaux pour les évaluations nationales de la sécurité alimentaire de cesproduits. Cet article fait le point sur les éléments en emergence concernant l’évaluation de la sécuritéalimentaire des produits issus des animaux génétiquement modifiés. Ces éléments devraient aider leschercheurs et les personnes en charge des aspects règlementaires à élaborer et à mettre en œuvre desrèglementations pour la commercialisation des produits alimentaires issus des animaux génétique-ment modifiés. Ces éléments doivent également constituer des références pour définir de outilsscientifiques capables de mieux évaluer les risques que peut engendrer la consommation de cesproduits. Cet article propose enfin des recommandations susceptibles de favoriser la mise en place denouvelles méthodes d’évaluation des risques alimentaires potentiellement induits par les produitsissus des animaux transgéniques.# 2007 Elsevier Ltd. All rights reserved.

Mots cles : Les évaluations de la sécurité sanitaire des aliments ; Animaux transgéniques ; Animaux à ADNrecombiné ; Applications non transmissibles ; Codex Alimentarius

1. Introduction

1.1. Animal transgenesis

1.1.1. Technical and political concept

A plethora of terms have been coined for referring to organisms carrying an insertion of

genetic material on its genome through the use of genetic engineering or recombinant-

DNA techniques. Examples of these terms are ‘‘genetically modified/engineered

organism’’, ‘‘transgenic/cisgenic organism’’, ‘‘recombinant-DNA organism’’, ‘‘living

modified organism’’, ‘‘organism obtained through the use of biotechnology’’, etc.

Moreover, each term may have different definitions in separate contexts. For the purposes

of this article we will use the term ‘‘recombinant-DNA’’ (r-DNA), as it is the one adopted

by the Codex Alimentarius, the source of the main documents referenced in this article.

It is important to note, however, that an influential definition of r-DNA organisms at the

international level has been adopted in the Cartagena Protocol on Biosafety [1] under the

Convention on Biological Diversity (CPB);

‘‘Living modified organism’’ means any living organism that possesses a novel

combination of genetic material obtained through the use of modern biotechnology;‘‘

‘‘Modern Biotechnology’’ means the application of:

M.A. Lema, M. Burachik / Comp. Immun. Microbiol. Infect. Dis. 32 (2009) 163–189164

(i) In vitro nucleic acid techniques, including recombinant deoxyribonucleic acid (DNA)and direct injection of nucleic acid into cells or organelles, or

(ii) fusion of cells beyond the taxonomic family,that overcome natural physiological reproductive or recombinant barriers and that

are not techniques used in traditional breeding and selection.’’

For practical purposes, item (i) above is more relevant, and reduces the field of ‘‘modern

biotechnology’’ to ‘‘transgenesis’’ within the context of international politics. A discussion

of the reasons why the more restricted term ‘‘transgenesis’’ was upgraded to

‘‘biotechnology’’ (or, conversely, why cloning, marker-assisted breeding, and a long

list of biotechnologies were not considered as such) is out of the scope of this article.

This ‘‘biotechnology’’ definition in the CPB was reflected in further documents of the

Codex Alimentarius Commission, and many national and international organizations. As a

consequence, a ‘‘recombinant-DNA animal’’ has been recently defined [2] as:

‘‘an animal in which the genetic material has been changed through in vitro nucleic acidtechniques, including recombinant deoxyribonucleic acid (DNA) and direct injection intocells and organelles’’.

The first r-DNA animal was obtained in 1980 by DNA microinjection of mouse embryos

[3]. Nowadays, many animals intended for food purposes like cows, swine, goats or fish,

can be successfully transformed by several technologies reviewed below.

Genetic engineering offers advantages over traditional selection and conventional

breeding by sexual crossing, the most relevant being the ability to deliberately introduce

any designed gene into an animal, even allowing the transfer of genes across species. This

leads to increased possibilities for the introduction of novel traits.

Moreover, as the main effects of a transgene can be predicted on the basis of previous

studies, and given that the desired trait can be introduced without significantly altering a

valuable pre-existing genetic background, the developer has increased control and a better

understanding of the process.

Also, once introduced in the first r-DNA animal and with the aid of simple molecular

markers, the transgene can be fast and easily introgressed into different genetic

backgrounds and/or stacked with other transgenes, by conventional breeding.

These advantages, among others, are similar to those realized for r-DNA crops, which

support the increasing use of the genetically modified crops for the past ten years, reaching

a global area of commercial cultivation of 102 million hectares in 2006 [4].

Potential practical applications of transgenesis in animals intended for food use include:

- Resistance to diseases, including human zoonoses; thus reducing the use of antibiotics

and other pharmaceuticals, enhancing animal welfare, simplifying breeding and

increasing production.

- Modification of carcass structure, milk or egg composition for nutritional or health

benefits; for instance optimizing nutrient content, incorporating nutraceuticals or

reducing the content of allergenic or toxic substances.

- Optimization of feed digestion to increase conversion efficiency and reduce

environmental pollution, in some cases allowing the use of alternative feeds.

M.A. Lema, M. Burachik / Comp. Immun. Microbiol. Infect. Dis. 32 (2009) 163–189 165

- Increase of growth rate, milk production, litter size, etc.

- Improvement of food quality attributes, like tenderness, taste, texture and flavor.

1.1.2. Technologies

Recombinant-DNA animal lines are generated through the introduction of a DNA

molecule in a cell that is capable of generating a whole animal, like a one cell embryo,

sperm, oocyte, blastomere, stem cell, an egg, or a somatic cultured cell which will be

subsequently used as a donor of genetic material for an enucleated egg. The DNA molecule

is integrated into the genome of such primordial cell, thus generating r-DNA animals

having all of their cells transformed. These animals are generally capable of transmitting

the transgene to their progeny.

Technologies for obtaining r-DNA animals are fully reviewed elsewhere [5]. The

most commonly used methods involve the physical microinjection of purified DNA into

pronuclei or cytoplasm of one-cell embryos, or into somatic cells to generate r-DNA

(and cloned) animals by nuclear transfer [6]. Alternatively, gametes can be

easily transformed [7–8], and then employed in standard assisted reproduction

techniques to generate the r-DNA animal. In other methods, pluripotent stem cells are

transformed and subsequently introduced into an early embryo, leading to the birth of

chimerical animals; some of the latter may be potentially capable to transmitting the

transgene to their progeny, hereby establishing a genetically stable (non-chimerical)

line [9–10].

In some cases, the movement and/or integration functions of transposable DNA

elements or retroviruses [7] have been employed in order to allow or enhance the

introduction of the DNA construct into the recipient genome.

Transposons consist of DNA stretches that can move to different locations within the

nuclear chromosomes of a single cell and replicate. Genomes of vertebrate animals

naturally contain many active transposons or sequences derived from them. These

mobilizable genetic elements contain particular flanking sequences which are recognized

by specialized enzymes encoded in the transposon or present in trans. Transposons

modified to carry transgenes have been used to generate r-DNA insects, fish, poultry and

mammals [11–15].

Retroviruses, on the other hand, have a RNA genome that is retro-transcribed upon

infection into a double-stranded DNA. The latter molecule then integrates randomly into

the chromosomal DNA of the host cell. Retroviruses have also been manipulated to carry

recombinant nucleic acid constructs.

Integrated retroviral DNA can reactivate spontaneously, leading to the production of

new integration sites within the DNA of the cell, or to new infection of other cells or other

individual animals. Such possibility generates expression instability and biosafety

concerns. However, retroviral vectors may be engineered to deprive them of the genetic

sequences required for a normal life cycle.

Strategies based on transposons or retroviruses pose concerns related to genetic stability

and biosafety. Therefore, their use has been discouraged in animals intended for food

production unless these vectors are ‘‘disarmed’’ in a way that will effectively hinder further

horizontal dissemination of genetic material to other locations in the genome or to other

organisms.


In order to avoid unintended effects derived from the random insertion of DNA into the

host genome (discussed below), potential non-integrative alternatives to obtain a reliable

transgene expression include the use of mammalian artificial chromosomes and episomal

circular vectors [16–17].

1.1.3. State of the art

Recombinant-DNA fishes carrying growth hormone genes are probably the products

closer to the market, and therefore they strongly contribute to the establishment of national

and intergovernmental regulations. These r-DNA fish have been produced by different

researchers [18–21], but the common design is the introduction of a fish growth hormone

gene under a constitutive fish promoter. As a result, fishes grow faster although usually

their final size is not altered. This reduces the time needed to grow the fish to commercial

weight, thereby reducing costs of aquaculture facilities. Recombinant-DNA fishes are

under consideration or close to be evaluated by at least five regulatory agencies in the

world.

Terrestrial animals like rabbits, pigs and sheep expressing foreign growth hormone

genes have also been generated for similar purposes [22–24].

Milk-related traits also constitute a flourishing field. The expression of bovine a-

lactalbumin gene in sow milk, for instance, has shown to increase milk production, and

significantly improves piglet growth and survival after weaning [25]. Recombinant-DNA

goats expressing lipid desaturase in their milk have lower levels of saturated fatty acids.

The milk containing either human lysozyme or lysostaphin is more resistant to bacterial

growth, and thus it is expected to protect consumers from bacterial infections and animals

from mastitis, and to reduce production costs [26–27].

Lactose content in milk can be reduced by secretion of transgenic lactase [28], which

could be helpful to reduce consumer problems related to lactose intolerance. Recombinant-

DNA cows overexpressing b-and k-casein genes produce protein enriched milk, which is

expected to improve cheese making techniques [29]. Many other genetic modifications

targeting milk have been envisaged [30].

Regarding other kinds of traits, for instance pigs expressing E. coli phytase in their

saliva are better able to hydrolyze phytates present in feed; as a consequence, phosphate

manure content is reduced up to 75%. Since pig manure is used as fertilizer, this

would avoid an excess of phosphorous ending up in watercourses. These ‘‘enviropigs’’

[31] also have an increased growth rate, probably due to the intake of released

phosphate and microelements (sequestered as phytate complexes). Phytase gene

has also been introduced in fish, pursuing similar environmental and production

benefits [32].

Another example is the increase of muscle growth, due to the expression of an

incomplete transgenic myostatin or the overexpression of Insulin-like Growth Factor 1

[33–34].

The discovery of post-transcriptional gene silencing or RNA interference has opened a

new avenue for the inactivation of genomic (or viral) genes. Inducing RNA interference in

animals is much easier than obtaining a gene knock out by homologous recombination.

Attempts to inactivate the PrP gene in order to prevent prion diseases, for instance, is under

study using gene knock out or the expression of interfering RNA [35–36].


1.1.4. Non-heritable applications

Other techniques, similar to those already mentioned, make use of physical and

biological processes that allow the introduction and expression of an r-DNA in a limited

number of cells, usually after birth of the animal. These techniques are not intended to

generate a stable line of r-DNA animals, but instead to modify a single animal, hence the

term ‘‘non-heritable’’.

‘‘Non-heritable applications’’ or ‘‘non-heritable constructs’’ and the food safety

assessment of animals modified by them are considered in detail elsewhere [37–38].

Typical r-DNA applications that could be considered ‘‘non-heritable’’ are DNA

vaccines, gene therapy, genetic enhancement and grafting of genetically modified cells

[38,39–41].

Methods relying on physical introduction include instillation into the airways, injection

into the bloodstream or muscle, immersion, spraying, gene gun, electroporation,

suppositories and liposomes vehicles; for a review on physical methods, see [42–43].

These applications usually involve simple constructs that do not contain homologous

recombination sequences or mobility/replicative/integrative functions from transposons

and retroviruses. It has been shown that these simple constructs will most likely remain as

extra chromosomal elements. Nevertheless, some authors anticipate that, if necessary,

deliberate integration in the genome by homologous recombination may be more feasible

in the future [44–45].

Alternatively, retro-, lenti-, adeno-, adeno associated-, and herpes viruses can be

recruited as vectors of non-heritable constructs.

In general, the methods and principles devised for assessing the food safety of r-DNA

animals bearing heritable constructs will be also valid for non-heritable applications, the

only obvious exception being the need for establishing the heritability of the transgene and

the stability of the trait in subsequent generations.

Nevertheless, animals modified by the use of non-heritable constructs pose additional

challenges from the perspective of food safety. On one side, there is the potential for

increased inter-animal variance in the expression of transgenes due to the particular

circumstances of each ‘‘DNA shot’’. On the other hand, a recent expert consultation [37]

has identified the potential for ‘‘excipient effects’’, a term coined to embrace risks from

‘‘materials’’ serving as vehicles of the non-heritable construct. These excipients may

include liposomes or gold beads in the case of physical methods; or viral sequences or

proteins used for packaging, cell entry, and nuclear targeting of the construct in the case of

biological methods. Finally, in some cases a characterization of the tissues directly affected

by the modification would be required for food safety assessment.

1.1.5. Cloning

Cloning is an artificial form of asexual reproduction, which produces offspring that is

genetically identical to a single parental organism. Original techniques developed in the

late 70s involved the use of embryos as parental organisms; as commercial applications

would usually require the reproduction of an adult of proven value, this technology had

limited usefulness for animal production.

In 1996, the sheep Dolly was the first success of somatic cell nuclear transfer (SCNT)

[46]. SCNToffers a better commercial application because it allows the cloning of valuable


adult animals, with no theoretical limitation to the number of clones that can be produced.

In addition, its flexibility allowed for a rapid adaptation to several species.

SCNT consists in the introduction of a cell nucleus into an unfertilized egg whose own

nucleus has been removed, and differentiated cells from adult animals can be used as nuclei

donors. Therefore, SCNT in fact usually produces an offspring that combines the nuclear

and mitochondrial genomes from two different individuals.

Usually after r-DNA animals are generated, they are cloned a few times for speeding

their multiplication. Therefore, cloning can be applied to both conventional or r-DNA

animals.

Cloning is considered a biotechnology in the academic field, and would also fit in the

broad biotechnology definition that can be found in the Convention on Biological Diversityand the FAO Glossary for Food and Agriculture: ‘‘Biotechnology means any technologicalapplication that uses biological systems, living organisms, or derivatives thereof, to makeor modify products or processes for specific use.’’ However, it has been considered that the

‘‘regulatory’’ definition of biotechnology, restricted to transgenesis as it has been discussed

before, may not encompass cloning [47]. Therefore, clones are not currently covered by

international standards or regulated per se at the national level. Despite this fact, some

organizations have worked towards clarifying the food safety implications and regulatory

status of cloning [48].

Three concerns have been raised in relation to the safety of food derived from cloned

livestock. One of them is the mixing of nuclear and mitochondrial genomes from different

animals, although no potential hazards have been identified from it. Furthermore, similar

mixing occurs on conventional sexual reproduction.

The second one is the hypothetical animal aging effect from using a cell with reduced

telomeres as a nuclear genome donor. Again, no clear novel food hazard, different from

those that might be related to the consumption of conventional food from old animals, has

been suggested.

Finally, the ‘‘large offspring syndrome’’ is probably the more resilient concern. It is

thought that in vitro culture process of embryos and cells can alter normal gene expression

patterns, leading to a newborn animal body size slightly greater than normal. This

syndrome can also be associated with minor alterations on the values of some indicative

animal blood metabolites, especially at early age. This phenomenon has been firstly

observed occasionally in calves and lambs following assisted reproduction techniques such

as in vitro fertilization. No actual hazards have been identified during the years of

commercial application of in vitro fertilization; moreover the symptoms seem to vanish

with age and are not found in the offspring of subsequent sexual reproduction (clones are

usually employed for conventional reproduction, because they are too expensive to be used

directly as food source, particularly at early age).

On the other hand, since cloning does not imply new genes and/or random insertions in

the genome, there is little basis for the ‘‘case by case’’ regulatory approach applied to r-

DNA organisms.

Recently, the Food and Drug Administration of the United States has released a risk

assessment document related to the meat and milk products derived from cloned animals

and their offspring, concluding they are safe for human consumption [49]. The conclusions

of this risk assessment, if widely adopted, may preclude the need for establishing specific


guidelines or frameworks for case-by-case regulations of food derived from cloned

animals, and therefore cloning is likely to be generally considered as safe as other assisted-

reproduction techniques.

1.2. Safety assessment

The first provisions for the safety assessment of r-DNA organisms were drawn up by

scientific experts in the mid-1980s [50–51]. The approach to the safety assessment of foods

derived from r-DNA animals was first discussed in 1991 during an expert consultation

organized by the Food and Agriculture Organization of the United Nations (FAO) and the

World Health Organization of the United Nations (WHO) [52]; later it has been elaborated

over time [37,53–54], leading to a standard framework described below.

There are no inherent risks in the use of recombinant DNA technologies, as deemed by

many international panels of experts [50,55], compared to mutagenesis and traditional

breeding. Moreover, this is one of the reasons why some countries have developed a ‘‘new

trait’’ regulation instead of a ‘‘transgene’’ regulation [56].

International guidelines [57–59] discriminate three kinds of interrelated activities: risk/

safety assessment, risk management and risk communication. Risk analysis is the

aggregate of these three activities.

Risk/safety assessment is a strictly science-based activity conducted by experts. It

involves identifying each hazard posed by the subject under study, and estimating the likely

degree of severity as well as the associated probabilities of realization, by establishing

cause-effect and dose-response relationships. Therefore, ‘‘risk’’ is a concept integrating the

hazard, its expected severity and the likelihood of realization.

Risk management should be conducted by regulatory authorities and can be defined as

‘‘the process of weighing policy alternatives to mitigate risks in the light of risk assessment

and, if required, selecting and implementing appropriate control options, including

regulatory measures’’. Risk management measures should be proportional to the risk and

based in the outcome of the risk assessment. Risk management strategies may involve

specific conditions for marketing, post-market monitoring of relevant safety aspects and

food labeling [59]. Finally, risk communication is defined ‘‘as the exchange of information

and opinion on risk between risk assessors, risk managers, other interested parties, and the

general public’’.

Two branches of scientific risk/safety assessment for r-DNA organisms can be

differentiated. One of them is food safety assessment, covered in this article for the animal

case. The other one is environmental risk assessment, which focuses on potential adverse

effects on the conservation and sustainable use of biological diversity at the potential

receiving environment. Guidance on environmental risk assessment can be found in annex

III of the CPB [1].

Assessments of other risks are theoretically possible. For example, regarding

occupational hazards; nevertheless, it has not been deemed necessary so far, perhaps

due to the nature of the r-DNA organisms intended for food trade to date.

Risk analysis should be the main factor in decision-making. Nonetheless, other kind of

studies are becoming emerging fields, for instance on the socioeconomic impact of the

incorporation of an r-DNA organism in the production chain, or on the trade exports impact


due to the r-DNA or its products not being authorized in customer countries; or on ethical

issues, particularly for animal products [60]. If implemented, this kind of studies should be

conducted separately from the ‘‘hard-science’’ safety assessments. This is due to

differences in the nature of the information and expertise required, in the development

stage of the methodologies and international guidance, on the expected rigor of the

conclusions, and finally on the different relevance for trade-related measures in the light of

applicable international agreements.

Finally, ‘‘trait efficacy’’ assessment perhaps will become another emerging field,

particularly for those r-DNA organisms modified to provide nutritional or health benefits.

Nevertheless, almost no guidance has been developed so far for this kind of assessment

which, if applicable, should be conducted separately from the food safety assessment from

a regulatory perspective.

Traditional toxicological risk assessment is applied to specific chemicals such as food

additives and pesticide residues. In those cases, there are only one or a few actual or

potential risks, which in many cases are well characterized in the prior art, and the risk

assessment can be regarded as ‘‘absolute’’.

In contrast, foods derived from r-DNA organisms are assessed as whole new foods, but

taking into account that they can be closely related to conventional analogues or

counterpart foods. Conventional foods have not been assessed scientifically to characterize

all risks associated with them, but in most cases a ‘‘history of safe use’’ is recognized

instead. Therefore, foods derived from r-DNA organisms are assessed relative to a

conventional counterpart having a history of safe use. Rather than trying to identify and

quantify every hazard associated with a particular food, the goal is to identify new or

altered hazards relative to the conventional counterpart. The outcome of such comparativeapproach is a conclusion regarding if the ‘‘new food’’ is as safe as its conventional

counterpart(s).

The concept of ‘‘substantial equivalence’’ [61–62] has been coined in order to support

this rationale; it corresponds to the starting point in the safety assessment process, and is

used to establish similarities between the new food relative to its conventional counterpart,

therefore highlighting differences which are subject to further investigation as to whether

they might have implications for human health. The expected endpoint of the global

assessment, therefore, will be a conclusion regarding whether the new food is as safe as the

conventional counterpart or not in the light of the best available scientific knowledge.

Furthermore, the concept of substantial equivalence in the context of the safety

assessment of foods derived from r-DNA animals and the emerging concept of comparative

safety assessment [63] was originally discussed at [53].

Another essential concept is ‘‘Conventional Counterpart’’. If applied to an animal, it

means an animal breed with a known history of safe use as food from which the initial r-

DNA animal was derived, as well as the breeding partners used afterwards in generating the

r-DNA animals ultimately used as food source. It may also refer to the food derived from

conventional counterpart animals. It is unlikely that r-DNA animals will be regarded as

appropriate conventional counterparts for other r-DNA animals, at least in the near future.

Assessments should not be ‘‘transgene based’’ i.e. performed once and for all for each

transgene or trait. Instead, assessments should be made for each transformation event, on a

case-by-case basis. This is an established concept in the assessment of r-DNA organisms


for two reasons. On one side, different insertion sites can have diverse influence on the

transgene expression levels. In addition, random integration may modify the pre-existing

local genetic information or the inserted DNA differently for each event.

In conclusion, the approach described is intended only for foods consisting of or derived

from r-DNA animals, which have been derived from conventional animals having a history

of safe use as sources of food.

Genetic modifications are intended to render a main, specific and useful trait. However,

the unintended effects of the genetic modification, i.e. those traits acquired, lost or modified

as a collateral effect, are also relevant for a food safety assessment.

Unintended effects may be deleterious, beneficial, or neutral regarding food safety.

They are a general phenomenon that also occurs, for instance, in conventional breeding.

For r-DNA animals, unintended effects may arise through the random insertion of the r-

DNA in the genome, which may cause modifications in the expression of pre-existing

genes located in the vicinity of the integrated foreign DNA, or even create new ones; or

from genetic rearrangements or reprogramming due to in vitro cell manipulation (similarly

to somaclonal variation in plant tissue culture), or from unanticipated metabolic

interactions of the gene product.

Many unintended effects are largely predictable based on knowledge of the region of

insertion and/or the biological functions of the elements used to build the transgene and

their analogues. Therefore, experimental data and background information submitted to a

safety assessment should suffice to evaluate the possibility that a food derived from the r-

DNA animal would have an overlooked, significant and adverse effect on human health.

1.3. Codex Alimentarius

The paramount international guidance on food safety assessment of r-DNA organisms

are the specific guidelines included in Codex Alimentarius (CODEX).

CODEX is a compilation of standards, methods and guidelines related to food products,

prepared by technical intergovernmental bodies which implement the Joint FAO/WHO

Food Standards Program. The objectives of the Program are to protect the health of

consumers, to ensure fair practices in food trade, and to harmonize food standards.

CODEX standards are elaborated after a sound scientific analysis of all relevant

information, and following a set of rules that guarantee the participation and endorsement

of governments, industry and consumers.

Although CODEX is not mandatory per se, its observance by governments is enforceable

in trade disputes under the World Trade Organization (WTO), particularly when its

Agreement on the Application of Sanitary and Phytosanitary Measures (SPS) is involved.

CODEX and its role in the context of the WTO agreements are described elsewhere [64].

Under the SPS Agreement, countries can establish new measures restricting

international trade if they are necessary to protect human, animal or plant life or health.

If a member country claim that its food products have been discriminated due to an

arbitrary or unjustifiable sanitary measure of another member county, a WTO panel will

analyze that measure, in the light of appropriate CODEX standards, in order to differentiate

legitimate sanitary measures from commercial protectionism. This mechanism has already

been utilized for r-DNA plant products [65].


To further support the CODEX elaboration, FAO and WHO usually assemble Expert

Consultations to provide independent scientific advice on issues of relevance to food safety.

The reports of these consultations are usually of high technical value, although they do not

necessarily represent a wide scientific or political consensus.

CODEX guidance for the food safety assessment of foods from r-DNA animals [2] has

been prepared by the Codex Task Force on Foods derived from Modern Biotechnology,

and it is currently in draft status. Nevertheless, it is expected to be released during

2008.

2. Framework of the safety assessment

Food safety assessment of foods derived from r-DNA animals should be framed on a

stepwise consideration of relevant information regarding different issues; this information

should begin with an overall description of the r-DNA animal and background information

on the biology of the conventional counterpart animal(s) and its use for food production.

Then, a description of the genetic modification, including the sources of the genetic

information is necessary.

Finally, the genetic modification should be characterized in the r-DNA animal

‘‘ultimately used for food production’’; the latter concept intends to address two

exceptional but potential situations. On one hand, occasionally r-DNA animals could be

initially obtained as chimeras or mosaic organisms; and almost certainly further breeding

will be applied to obtain a line of genetically homogeneous r-DNA animals. On the other

hand, r-DNA animals in particular cases could be initially generated from non-

commercial or wild races which are more amenable for genetic modification but lack a

history of safe use. In such cases further breeding will likely be applied to introgress the

transgene into a commercial line with an established history of safe use. Regardless of

these exceptional situations, in any case the initial r-DNA animal will be hemizygous for

each r-DNA insert, and backcrossing will likely be applied for obtaining homozygous

animals.

Therefore, the animal which will be actually used to introduce the event in the market

should be a more appropriate subject for the final characterization of the genetic

modification. This does not imply that a new assessment is necessary if an r-DNA animal

line previously assessed to be safe is sexually crossed afterwards with a conventional race

already in the market.

2.1. Information required

The food safety assessment of r-DNA animals should follow a stepwise procedure for

addressing relevant factors. Therefore, it is important to proactively obtain the necessary

information and organize it in order to allow for early identification of any potential risk,

and with a view of facilitating the future regulatory process.

Procedures and experiments which will be used as sources for information should be

properly documented, and primary data should be recorded in case it is requested later by

regulatory authorities.


2.1.1. On the (recipient) animal prior to the modification

The following information, regarding conventional animals that have made a significant

contribution to the genetic background of the initial r-DNA animal, is relevant for a food

safety assessment:

� Common or usual name(s), scientific name and taxonomic classification.

� History of breeding, information on the animal’s genotype and phenotype related to its

safety as a source of food, including any known toxicity, allergenicity, presence of toxin-

producing organisms or human pathogens.

� Information on the effect of different husbandry conditions (feed, exercise, growth

environment) on food products.

� History of safe use as food or for food production. It should include information on how

the animals breed and grow, how its food products are obtained, and the conditions under

which those food products are delivered to and used by the population (e.g. storage,

transport, processing and home preparation). This description should be sufficient to

understand the nature and types of foods being submitted for safety assessment.

� Nutritional relevance of the main food products to the general population and/or

particular subgroups, including what important macro- or micronutrients they contribute

to the diet.

2.1.2. On the sources of the r-DNA

For each DNA sequence in the r-DNA insert which was derived (directly or through

optimization, e.g. for codon usage) from an organism other than the recipient animal, the

following additional information on that organism is considered necessary for a food safety

assessment:

� Common or usual name(s), scientific name and taxonomic classification.

� For animals: history of breeding, known genotype and phenotype information relevant to

its safety as a source of food, including any known toxicity, allergenicity and presence of

toxin-producing organisms or human pathogens.

� For microorganisms: additional information on pathogenicity to humans or the recipient

animal, as well as any known phylogenetic relationship or natural association to human

or animal pathogens.

� For donors of animal or viral origin: information on the source materials used (e.g. cell

culture), to allow for the assessment of potential risks derived from unexpected

pathogens in those source materials.

� If applicable, information on the presence of the source organism or its derivatives in the

food supply (e.g. food additives, contaminants).

2.1.3. On the r-DNA construct and the initial genetic modification

It would be expected a description of how the r-DNA construct was assembled from

natural or synthetic fragments of DNA. If the sequence of a DNA fragment was designed

(artificial optimization of natural sequences, de novo protein design [66]), the rationale

behind the design should be provided along with any separate experiment, if available,

demonstrating its efficacy.


The following information regarding the DNA construct is essential for a safety

assessment:

� Complete nucleotide sequence, and a map of the final vector/construct indicating the

location and orientation of the genetic components.

� Characterization of every individual genetic component, including open reading frames,

regulatory sequences and any other one affecting DNA expression and/or function. For

all these elements, information should include relevant details like source (see above),

size, biological function, analysis of the potential for mobilization or recombination and

literature references.

� An explanation of the expected function of the transgenes in the r-DNA animal.

� The protocol followed in order to introduce the r-DNA into the recipient animal,

including a description of the specific methodology used for the transformation. If

pathogenic organisms have been used as vectors or during r-DNA assembly, information

on their natural hosts, target organs, transmission mode, pathogenicity, and potential for

recombination with other pathogens.

Sufficient information should be provided on the genetic modification to allow

identifying any genetic material potentially delivered to the recipient animal, intentionally

or not.

2.1.4. On the r-DNA animals ultimately used for food production

Information should be supplied on how the initial r-DNA animal was used to breed the

animals ultimately used as food or for food production, including:

� Further methods used to obtain the r-DNA animals ultimately used as food or for food

production (e.g. the steps of a traditional breeding scheme, the use of marked-assisted

selection, the use of cloning or other assisted reproduction techniques, etc.). If

applicable, how heritability is attained (e.g., breeding chimeras or mosaic animals to

obtain genetically homogeneous lines, self-breeding for homozygosis).

� Descriptions of other animals used to generate the animals ultimately used for food

production (breeding partners, surrogate mothers) including relevant information on

genotype, phenotype and husbandry conditions under which they were raised or

harvested.

Regarding the latter, whenever information on the history of use of food products from

other animals involved (e.g. breeding partners, surrogate dams) differs from that applicable

to the recipient animal prior to the modification, such information (as described above)

should be included.

2.1.5. Characterization of the genetic modification(s)

In order to provide clear understanding of the impact on the composition and safety of

foods derived from r-DNA animals, a comprehensive molecular and biochemical

characterization of the genetic modification in the r-DNA animal ultimately used as food or

for food production should be carried out.


Relevant information on the genetic modification of the animal genome includes:

� Number of insertion sites;

� Organization of the inserted genetic material at each insertion site, including copy

number for tandem insertions and sequence data of the inserted material and the flanking

regions.

This information should suffice to determine if the original arrangement of the genetic

material used for transformation has been conserved or whether significant rearrangements

have occurred upon integration. Particularly, it should be useful to assess if the expression

of other polypeptides has been generated (fusion proteins or novel open reading frames),

suppressed (disrupted endogenous pre-existing genes) or otherwise modified as a

consequence of the insertion and/or rearrangements.

Any novel substance, in the context of the r-DNA animal and/or derived foods, whose

expression is a direct or indirect consequence of the genetic modification, should be

identified. This could include proteins or untranslated RNA expressed from transgenes in

the original construct or created due to the insertion of the r-DNA in the genome, and new

metabolites produced by catalytic or other biochemical activities of these substances.

Further characterization may require the isolation of the new substance from the r-DNA

animal; when sufficient amounts of test protein cannot be readily extracted, production of

the substance from an alternative source (e.g. recombinant bacteria) may be accepted. In

the latter case, the material should be shown to be biochemically, structurally, and

functionally equivalent to that produced in the r-DNA animal. Tests to be done in this

regard for proteins may include, as appropriate, sequence analysis, activity measurements,

electrophoresis patterns of full-length and trypsinated forms, immunoreactivity analysis,

glycosilation patterns and determination of other posttranslational modifications.

Relevant information on newly expressed substances (either intended or not) and new

traits comprise:

� A comprehensive molecular and biochemical characterization. For proteins this may

include biological activities (e.g. receptor binding, enzymatic activity, immunological

response, etc.), significant sequence homology with known proteins and biological

activities of those proteins, and degradation studies under expected conditions of storage

and processing, and under simulated human digestion. For proteins from distantly

related organisms or carrying modifications to the original amino acid sequence, if there

have been changes in the protein post-translational processing or if critical sites for its

structure or function have been affected.

� A description of the expected function or potential effect in the r-DNA animal, including

a phenotypic description of the derived new trait(s), if applicable.

� A characterization of the expression level and tissue specificity, if applicable, in the animal

and the derived food. For proteins, whether expression timing, level, tissues, etc., are

consistent with the regulatory sequences driving the expression of the corresponding gene.

In addition, whether this expression correlates with the expected phenotype or trait.

� If the function of the substance is to alter the accumulation of a specific endogenous

substance, the amount of that endogenous target should be reported. Any evidence


suggesting that endogenous genes in the r-DNA animal have been, intentionally or not,

affected by the transformation process is relevant. This could be done through analysis

of transcripts or expression products.

� Whether the intended effect of the modification has been achieved and all traits are stable

and expressed as expected. It may be necessary to examine the inheritance of the DNA

insert itself or the expression of the corresponding RNA, if the phenotypic

characteristics cannot be assessed directly.

Information required for the allergenicity assessment may require a separate report.

A detailed description of this point can be found in the specific section (Section 2.2)

below.

2.1.6. Compositional analysis

‘‘Key components’’ are relevant substances for food safety, which are inherently present

in the foods derived from the kind of animals under assessment. On one side, key nutrients

or anti-nutrients are components in a particular food that may have a substantial impact in

the overall diet. Nutrients may be major constituents (fats, proteins, carbohydrates) or

minor compounds (minerals, vitamins). Anti-nutrients are substances that impair nutrient

uptake, as digestive enzymes inhibitors. Toxicants are those food components that inhibit

or block important pathways in the human metabolism other than nutrient uptake; while

food allergens are proteins that have shown to induce allergic sensitisation and/or reactions

in certain individuals.

The levels of key components of the r-DNA animal should be measured, and reported

along with those of a conventional counterpart grown under equivalent (or as close as

possible) husbandry conditions. If available, the range of variation in key components for

the species and/or animal breeds involved in obtaining the animal ultimately used as food,

or for food production, will also be reported.

2.1.7. Health status

Food products derived from animals developed through conventional breeding are not

systematically subjected to rigorous and extensive food safety testing. Instead, food has

generally been regarded to be safe for human consumption when derived from animals

with an acceptable health status belonging to a species with a history of safe use. Health

status has proven to be a robust and broad indicator of safety, which is also applicable for r-

DNA animals.

Additionally, the evaluation of the health status of an r-DNA animal could provide an

additional insight about possible toxicity and bioactivity of the newly expressed

substances. Finally, health parameters can be considered as traits simultaneously

influenced by many genes, therefore they could constitute an additional assurance on the

absence of unknown negative changes in the metabolism leading to unintended effects.

In conclusion, a comparison of the health status of the r-DNA animal to the health status

of an appropriate conventional counterpart should be an important step towards ensuring

safety of food derived from r-DNA animals.

Data on this topic should comprise overall health and performance indicators (regarding

anatomy, behaviour, growth, development and reproductive aptitude), physiological


measures (including clinical parameters) and other species-specific indicators or

considerations, as appropriate.

2.2. Allergenicity assessment

Food allergies are abnormal immune responses to a food component, with adverse

effects that can escalate from a mild local irritation to lethal anaphylactic shock. Ordinarily,

food allergy is mediated by immunoglobulin class E (IgE) antibodies [67–68].

CODEX includes a list of the eight most common allergenic foods, accounting for over

90% of all allergic reactions on a worldwide basis: peanuts, soybeans, milk, eggs, fish,

crustaceans, wheat, and tree nuts. Nevertheless, no less than 160 foods are associated with

sporadic allergic reactions [69].

Any new protein that could be present in the food due to the genetic modification should

be assessed for its potential to cause allergic reactions. This should include the potential of

cross reactivity with IgE raised against the same or similar proteins by sensitized

individuals, as well as whether a protein which is completely new or is presented in a

different way to the food supply is likely to induce allergy in some individuals, leading to

an adverse reaction after subsequent dietary exposure to the same or a similar protein.

No single decisive factor is sufficient to rule out potential allergenicity. Therefore a

stepwise approach, relying upon the combination of various criteria, is used to ascertain the

likelihood of the protein being a food allergen. The first decision tree was presented by the

International Life Sciences Institute (ILSI) [70], and alternative schemes have been

developed afterwards along three different FAO/WHO expert consultations [62,71–73].

2.2.1. Source of the protein

If any, every report of oral, respiratory or contact allergy associated with the donor

organism should be considered; furthermore, data should be seek regarding type of allergy,

severity and frequency of allergic reactions. This information should be gathered even

before attempting to obtain an r-DNA animal, since the transfer of known allergens to r-

DNA organisms intended for food production must be avoided.

When the r-DNA animal is finally considered for a food safety assessment, this

information should be updated and used to further lead to the identification of specific tools

and relevant data for the allergenicity assessment, including sera for screening purposes

and biochemical information regarding known allergenic proteins from relevant gene

sources.

2.2.2. Amino acid sequence homology

A sequence homology comparison between the newly expressed protein and the best

available database of known allergens should be performed, in order to search for

similarities suggesting that the new protein is likely to cross-react with known allergens.

Examples of possible criteria for a positive result, using standard alignment tools like

FASTA or BLASTP, are more than 35% identity over a window of 80 amino acids using a

suitable gap penalty, or complete identity across a stretch of 6–8 contiguous amino acids.

Two Internet servers to facilitate this analysis, including allergen databases and ad hocalignment tools, have been recently launched [74–75].


2.2.3. Immunoassays

In vitro assays of specific binding to IgE class antibodies should be performed for those

proteins from a source known to be allergenic or displaying significant sequence homology

with a known allergen, as long as sera from individuals with a clinically validated allergy to

that source or allergen are available.

A negative result from in vitro immunoassays might be further confirmed by additional

testing. Current options in this regard are skin prick testing [76], or ex vivo protocols using

cells or tissue culture from allergic human subjects, as the basophil histamine release assay

[77].

2.2.4. Biochemical characteristics

Some biochemical and physicochemical characteristics have been frequently observed

in certain relevant food allergens; although none of them is a definitive indicator neither

constitutes a sine qua non condition for allergenicity.

Among the more important ones is digestive stability, i.e. the resistance of the protein to

degradation in gastric juices. Therefore, measures of the protein resistance to degradation

in the presence of pepsin under realistic gastric conditions [78–79] has become of

widespread application.

For similar reasons, data on stability to food processing, in particular to storage and heat,

is relevant. Labile proteins that are degraded by cooking or any processing needed before

consumption are less likely to become food allergens.

Another important consideration should be post-translational modifications, particu-

larly glycosylation. The degree of glycosylation may affect the susceptibility of the protein

to processing and digestive degradation; in addition, glycan epitopes can be highly cross-

reactive. For transgenes from distantly related organisms, different glycosylation pathways

could alter potential epitopes; hereby having the potential of turning non-allergenic

proteins, even those with a history of safe use, into prospective allergens.

Yet other characteristics (molecular size, repetitive substructures, ability to form

aggregates, rheomorphism, binding to ligands or to lipid membranes) have been suggested

to be potentially related to the allergenicity of particular proteins [80], hence they should be

reported. However, their relevance for new proteins, if any, should be carefully considered

in a case-by-case basis.

2.2.5. Other considerations

A history of safe use, if applicable, should be taken into account for those proteins from

a source not known to be allergenic. This would hold true as long as the protein expressed

in the r-DNA animal is equivalent in terms of sequence, structure and posttranslational

modifications; and if the expected consumption level and food processing are similar.

As the state of the art in this field evolves, other methods and tools may be applicable in

the near future to complement the assessment strategy [81]. Therefore, it is particularly

important to keep updated on the current state of the art in validated methodologies,

because allergenicity assessment is a rapidly evolving field.

For instance, targeted serum screening (TSS) has been proposed to substitute for

specific serum screening when the protein is not from an allergenic source nor it exhibits

sequence homology to a known allergen. TSS utilizes serum samples from individuals with


allergies whose specificity is broadly related to the gene source. For instance, if the protein is

derived from an invertebrate, it is proposed to test it against serum samples from patients with

different invertebrate-related allergies such as to mites, cockroach, shrimp, chironomids or

silk. For those cases when the source is not a species with a history of safe use as food, TSS

may actually become a standard reassure as long as such tests are available.

In addition, several research groups work on the development of different animal

models [82–85]. Although animal models could provide additional information on

potential immune reactivity of novel proteins, they still do not reflect many important

aspects of IgE-mediated food allergies in humans.

Other important avenues to be explored are the prediction of T-cell epitopes,

complementing current methods intended to predict IgE cross-reaction, and the

development of algorithms based on protein structure information to identify three-

dimensional motifs associated with allergens and potential non-linear epitopes.

Since the strategies depicted here were devised for newly expressed proteins of

unknown allergenicity, it was not intended to consider the modification of an animal whose

derived foods were allergenic prior to the modification. Therefore, it is not proposed for the

evaluation of potential unintended effects in the expression and presentation of previously

present allergens. Neither is it aimed to foods where gene products are down regulated for

hypoallergenic purposes. There is little development in specific methodologies for both

cases, but for the latter it is anticipated that skin prick testing and double-blind, placebo-

controlled food challenges would be required.

More details on the methodologies mentioned and proposals for their standardization

can be found elsewhere [72,86–87]. Guidance regarding the assessment of potential

allergenicity is practically the same for all r-DNA organisms. In fact, the annex for the

assessment of possible allergenicity of the current CODEX draft guideline for the

assessment of r-DNA animal food products [2] is identical to the analogous annex of the

guideline for r-DNA plant products [88], except for the elimination of references to gluten-

sensitive enteropathy, since this substance is exclusively found in some plants. Therefore,

the expertise gained in the future regarding foods derived from r-DNA plants or other

organisms will likely be also valuable.

2.3. Toxicity and bioactivity assessment

As it is the case for allergens, it should also be verified that genes involved in the

expression of toxins or anti-nutrients present in donor organisms, vectors or other

biological materials used are not transferred to r-DNA animals.

The traditional assessment of toxicological endpoints using test animals usually

involves substances whose chemistry is relatively simple, which are available in purified

form, that have no particular nutritional value, and human exposure to it is expected to be

low. Many toxic effects induced by chemicals exhibit a lower effect threshold, a No

Observed Adverse Effect Level (NOAEL) dose that can be determined in toxicity studies

performed in laboratory animals. The conclusion of many safety assessments of food

chemicals is the establishment of a level of lifetime Acceptable Daily Intake (ADI) that

should not cause an appreciable risk; ADI is usually derived from the NOAEL after the

application of a safety margin.


Nevertheless, this kind of studies cannot be readily adapted for testing the risks

associated with whole foods, which are complex mixtures of compounds, and often

characterized by a wide variation in composition and nutritional value. Due to their bulk,

effect on satiety and possible interference with the nutritional balance of the test animal, the

amounts of test foods that can be administered are usually limited. A key factor to consider

in conducting animal studies on whole foods is the nutritional value and balance of the diets

used, in order to avoid the induction of adverse effects that are not related to the genetic

modification.

Conventional toxicology studies may not be regarded necessary when the substance or a

closely related substance has been consumed safely in food, as long as function and

exposure are similar in both cases. In other situations, the use of appropriate conventional

toxicology or other studies on the new substances may be necessary, with the limitations

indicated above. Furthermore, the whole food may be tested for safety using feeding

studies when the composition of the food is altered substantially, or if the available data are

insufficient for a thorough safety assessment, or if there is a sound hypothesis on the

potential for a defined unintended effect. An animal feeding trial of 90 days is generally

regarded appropriate for these particular cases.

The toxicology and bioactivity safety assessment should take into account the chemical

nature and function of any newly expressed or up-regulated substances, the concentration

range in edible tissues and other derived food products of the r-DNA animal and the usual

dietary exposure to the conventional counterpart foods. Consideration should be given to

the possibility of differential risks to specific population subgroups such as infants,

pregnant women, the elderly and those with relevant diseases.

Regarding proteins, the assessment of potential toxicity should focus on amino acid

sequence similarity between the protein and known protein toxins, as well as stability to

heat or processing and to degradation in appropriate representative gastric and intestinal

model systems. Additional consideration should be given in cases where food derived from

the r-DNA animal is expected to be processed differently from the conventional

counterpart. For instance, less stringent food processing techniques may fail to deactivate,

degrade or eliminate some anti-nutrients or toxicants.

Appropriate oral toxicity studies may need to be carried out in cases where the protein is

not similar to proteins that have been consumed safely in food. The toxicity of novel

proteins is normally tested in laboratory animals, using appropriate dose-regimes and a

duration of 28 days.

The potential toxicity of a non-protein substance that has not been safely consumed in

food should be assessed under an appropriate case-by-case rationale, considering its

chemical nature, biological effects and expected dietary exposure. The type of studies to be

performed may include toxicokinetics, acute/sub-chronic/chronic toxicity and carcino-

genicity, and immunological, reproductive and developmental toxicity. Guidelines and

protocols for oral toxicity studies have been developed, for example the Guidelines for the

Testing of Chemicals of the Organisation for Economic Cooperation and Development

(OECD) [89]. The identification of hazards by methods of animal-based toxicology is fully

reviewed elsewhere [90].

In the case of newly expressed bioactive substances, r-DNA animals should be evaluated

for potential effects of those substances as part of the overall animal health evaluation.


Besides, consideration should be given to the potential dietary exposure where the

substance is likely to be bioactive following consumption.

2.4. Compositional assessment

The levels of key components (nutrients, anti-nutrients, toxicants and allergens) of the r-

DNA animal which are typical of the food should be compared with a conventional

counterpart; the latter grown under similar husbandry conditions. Relevant husbandry

conditions may include breed, age, sex, parity, lactation, or laying cycle.

The purpose of such comparison, in conjunction with an exposure assessment as

necessary, is to establish that substances that are nutritionally important or that can affect

the safety of the food have not been altered in a manner that would have an adverse impact

on human health. The statistical significance of any observed difference should be assessed

in the context of the range of natural variations for that parameter, in order to determine its

biological significance.

Although a particular component may be individually assessed as safe, intended or

unexpected alterations in the content of nutrients could affect the nutritional status of

individuals consuming the food.

2.5. Other considerations

2.5.1. Accumulation of xenobiotics or microorganisms

While reviewing the traits acquired by the r-DNA animal, it is important to analyze the

information with regard to traits that could represent an increased risk of zoonoses,

considering either the potential for new or increased colonization and/or shedding of

human pathogens or toxin-producing organisms. It is also advisable to examine the

possibility of alterations in the accumulation or distribution of xenobiotics (e.g. veterinary

drug residues, metals), which may be relevant for food safety.

2.5.2. Use of antibiotic resistance genes

Actual gene transfer from animals and their food products to microorganisms or

human cells in the gastrointestinal tract is highly unlikely [91–92], because of the

many complex and rare events that would need to occur consecutively. Such

events would include DNA escape from degradation during processing and food

digestion, then uptake and incorporation into the genome, then alteration of regulatory

sequences for appropriate expression, then positive selection and stable inheritance, and

so on.

Nevertheless, the hypothetical consequences of such a transfer, which are assessed

elsewhere [93–94], cannot be completely ruled out. The most raised concern is the transfer

of antibiotic resistance ‘‘marker’’ genes, which enable the selection of transformed cells in

most transformation methods, to pathogenic bacteria in the intestines of humans

consuming the food; thus compromising therapies for infectious diseases.

Therefore, in assessing the safety of animal foods harbouring antibiotic resistance

marker (selection) genes, it is important to consider if the antibiotic(s) involved have

clinical and veterinary relevance.


In particular for animal transgenesis, if the antibiotic is effective for selection purposes,

it should be highly toxic for animal cells; therefore its clinical or veterinary usefulness, if

any, would be limited in many cases.

Another issue related to the use of antibiotic resistance genes is the potential for the

relevant protein, when present in food, to interfere with the effectiveness of orally

administered antibiotics. Nevertheless, even if the protein is an antibiotic-degrading

enzyme, it may probably be degraded fast in the digestive tract, which is tested in the

context of the allergenicity assessment.

Alternative or complementary technologies have been considered [37]. On one hand,

reporter genes like those coding for Green Fluorescent Proteins have limited usefulness in

this field, and usually they are quite exotic genes whose protein products would still need

comprehensive allergenicity and toxicity studies. On the other hand, gene excision

technologies like the Cre-LoxP and Flp-FRT systems [95–96] demand the presence of an

additional recombinase gene. Again, these recombinases lack appropriate allergenicity and

toxicity studies to date, or they should be also eliminated from the animal intended for food

production. In addition, gene excision technologies are complex to use and not so readily

available.

2.5.3. Intended nutritional modification

Animals could be genetically engineered to improve the nutritional quality or

functionality of the foods derived from them, usually through increasing the content of a

nutrient or incorporating a new one. In such cases, it should be further analyzed how

nutritional intakes are likely to be modified by the introduction of the derived food products

into the food supply.

This may involve assessing different consumption scenarios against relevant historical

or recommended reference values, while considering the ranges of dietary exposure due to

geographical and cultural variations in food consumption.

Dietary exposure assessment should consider the concentration of the nutrient(s) in the

r-DNA animal derived food and any factors that could impact its final bioavailability, as well

as the usual consumption level of the conventional counterpart and/or other foods that are

likely to be displaced. Finally, exposure should be evaluated in the context of the total diet.

Guidance for the fortification of foods is available [97] to be considered when

applicable. However, in contrast with conventional fortification of food (where a single

chemical form of a nutrient can be added at an exact proportion), foods derived from r-

DNA organisms require characterization of the concentration ranges, the chemical forms

involved and their combined bioavailability.

In many cases, foods derived from this kind of r-DNA animals would exhibit a

composition significantly different from their conventional counterparts, at least

concerning the relevant nutrient. Therefore, the additional use of other comparators

(conventional whole foods, food components or fortified foods), whose composition is

more akin to the r-DNA animal derived food, will be useful for nutritional impact studies.

Animal feeding studies on the whole foods may be required in addition where the

modification has the potential to change the bioavailability of a nutrient.

In particular cases, foods that have been modified to benefit the general population or a

specific subgroup might pose a risk for a different subgroup. Thus, the assessment should


consider, in relation to the relevant nutrient(s), the particular physiologic and metabolic

characteristics and dietary needs of specific population groups, such as infants, pregnant

women, the elderly and those with relevant diseases.

Finally, most of this rationale would be also applicable in cases where the genetic

modification has been designed to provide health benefits. Potential examples are the

expression of a health promoting substance that is not regarded as a nutrient, or a reduction

in the level of an endogenous toxicant. Nevertheless, additional ad hoc studies or

considerations may be necessary for such cases.

2.5.4. Food storage and processing

The potential effects of food storage and processing, including home preparation,

should be carefully considered in the assessment of the aforementioned risks.

On one side, the implications of the expected range of food storage and processing

conditions should be analyzed. For example, heat may reduce the stability of a toxicant or

the bioavailability of a nutrient; however, cooking might not be included in all the realistic

processing scenarios for a given food.

On the other side, the genetic modification may be intended to change the customary

way the food is processed or its shelf life. Therefore, the impact of such changes in

previously existing toxicants, allergens, nutrients and other relevant substances should be

evaluated.

Finally, in particular cases the newly expressed substance may interact with key food

components, altering their storage and processing stability.

3. Future trends and conclusions

Both the technology for obtaining r-DNA animals and the safety assessment of foods

derived from them are rapidly evolving fields. The present article is intended to show a

preliminary view of the current status.

The search of new tools for assessing potential allergenicity will probably become a

highly active field. Suggested avenues in this regard are the development of animal models,

the creation of ad hoc international serum banks, the use of 3-D structural protein

information in similarity searches against allergen databases and the prediction of T-cell

epitopes using bioinformatics.

Another field that may become a target of immediate development is the search of

unintended effects by the comparison of relevant compounds in r-DNA animals and in the

conventional counterparts. For instance, non-targeted gene expression profiling analysis

using microarray technology [98], or chemical fingerprinting of the metabolome by gas

chromatography, high performance liquid chromatography, and/or mass spectrometry (in

particular MALDITOF) are promising technologies.

Also, there will be a need for incorporating animal information in peer reviewed

databases and consensus documents (inventories of food key constituents and critical

safety considerations for particular organisms), which at present are mostly available for

plant products [99–100]. In this way, the natural variation range of key components can be

used to determine the significance of observed differences in the comparative


compositional analysis. The OECD is undertaking a pioneer work of this kind on the

Atlantic salmon [100].

There are still gaps in the technical guidance for the food safety assessment of certain

products that may become relevant in the future, such as non-heritable applications or

hypoallergenic foods.

Although it is recognized that most of current guidance can be applied to non-heritable

applications/constructs, there is still a need of developing a complete framework for this

field due to the active development of future commercial products [38]. The harmonization

of statistical analysis and the standardization of transformation protocols would probably

become the challenges of this area. Excipient effects, although a novelty, would probably

be adequately handled by common sense and may not require specific guidance.

Conversely, even though the development of alternatives to antibiotic resistance marker

genes has been recommended, the actual need and safety of such technologies are still

uncertain.

In conclusion, the safety assessment of r-DNA animals should be carried out on a case-

by-case basis. It should focus on the characterization on the genetic modification, and also

compare the properties of the derived food with those of a conventional counterpart with a

history of safe use.

This rationale, as developed by CODEX, FAO/WHO, OECD, ILSI and other

organizations, has proven its validity on the assessment of other types of r-DNA organisms,

and still contributes to the construction of adequate safety assessment strategies.

Finally, it is important to stress that food safety assessment is an activity restricted to

food safety and nutritional issues. Therefore, it should not address a variety of other matters

related to the use of either conventional or r-DNA animals for human purposes, like animal

welfare, ethical, moral and socio-economical aspects, environmental biosafety or feed use.

In memoriam

This article is devoted to the memory of Carlos Camano, one of the leading Argentine

governmental experts in the field of food safety assessment of transgenic organisms, who

was also an outstanding delegate to Codex Alimentarius meetings on biotech products. He

passed away in 2006, but his colleagues will treasure his memory.

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Food Safety Assessment of Foods derived from Recombinant-DNA Animals; 2007.[3] Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos

by microinjection of purified DNA. Proc Natl Acad Sci USA 1980;77:7380–4.[4] James C. Global Status of Commercialized Biotech/GM Crops: 2006. International Service for the

Acquisition of Agri-biotech Applications (ISAAA) Briefs, 2006; 35.[5] Houdebine LM. Generation and use of genetically modified farm animals. Food and Nutrition Division,

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