Risk Assessment and Regulation of Molecular Farming

18 Pages·2013·0.26 MB·English

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Current Pharmaceutical Design, 2013, 19, 000-000 1

Risk Assessment and Regulation of Molecular Farming - A Comparison between
Europe and US
Penelope Sparrow1*, Inge Broer2, Elizabeth E. Hood3, Kellye Eversole4, Frank Hartung5 and Joachim Schie-
mann5
1John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK; 2University of Rostock, Justus von Liebig-Weg 8, D-18059
Rostock, Germany; 3Arkansas State University College of Agriculture, PO Box 639, State University, AR 72467, United States;
4Eversole Associates, 5207 Wyoming Road, Bethesda, Maryland, 20816, United States; 5Julius Kühn-Institut, Federal Research Cen-
tre for Cultivated Plants (JKI), Institute for Biosafety in Plant Biotechnology, Erwin-Baur-Str. 27, D-06484 Quedlinburg, Germany
Abstract: In this article, the general principles of genetically modified (GM) plant risk assessment and the regulatory framework for con-
tained use and open field production of plant-made pharmaceuticals/plant-made industrials (PMP/PMI) are described. While significant
progress has been made for the containment grown (plant cell culture) production of PMPs, with the first regulatory approval made by the
FDA in 2012, the commercialization of medicinal or industrial products produced in the field has yet to emerge in either Europe or the
US. In the current paper, we discribe the regulatory environment in Europe and the US surrounding GM crops, and provide case studies
for experimental field releases of PMP and PMI producing plants in both regions. Suggestions for reducing the regulatory burden for GM
plants will be discussed, also in light of the emerging new technologies to modify the genetics of plants. Since regulations surrounding
the commercialization of GM crops are very costly and not appropriate for most of the PMP/PMI applications in Europe, we propose that
amendments to the EU Directive 2001/18/EC are necessary to allow for the commercialization of products from GM plants without the
need of an ‘authorization’. To fully acknowledge the overall outcome of adopting plants to produce PMP/PMI, the conclusion is that
broader and more balanced legislative oversight is needed in Europe; while specific legislation is not needed in the US.
Keywords: Plant molecular farming (PMF), plant made pharmaceuticals (PMP), plant made industrials (PMI), genetically modified plants,
GM plant regulation, GM plant risk assessment.
INTRODUCTION move ahead to the field or production arena. As with all GM plants,
Plant genetic modification (GM), developed over the past those intended for molecular farming must go through a thorough
twenty five-years, has been applied to input traits, to improve plant risk assessment before they can be used in the field. In that respect,
whereas existing guidance on the environmental risk assessment of
production in the form of herbicide and pest resistant crops, as well
as to output traits in the form of nutritional improvement (such as GM plants for food/feed uses is considered adequate to also cover
Golden Rice), industrial proteins (e.g. cellulase, and trypsin) and GM plants for non-food/feed uses [3], PMF raises novel questions
that might trigger a need for specific biosafety considerations due to
pharmaceuticals and vaccines (Plant Molecular Farming - PMF).
The benefits of input and output traits are now extensively docu- the nature of the proteins to be expressed.
mented, yet the full exploitation of this technology remains hin- Commercialization has many hurdles including not only the
dered by the long lead times associated with such emerging R&D science and associated intellectual property to protect the inven-
technology combined with the timescales involved in introducing tions, but also market realities and government regulations. As each
these new traits into local adapted varieties. One of the biggest GM plant moves from the laboratory to scaled-up greenhouse pro-
barriers to the adoption of this technology into the commercial sec- duction or to the field, issues emerge that trigger additional over-
tor is the intense scrutiny of these new plant varieties by regulatory sight for environmental release or ultimate use by humans. In this
agencies and the public, and the costs associated with regulatory review, we outline the current state of play regarding the risk as-
approval. The average cost for having GMOs approved in Europe sessment and regulations surrounding GM plants and how the cur-
has been estimated at (cid:1)7-10 million Euros per event [1]. These rent regulatory systems are adapting to accommodate PMF.
costs mainly accrue from the large number of studies which the
applicant companies have to present to the European Food Safety 2. GENERAL PRINCIPLES OF GM PLANT RISK ASSESS-
Authority (EFSA). The 30 approvals (including for imports) having MENT
been granted by April 2011 represent total costs to companies of 2.1. Interplay of Risk Assessment, Risk Management and Risk
between (cid:1)210 and 300 million [2]. Communication
PMF consists of using plants as production platforms for the In different countries across the world (termed hereafter as “ju-
synthesis of compounds for pharmaceutical or industrial purposes. risdictions”), GM plants and their derived food and feed products
PMF has been presented as a convenient way to produce molecules are subject to a risk analysis before they can be used for experimen-
of interest on a large scale at low costs. Other benefits associated tal or commercial uses. The case-by-case risk analysis encompasses
with the use of plants are; rapid scaling up, convenient storage of all components of risk, and consists of assessment, management,
raw material (in the form of seed) and less concern over human and risk communication. In risk assessment, potential adverse im-
pathogen contamination. However, in order for any impact to be pacts associated with a specific activity are scientifically character-
realized from this research, these laboratory experiments must ized on a case-by-case basis. Risks are identified from plausible sets
of circumstances that may result in harm to human and animal
health or the environment, and subsequently the level of risk is
*Address correspondence to this author at the John Innes Centre, Norwich
Research Park, Norwich, NR4 7UH, UK; Tel: [phone]; estimated on the basis of the seriousness and chance of harm. Risk
Fax: [phone]; E-mail: [email protected] management evaluates, selects, and implements plans or actions to

1381-6128/13 $58.00+.00 © 2013 Bentham Science Publishers
2 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 Sparrow et al.
ensure risks are appropriately managed. Thereby, policy alterna- with the abiotic environment; and (13) the scientific quality of the
tives to accept, minimize, or reduce the characterized risks are proposed post-market (environmental) monitoring plan [8, 9, 10].
weighed and, if needed, appropriate prevention and control options Moreover, an assessment of direct and indirect, as well as immedi-
are selected. Because risk managers and regulators rely on risk ate and delayed, including cumulative long-term effects is required
assessments to make an informed decision on whether or not to in several jurisdictions. Now it could be argued that a lot of the
approve a certain use of a GM plant, it should explain clearly what criteria set out above, would not be applicable to PMF as these
assumptions have been made during the risk assessment, and what crops are not designed to enter the food chain. However, if they are
is the nature and magnitude of scientific uncertainties associated engineered into a food crop then these assessments will be neces-
with the characterized risks. The decision whether a certain risk is sary, as part of the precautionary approach – i.e. what if they did
acceptable and/or tolerable under a particular set of conditions is become mixed in the food chain. EFSA have produced guidance
not part of the risk assessment itself, but part of the wider risk notes on the Risk Assessment for GM crops for non-food/feed uses
analysis, as this choice is not only based on scientific criteria, but to address some of these differences [3].
also involves political, social, cultural and economic considerations
Even though there are considerable differences in regulatory
- for example in the case of PMF it will be critical that these crops requirements for GM plants and derived products, priorities in
should not become mingled with commodity crops. While this is terms of protection goals, as well as risk terminology among juris-
also the case for agricultural GM crops, the perceived public back-
dictions globally, most risk assessments: (1) follow sequential
lash is likely to be greater for a pharmaceutical crop. Risk manage- steps; (2) are science-based, where quantitative information is
ment is also functionally and temporally separate from risk assess- available, and use qualitative information in the form of expert
ment in order to reduce any conflict of interest and to protect the
judgment; (3) use a comparative approach whereby the level of risk
scientific integrity of risk assessment [4]. Risk communication is is estimated through comparison with a non-GM counterpart; (4)
defined as an interactive exchange of information and opinions on are case-specific; (5) are iterative and, in a transparent manner,
risk throughout risk analysis, between risk assessors, risk managers
examine previous conclusions in light of new information; and (6)
and other interested parties. It includes the explanation of risk as- follow a tiered approach. For a more detailed account of GM risk
sessment findings and of the basis upon which risk management assessment we refer you to [11].
decisions are made [5].
Harm refers to adverse effects to the human and animal health 3. NEW BREEDING TECHNIQUES IN MOLECULAR
or the environment, while risk is recognized as a function of the FARMING AND IMPLICATIONS FOR THE CURRENT EU
probability of an adverse effect occurring to human and animal REGULATORY SYSTEM
health or the environment and its seriousness, following exposure to Several new breeding techniques (NBT) are currently under
a hazard due to an activity i.e. the possible intended use(s) of a GM way which differ substantially from the transgenic (GM) techniques
plant. Without hazard (i.e., the potential of an agent or situation to that have been used in the last two decades. For some of them the
cause harm), there is no harm and thus no risk. It is therefore crucial regulatory framework in the EU may no longer be feasible, result-
to distinguish risk from hazard in risk assessment, as both terms ing in a legislative/regulatory uncertainty concerning these tech-
have different meanings. Various studies, assessing the impact of niques. In 2007, due to a mandate of the EU commission a new
GM plants, have postulated dire risks when all they have done is techniques working group (NTWG) was established in which ex-
characterized either a hazard associated with the use of GM plants, perts from competent authorities of several European countries
or an exposure to the GM plant without demonstrating whether this evaluated the following eight NBTs: Oligo-directed mutagenesis
exposure is hazardous (reviewed by [4]). Such studies rarely link (ODM); Zinc finger nuclease technology (ZFN); Cisgene-
hazard to exposure, and in effect confuse hazard or exposure with sis/intragenesis; RNA-dependent DNA methylation (RdDM); Graft-
risk. ing (on GM rootstock); Reverse breeding; Agro-infiltration; and
Synthetic genomics. In 2012, the working group finished the
2.2. Risk Assessment Principles and Concepts evaluation and circulated its opinion for comments. In 2010, a
Regulatory requirements involve the consideration of a range of working group led by the Joint Research Centre of the EU commis-
issues (areas of risk) relevant to the overall risk assessment, in order sion started to develop a study concerning the same eight tech-
to determine the impact of the GM plant on human and animal niques. The final report of this study was published last year [12].
health or the environment relative to the comparable non-GM crop, In 2011 on request of DG SANCO, the EFSA also set up a working
and thus its relative safety [6, 7, 8, 9]. These include amongst others group to determine whether the existing guidance on risk assess-
(and differently required in different jurisdictions): (1) molecular ment is appropriate concerning NBTs. The EFSA working group
characterization of the GM plant, which provides information on will be providing its opinion one by one, starting with cisgenesis
the structure and expression of the insert(s) and on the stability of and intragenesis [13].
the intended trait(s); (2) the comparative analysis of compositional,
Cisgenesis/intragenesis technique refers to the integration of
phenotypic and agronomic characteristics to identify intended and genes into a plant genome: a transgenic (GM) approach, but re-
unintended changes in the GM plant; (3) the toxicological assess- stricting the source of the genes to sexually compatible crossing
ment of the genetic modification, which addresses the impact on
partners of the recipient plant. By this approach only cisgenesis
human and animal health of biologically relevant change(s) in the produces progeny which can arise by chance by a conventional
GM plant and/or derived food and feed resulting from the genetic breeding process (CBP), whereas intragenesis always produces
modification; (4) the assessment of the allergenic potential of the
progeny which cannot be achieved by CBP. As the process itself
novel protein(s) (in the case of PMF, this assesment would deter- introduces foreign DNA which is capable of replication and inherit-
mine whether approval would be granted for a field release); (5) the able, the technique might be considered to be regulated by GMO
nutritional assessment, which aims to demonstrate that the food and
legislation. If a cisgene without a selection marker or other foreign
feed derived from a GM plant is not nutritionally disadvantageous DNA-sequences is transferred using nuclease technique (see ZFN-
to humans and/or animals; (6) potential changes in the persistence 3), the resulting plant is nearly indistinguishable from the one de-
(weediness) and invasiveness ability of the GM plant; (7) potential
rived through a CBP and meets the criteria of self-cloning. In this
for gene transfer; (8) interactions between the GM plant and target case, which is described in Annex II, part A of Directive
organisms; (9) interactions between the GM plant and non-target 2009/41/EC it might be falling outside the scope of the GMO legis-
organisms; (10) potential adverse effects on biogeochemical proc-
lation.
esses; (11) impacts of altered farm management practices associ-
ated with the cultivation of the GM plant; (12) potential interactions
Risk Assessment and Regulation of Molecular Current Pharmaceutical Design, 2013, Vol. 19, No. 00 3
Oligo-directed mutagenesis is a technique using short oli- integration by homologous recombination, long DNA sequences
gonucleotides as a tool to induce targeted alterations in the genome (500-1000 nt) with homology nearby the DSB site have to be pre-
ranging from one to a few adjacent nucleotides. The alterations are sent on the targeting construct.
caused by mispairing of nucleotides and the subsequent DNA repair
Two other nuclease techniques have been developed to achieve
by the host cell. The ODM technique can be used, e.g., for induc- the same task by slightly differing means, meganucleases (MN) and
tion of herbicide resistance in tobacco due to point mutations [14]. transcription activator like effector nucleases (TALENs) [17, 18].
However, because the ODM technique does not introduce recombi-
In general all three types of site directed nucleases (SDN) perform
nant DNA into the plant which is capable of replication, it is dis- two functions: i) specific DNA recognition and binding and ii) in-
cussed by the NTWG to be not regulated by the GMO legislation or troduction of a DSB in or nearby the bound sequence. This DSB
to be excluded from Directive 2001/18/EC (Annex IA, Part 1).
greatly enhances the chance for homologous recombination and
The aim of RNA-dependent DNA methylation is the induc- thereby offers the opportunity to integrate any DNA sequence spe-
tion of gene silencing via promoter methylation of target genes. cifically at a targeted locus instead of elsewhere in the genome [19,
Depending on the delivery method of the DNA which encodes for 20]. Therefore, SDN technique allows choosing or designing a ge-
the silencing RNAs, the expression can be transient thus only in- nomic locus which possesses the best performance for stable and
ducing epigenetic changes of the genome by the RNA silencing high expression of, e.g., PMP genes.
pathway. If the DNA coding for the silencing RNA is stably inte- The nuclease techniques described under ZFN-1 and -2 used for
grated into a plant genome it is regulated by current GMO legisla-
gene alteration with only point mutations or a few nucleotide
tion. If only transient expression occurs, it should not be in the
changes, might be considered to be not regulated by GMO legisla-
scope of the GMO legislation because methylation of nucleotides is tion as far as no foreign DNA is integrated in the plant cell. The
not considered to be an alteration of genomic material.
outcome of ZFN-1 and -2 is indistinguishable from natural muta-
Grafting with GM scions or rootstocks can be used for the tions. ZFN-3 technique used for gene integration is covered by the
transient expression of proteins or other effectors like RNAs to existing guidance for risk assessment and GMO legislation as it
induce, e.g., enhanced pathogen resistance. This technique can be does not differ in its mode of action from the integration of genes
performed using a non-GM scion onto a GM rootstock or vice by transgenesis. However, nuclease technique in general greatly
versa. In both cases the resulting plant is a chimeric plant which enhances the accuracy of gene integration in a preselected target
falls within the scope of the GMO legislation. The fruits, seeds or locus, thereby minimizing unattended side effects which might
offspring of a GM scion are also regulated by GMO legislation, result in an easier risk assessment.
whereas the fruits, seeds or offspring derived from a non-GM scion
The term Agro-infiltration (or Agro-inoculation) means that a
grafted onto a GM rootstock might not fall under the scope of Di- plant or plant tissue is infiltrated with a liquid Agrobacterium sp.
rective 2001/18/EC. suspension. The Agrobacterium delivers T-DNA from which ge-
The Reverse breeding technique is able to reconstitute the netic material is expressed locally and transiently at high levels in
parental lines starting with an elite F1 hybrid whose genetic mate- the plant to produce a specific protein. Agro-infiltration has been
rial is unknown [15, 16]. Reverse breeding combines several other used in research for, e.g., the transient expression of resistance
techniques, i) RNAi to suppress meiotic recombination; ii) tissue genes from wild grape in susceptible grapevine cultivars or for the
culture to regenerate plants from cells; and iii) the double haploidi- expression of artificial micro RNAs conferring resistance to potato
zation technique to create double haploid plants which are used as virus X and Y [21]. Concerning the production of pharmaceutical
the respective parental lines to produce new elite F1 hybrids. Dur- proteins in plants, e.g., Artemisinin precursors as well as IgG or
ing this procedure a transgenic step can be included but the final IgM antibodies have been produced in Nicotiana benthamiana re-
plant lines are non-transgenic and therefore might be considered as sulting in yields of 40 to about 600 mg/kg of fresh weight [22-25].
falling outside the scope of the GMO legislation. As the technique described does not aim for progeny of the
Synthetic genomics aims to synthesize large DNA molecules inoculated plant tissue, the only GMO involved in the process is the
followed by transformation in a recipient structure. As the applica- Agrobacterium. Therefore, Agro-infiltration is covered by the cur-
tion of this technique to genetically modify plants is in a very early rently existing legislation and risk assessment as GMM in plants.
stage, possible future implications have been evaluated. Only if the Usually the plants or the plant tissue are destroyed after the experi-
synthesized DNA is assembled to result in an entity which is a mi- ment. Progeny derived from this plant material in which absence of
croorganism will it fall under the scope of Directives 2009/41/EC stably integrated DNA is proven might not be considered a GMO
(Annex I Part A) and 2001/18/EC (Annex 1A Part 1). and therefore might not fall under the scope of the GMO legisla-
Several of the NBTs are already in use or planned to be used for tion. Agro-infiltration approaches for PMP production, of contain-
ment grown plants, are beginning to see investment. Two compa-
general plant breeding in the near future. The following two NBTs
especially, may have great benefits for molecular farming purposes: nies leading in this area are Kentucky Bioprocessing
Zinc finger nuclease technology and Agro-infiltration. (www.kbpllc.com/) and Medicago (www.medicago.com/), the later
now being in scale up for phase II clinical trials of their avian flu
Zinc finger nuclease technique was the original term describ-
vaccine, produced using transient expression systems.
ing a new technique which uses an unspecific nuclease (usually
FokI) coupled to a sequence specific DNA binding element, the 4. CONDITIONS FOR MOLECULAR FARMING IN
zinc finger. The restriction activity is directed to a certain locus in EUROPE
the genome of an organism by the zinc finger binding activity. Af-
4.1. Regulatory Framework for Contained and Open Field use
ter induction of a double strand break (DSB), the repair is done
of GM Plants
completely by the host cell. The working group of new techniques
discriminated between three different ways how ZFN can be used. The European Union (EU) is considered to have the toughest
ZFN-1: Without any additional DNA homology, the DSB will be and most stringent legislation on GM products and commodities in
repaired in almost all cases by the non-homologous end joining the world. The enforcement of such legislation has three main ob-
pathway of the cell. This will result in point mutations or small jectives: to protect human and animal health and the environment;
InDels at the DSB site. ZFN-2: Adding short DNA sequences to guarantee consumers’ freedom of choice without misleading
which are differing only in a few nucleotides to the genomic se- consumers/users; and to create an internal market allowing the free
quence around the DSB can result in a repair which copies these movement of safe and healthy GM products within the EU, thus
differences in the genomic sequence. ZFN-3: To enable targeted
4 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 Sparrow et al.
avoiding unequal competition and trade impediments between and - All waste material generated in containment is steam
within EU Member States. treated/autoclaved or incinerated (soil, plant debris, pots and
The EU regulatory approach is precautionary, process-related, disposable protective clothing like gloves, hair nets, etc.);
and includes mandatory labelling and traceability requirements for - Personnel wear protective clothing and hand washing facilities
food and feed products derived from GM plants, unprocessed or are used where appropriate, again to minimize or prevent pol-
processed. Only non-food GM products (unseeded), such as textile len transfer and insect movement into and out of the facility;
or other industrial products are not subject to any requirements for - Live tissue leaving the premises (e.g. leaf tissue, or seeds) is
labelling.
moved in double contained vessels, with an appropriate risk
EU legislation is adopted through a system of interactions be- assessment, to ensure that no unintended release into the envi-
tween the three main EU institutions: the European Parliament, the ronment occurs.
Council of the European Union (i.e., representatives of all the EU It should be noted that these plants in general will not pose any
Member States at the ministerial level), and the European Commis- health or environmental risks, but EU legislation follows a precau-
sion. In most cases, the European Commission initiates legislative
tionary principle and all ‘what if’ scenarios need to be addressed.
proposals that are decided jointly by the Council and the European Implementation of these regulations is very straightforward, and
Parliament; the most common legislative measures are Regulations indeed have become common practice for all EU universi-
(acts that are binding in their entirety and are immediately applica-
ties/institutes and organizations. For PMPs and PMIs these meas-
ble throughout the EU) and Directives (acts that require the modifi- urements would also ensure that plants are as clean as technically
cation or establishment of national measures, generally for har- possible (for cultivation in a non-sterile environment) before they
monization purposes).
can be processed prior to entering cGMP facilities (see Fischer et
In the early 1990s, two European Directives for the use of al., this issue).
GMOs were adopted: Directive 90/219/EEC (which has since been
When GM plants are used to produce therapeutic or industrial
amended by Directive 98/81/EEC, and more recently 2009/41/EC), proteins in plant suspension cell systems, containment in enclosed
which regulated the contained use of GM micro-organisms; and
sealed bioreactors would in some cases simplify the risk assessment
Directive 90/220/EEC (which has since been replaced by Directive
and biosafety issues. These types of culture systems are likely to
2001/18/EC) which regulated the deliberate release of GMOs into also be more familiar to the current pharmaceutical industry (cf.,
the environment, covering both the release for research purposes
use of mammalian and bacterial cell culture systems). In 2006, the
(part B) and for commercial use as or in products (part C). This
first PMP, a vaccine for poultry (against Newcastle disease) gained
triad reflects the stepwise process GM plants go through, beginning regulatory approval from the US Department of Agriculture. Al-
with experiments under contained use (e.g., laboratory, green-
though this poultry vaccine remained a proof-of-concept and was
house), through experimental release, up to the placing on the mar-
never sold, it paved the way for future PMF. Indeed, the first plant-
ket. According to the step-by-step principle, the containment of made biologic, for human use, to gain [US] regulatory approval was
GMOs can be reduced and the scale of release increased gradually,
produced in a carrot cell system (for the production of Elelyso, an
if assessment of earlier steps indicated that the next step can be
enzyme to treat type 1 Gaucher’s disease) by the Israel-based Pro-
taken. Thus broadly speaking there are two categories of authoriza- talix BioTherapeutics, which gained FDA regulatory approval in
tion: (1) for the contained use (i.e., those grown in laboratories,
May 2012 (http://www.protalix.com/product-
glasshouses, or fully contained facilities such as cell culture sys-
development/elelyso.asp).
tems); and (2) for the ‘deliberate release into the environment’,
Meanwhile, in Europe an EU-funded project that ran from
either for experimental purposes or for commercial cultivation. If
2004-2011 (www.pharma-planta.net) saw the first regulatory ap-
the GMO is intended for food and/or feed uses, its potential uses are
proval for a PMF product to enter phase 1 clinical trial in humans.
regulated under the scope of GM food and feed Regulation (EC)
The product was an anti-HIV monoclonal antibody; using whole
1829/2003.
tobacco plants grown in containment as the platform. The choice of
When the end product of a molecular farming activity is a host plant and growing environment was somewhat determined as
pharmaceutical, the safety or toxicological aspects of the product
the EU program developed, in order to meet the least regulatory
and its uses fall under Regulation (EC) 726/2004 on medicinal resistance, and is further discussed later. Although plants have been
products. shown to be potentially useful for the production of therapeutic
4.1.1. Contained use of GM Plants proteins, they are at a relatively early stage of development and
more needs to be done so that they can be used to produce proteins
Within Europe the contained use of all GMOs (which includes
in a consistent manner. Another EU funded project,
GM bacterial, mammalian and plant cultures) as previously men-
tioned, is covered by Directive 2009/41/EC. Contained use covers (www.comofarm.org), was established to address these issues, and
to look at a range of containment platforms (hydroponic plants, root
the initial production of a GMO in the laboratory, as well as the use,
culture, cultivated moss and cell suspension culture) across a range
storage, transport and destruction of the GMO. Any organization
wanting to produce or handle GMOs must notify the relevant com- of plant species. Ultimately, the results from the CoMoFarm (con-
tained molecular farming) project, which concludes at the end of
petent authorities of their intention to use their premises for con-
2012, aims to help reduce the costs involved in the production of
tained use activities for the first time. It then becomes the responsi-
bility of the organization to ensure that the appropriate risk assess- pharmaceutical and industrial proteins and ensure that pharmaceuti-
cals from plants are produced to the highest possible standards.
ments are in place, and the appropriate level of containment is se-
lected to minimize any potential risks to negligible or low. In the However, to fully exploit the claims of using plants as produc-
case of growing a GM plant in glass house containment, for exam- tion vehicles for cheap production of therapeutic and industrial
ple, measures would be put in place: proteins (especially the lower value proteins) it may be desirable for
- To reduce the likelihood of, or prevent insects or rodents from plants to be cultivated on a larger scale than containment-grown
approaches. For example, it has been estimated that one acre of
entering the glasshouse - thereby minimizing any potential
transfer of material, i.e., pollen or seed out of the glasshouse safflower engineered to produce insulin by the Canadian company
and reducing any potential unintended effects resulting in ex- SemBioSys, could produce more than one kilogram of insulin,
enough to treat 2,500 diabetic patients for one year. That means just
posure of these groups to the plants;
16,000 acres of safflower could meet the world’s total demand each
year.
Risk Assessment and Regulation of Molecular Current Pharmaceutical Design, 2013, Vol. 19, No. 00 5
4.1.2. Open field use of GM Plants tion information format). The SNIF document is then made public,
Growing GM plants in the field for experimental purposes con- which in turn supports the transparency (openness) of the activities
stitutes a ‘deliberate environmental release’ and as such requires proposed – although this openness of precise field locations may
then render field trials vulnerable to abuse by potential protestors.
prior notification under the Directive 2001/18/EC to the local com-
petent authority of the EU Member State in which the proposed Information about all experimental releases authorized by the EU
field release was intended. Such notifications must include details member states is available to all and can be accessed at the follow-
ing URL: http://gmoinfo.jrc.ec.europa.eu/. According to Article 6.5
about the host plant, the introduced trait, the purpose of the release
and details of the exact location and size of the trial, an environ- of Directive 2001/18/EC, the local (national and/or regional)
mental risk assessment and details of the risk management strate- authority must respond to the notification within 90 days by ap-
proving or rejecting it, if no additional information is requested
gies [26]. Once the notification has been sent to the competent na-
tional authority it is passed on to a scientific advisory committee, from the notifier. Field trials are then subject to [annual] inspection
who will look at the risk assessments and potential environmental throughout the life time of the trial by the GM Inspectorate; and for
a set period of time post trial to ensure compliance.
impacts and assess if the trial would pose a threat to health or the
environment. Although the regulations cover the whole of the EU, Currently many EU countries have a negative position on GM
the power to approve or reject a notification rests solely with the plants and do not allow experimental field trials in their territory.
national competent authority, in some cases with additional input Other countries are more open to the evaluation of this technology
from national scientific advisory bodies, other member states and and while they allow field trials they impose strict compliance
the European Commission. It is also mandatory for the notifier to measures to avoid potential risks to the environment or human and
advertise the proposed release (usually in a national paper) within animal health. Usually, specific conditions are imposed under
10 days of submission, and for the national authority to provide the which the field trial may go ahead, and these include: isolation
European Commission with a summary communicating the most distances and buffer strips to minimize outcrossing with the con-
important information in the application (SNIF, summary notifica- ventional counterpart or hybridization with sexually cross-
Table 1. Overview of field trials in Europe (1995-2012) with GM plants used as production platforms for pharmaceutical or in-
dustrial products
Crop Trait / Molecule of Interest No. of Trials
Barley Thaumatin (sweetner) 1
Barley Human serum albium 1
Barley Growth factor 1
Flax Biodegradable plastic 1
Maize Rabies virus G glycoprotein 1
Maize Dog gastric lipase 6
Maize Human collagen 2
Maize Human lactoferrin 2
Maize Human albumin 1
Maize Antibodies 1
Rape Dog gastric lipase 2
Rice human enzyme acid (cid:1)-glucosidase (GCase) 1
Pea Alpha-amylase 1
Feed pea Alpha-amylase 1
Potato Spider silk 1
Potato Non-plant carbohydrates 1
Potato Spider silk-elastin fusion protein 1
Potato Expression of Cyanophycin synthetase 5
Potato Expression of the viral coat protein VP60 and choleratoxin B 6
Tobacco Human alpha-1 anti-trypsin 1
Tobacco Dog gastric lipase 7
Tobacco Rabies virus G glycoprotein 1
Tobacco Technical enzymes 1
Tobacco Human collagen 3
Tobacco Human glucocerebrosidase protein 1
Tobacco Taxane diterpenoid 1
(Source: Adapted from Breyer et al. (2009). Joint Research Centre of the European Commission, 2012: http://gmoinfo.jrc.it/)
6 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 Sparrow et al.
compatible wild relatives; confinement measures to avoid the oc- rounding master and working cell banks, compliance with GMP
currence of volunteer plants in subsequent years originating from and particularly issues regarding batch-to-batch consistency, stan-
spilled seeds and/or vegetative plant parts during the trial and mix- dard operating procedures for different production systems and
ing of plant material in machinery during sowing, harvest and/or downstream processing requirements need to be addressed. The
post-harvest operations [27]; post-release follow-up to control po- European Medicines Agency (EMEA) that oversees the assessment
tential volunteer plants occurring in subsequent years; and crop of biopharmaceuticals and vaccines, published guidance notes in
destruction. Plant material from these trials cannot enter the food 2006 on the ‘quality of biological active substances produced by
and feed chain and has to be destroyed at the end of the trial [28, stable transgene expression in higher plants’ [34], which looks at
29, 30]. such issues. As with all emerging technologies, it will be the early
and first products to enter the approval/regulatory systems that will
In the EU, 51 field trials with GM plants serving as production
platform for pharmaceutical or industrial products have been noti- ultimately help shape and evolve the regulation surrounding PMPs.
fied since 1995 (see Table 1). However, Europe has yet to witness When considering host plants in the context of biosafety and
the commercial application of PMP technology, although some risk assessment, the better scientific option (e.g., plants with the
pharmaceutical proteins (gastric lipase, lactoferrin) have entered highest yield, best protein stability, and better downstream process-
phase I and II clinical trials [31, 32]. ing ability) may not be the best choice of host from a biosafety and
regulatory perspective.
4.2. Regulatory Requirements for PMP in Europe
The choice of host crop falls into three main categories: non-
There is currently no ‘natural’ home for the start to finish regu- food crops, food crops and non-cultivated crops (reviewed in [35].
lation of PMP/PMIs. Their regulation in Europe therefore falls to a Desirable crops from a biosafety perspective could include male
number of regulatory bodies (Table 2). PMPs will need to adhere to sterile plants (such as male sterile lines of potato), crops with no
the regulations that cover the contained use of GMOs wild relatives within the growing region (such as maize, tobacco or
(2009/41/EC), for the early laboratory R&D phase, as well as cov- potato), or plants with heavy pollen (such as maize, barley and rice,
ering GM material used in bioreactors and the growth of whole again in countries with no wild relatives). A wealth of knowledge
plants for PMF in contained facilities such as glass-houses. For surrounds the cultivation of food crops and transformation proce-
some PMP products, where a therapeutic protein is considered dures are well defined for a number of major crop species. How-
‘high risk’ because it could result in adverse effects if inadvertently ever, the regulators may view such crops as less desirable because
eaten, regulatory approval would only be granted if crops were they represent major food crops, and therefore the risk of PMPs
grown under containment conditions. PMP producing plants grown entering the food chain (even if only a perceived risk) would be
under containment conditions are likely to face fewer regulatory higher than it would be when using non-food crops such as tobacco
hurdles than field-grown PMP producing plants because the risk to or non-cultivated crops, such as duckweed, that could be grown in
the environment is lower. containment. Nevertheless, the lack of toxic ingredients is an im-
Regulatory oversight for PMF in the field is covered by Direc- portant advantage of food crops which should be taken into ac-
tive 2001/18/EC, which regulates the deliberate release into the count.
environment of GMOs. Part B of this Directive regulates the ex- The choice of expression site within the plant can also have
perimental field release (for which Europe has seen a number of biosafety implications. Targeting expression to the seed reduces the
notifications for PMF experimental release (see Table 1), although potential exposure of herbivores and other non-target organisms to
far behind the numbers for US). Part C of this Directive would pharmaceutical products expressed in the leaves as well as leaching
regulate the import, cultivation, processing and marketing for of recombinant proteins into the environment from roots. However,
commercial purposes. The EFSA, who oversees the assessment of one advantage of harvesting leaf tissue, rather than seeds or fruit, is
GM plants to be deliberately released into the environment, pub- that the plants do not need to flower, thereby reducing the risk of
lished guidance notes in 2009 on the risk assessment of genetically pollen and seed dispersal.
modified plants used for non-food or non-feed purposes [3] includ-
Some plants have already been recommended as not suitable for
ing GM plants as production platform for PMI/PMP.
field production by regulatory bodies such as the US Animal and
Europe has yet to see any Part C application for a commercial Plant Health Inspectorate (APHIS) and the Canadian regulatory
scale field release of a GM crop for PMF. Indeed, Europe in general authorities, these included oilseed rape (canola) and alfalfa. These
falls far behind the rest of the world in approvals of GM crops for plants are considered to be unsuitable because they are bee-
cultivation, with only two products being approved in the last 14 pollinated, sexually compatible with local weed species and have
years for cultivation (insect resistant maize (MON810) and a modi- multiple year seed dormancy. These traits therefore increase the
fied starch potato (Amflora)). The maize MON810, and derivatives risk of transgene spread and volunteer plants in subsequent crops.
thereof (i.e., subsequent introduction of the insect resistant trait into However, such crops may be considered as beneficial hosts if
maize breeding programs) are the only GM crops currently being grown in containment.
cultivated in Europe. However, across Europe the political view
point of cultivating GM crops is far from harmonious, with a num- 4.3. Case Studies for Experimental Field Releases of PMP and
ber of Member States banning such cultivation PMI Producing Plants
(www.greenbiotech.org). Given the state of play surrounding the In October 2008, the University of Rostock applied for the
cultivation of agricultural GM crops, it is unlikely that we will see a permission to release potatoes each carrying one of three different
pharma crop grown commercially in Europe any time soon.
transgenes in a field trial planned from 2009 to 2011 at two sites
When the plant cells/plants are harvested they then need to (Thulendorf in Mecklenburg Pomerania and Üplingen in Saxony
enter downstream processing in order to isolate the active sub- Anhalt). For each transgene, different events in different cultivars
stance, which can then enter cGMP facilities (discussed in detail in should be released in order to identify effects caused by somaclonal
Schillberg et al., this issue). It is under cGMP that the formulation variation or cultivar, instead of the specific transgene (Table 3). The
and assembly of a finished product, to enter clinical assessment, first transgene (35SctxBSEK) codes for the non-toxic B subunit
will take place and this will be in line with Regulation (EC) (CTB) of Cholera toxin (CTA-CTB). CTB is one of the most potent
726/2004 on ‘Biopharmaceuticals and vaccines for human and vet- natural adjuvants which functions as both mucosal and parenteral
erinary use’. This Directive was originally set up for cell lines (e.g. adjuvant [36, 37]. The natural CTB of Vibrio cholerae contains a
CHO and bacterial cells), and it is argued that it needs to be refined specific signal peptide at its amino terminal end [38] that facilitates
to be more specific for plants [32, 33]. In particular, issues sur- transport into the periplasmatic space, where the CTB monomers
Risk Assessment and Regulation of Molecular Current Pharmaceutical Design, 2013, Vol. 19, No. 00 7
Table 2. Overview of EU Regulations Currently Surrounding PMF
Directive / Regulation Covers Relevance to Pharma - covers Authority
Directive 2009/41/EC Contained use of GMOs • Early laboratory R&D phase Member States National compe-
tent authorities
• GMOs grown in enclosed cell culture systems
(Bioreactors)
• GM plants grown in containment, i.e. glass-
houses or controlled environment rooms
Directive Deliberate release to the environ- Part B of the directive covering experimental field Member States National compe-
2001/18/EC ment release tent authorities
Part C covering cultivation at the commercial level
Member States National compe-
tent authorities, European Com-
mission, EFSA
Regulation (EC) Regulation on GM food/feed Applies only if GM plant parts are to enter the European Commission, EFSA,
1829/2003 food or feed chain (highly unlikely for Pharma) or Member States National compe-
may also apply if a ‘food’ crop is used for produc- tent authorities
tion even if for non-food/feed purposes
Directive 2001/10/EC Safety and efficacy testing of drugs Clinical trials Overseen by the Member States
National competent authorities,
at the R&D stage and by EMEA
at the marketing stage
Regulation (EC) ‘laying down Community proce- Would follow the existing regulations developed for EMEA (assessment only), Euro-
726/2004 dures for the authorization and su- mammalian/ bacterial systems and draw guidance pean Commission, Member
pervision of medicinal products for from EMEA guidance notes published on the ‘qual- States National competent
human and veterinary use and estab- ity of biological active substances produced by authorities
lishing a European Medicines stable transgene expression in higher plants’ [34]
(Market authorization)
Agency
EFSA = European Food Safety Authority; EMEA = European Medicines Agency
oligomerize [39]. In plants, both secretory and chloroplast-derived cyanophycin synthetase, encoded by cphA, is necessary to catalyze
CTB expression have been demonstrated [40, 41]. Plant-derived the ATP dependent elongation of the cyanophycin primer by the
fusion proteins of CTB expressing different antigens via the secre- consecutive addition of aspartic acid and arginine. All transgenic
tory pathway have been successfully used to induce specific im- events carried the nptII gene as selectable marker. As controls, a
mune responses and therapeutic effects [42-48]. No harmful effects transgenic line transformed with the empty vector carrying only the
could be detected when CTB was applied to mice [36, 40, 49, 50]. nptII gene was selected. In addition the cultivar ‘Albatros’ was used
A very similar protein, Lt-B, was applied to humans without any as near-isogenic variant (niv) and ‘Desiree’ as second potato culti-
deleterious effect [51]. The second transgene (35Svp60SEK) codes var.
for an antigen supposed to lead to immunogenicity against the rab-
The aim of the release was to identify potential effects of the
bit haemorrhagic disease (RHD) virus. RHD is an acute and highly three transgenes on the environment, to analyse the variability of
infectious viral disease of the European rabbit (Oryctolagus cunicu-
transgene expression in comparison to greenhouse trials and to
lus). Its capsid protein VP60, which forms a 27-40 nm virion with a
produce material for feeding studies. The number of tubers released
structured surface of regularly arranged cup-shaped depressions is given in (Table 3).
[42], was chosen as antigen. The third transgene (pPsbY-cphA)
In October 2008 the application was sent to the German Federal
codes for a cyanophycin synthetase. Cyanophycin is a cyanobacte-
Office of Consumer Protection and Food Safety (BVL) which is
rial reserve polymer composed of a poly-(cid:1)-aspartic acid backbone
responsible for the approval of the trial. The application was a fol-
with arginine residues linked via their (cid:1)-amino group to the (cid:2)-
low-up of a release that was carried out in 2005 to 2008, where
carboxyl group of each aspartate residue [52-54]. Mild hydrolysis
transgenic potatoes carrying the same transgenes have already been
of cyanophycin [55] results in homo- and copolymers of polyaspar-
released at the same sites. No signs of any detrimental effect have
tate and additionally L-arginine as products. The latter one has a
been observed. In December, the application was declared to be
broad range of technical and medical applications, amongst others
complete by BVL and this was announced in the federal bulletin as
as a stimulator of the immune system [56-60], growth increase by
well as in local newspapers at the sites of release. In addition, the
feeding [61, 62], and as an inhibitor of tumour cell growth [63].
complete application was displayed for one month to the public at
Polyaspartate is a soluble, non-toxic and biodegradable polycar-
both locations and in Berlin in order to give everybody the chance
boxylate [64], which could replace the non-biodegradable polyacry-
to be informed. The whole process is expected to be finished in 90
lates in many of its industrial, agricultural, and medical applications
days after the submission of the application, the time needed for
[55, 65, 66]. Cyanophycin is synthesized via non-ribosomal poly-
corrections by the applicant and the month of public display is
peptide synthesis in many cyanobacteria and some other non photo-
added. The approval was expected at least at the beginning of
synthetic bacteria [67, 68]. For cyanophycin synthesis only the
March 2009 in order to allow a timely release of the potatoes.
8 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 Sparrow et al.
Table 3. Events and number of tubers predicted to be released in the field trial
Vector Cultivar Event No. of tubers (in year)
2009 2010-2012, each
35Svp60SEK Desiree 6 458 458
35Svp60SEK Fasan 201 38 38
35Svp60SEK Albatros 204 143 437
35SctxBSEK Albatros 204 38 38
pPsbY-cphA Albatros 12 458 458
pPsbY-cphA Albatros 23 38 38
pPsbY-cphA Albatros 9 38 38
pPsbY-cphA Albatros 10 38 38
pPsbY-cphA Albatros 24 38 38
pPsbY-cphA Albatros 31 38 38
pPsbY-cphA Albatros 35 38 38
35S (nptII) Desiree 6 458 458
35S (nptII) Fasan 203 38 38
35S (nptII) Albatros 205 458 458
near-isogenic variant (niv) Desiree niv 458 458
near-isogenic variant (niv) Fasan niv 38 38
near-isogenic variant (niv) Albatros niv 547 547
Amazingly, only a partial permission was given in May 2009. A
5. CONDITIONS FOR MOLECULAR FARMING IN THE US
prerequisite for this permission was the agreement to postpone the
release of the 76 CTB tubers to 2010. Hence the application for the 5.1. Regulatory Framework for Contained and Open Field use
permission to release the CTB event 35SctxBsek 204 was repeated of Genetically Engineered (GE) Plants
in 2009. The approval was provided in time to add these tubers to In the US, laboratory practices using recombinant DNA
the field experiment in 2010. No additional information confirming (rDNA) must comply with National Institutes of Health (NIH)
the safety of the release of these potatoes was necessary. The loca- guidelines for recombinant DNA and these guidelines are adminis-
tion of the release had to be announced in the German ‘Standortreg- tered by the Institutional Biosafety Committees of whatever entity
ister’ (http://apps2.bvl.bund.de/stareg_web/showflaechen.do) in is performing the research. However, in order for any impact to be
detail that it was easy to identify the place of release. realized from this research, these laboratory experiments must
Twelve additional measures were ordered by the BVL including move ahead to the field or production arena. Commercialization has
that 1) during the release and the monitoring period, a 20 m wide many hurdles including not only the science and associated intellec-
area surrounding the trial had to be controlled every month for po- tual property to protect the inventions, but also market realities and
tential transgenic progeny; 2) after the release the area had to be government regulations. As each engineered plant moves from the
loosened up 15 to 20 cm deep. Plugging was not permitted; 3) the laboratory to scaled-up greenhouse production or to the field, issues
area had to be monitored once a month in the following year in emerge that trigger additional oversight for environmental release
order to identify volunteers. If any transgenic volunteer were found or ultimate use by humans.
in the area where the trial occurred, the monitoring period had to be The regulatory framework for genetically engineered plants in
prolonged for a second year. The tubers were planted by hand to- the United States has been discussed in many articles from several
gether with the controls and the area was surrounded by a 2 m high perspectives [69-71]. In contrast to the development of regulations
fence buried 30 cm deep into the soil. Watchmen were employed to in Europe, no specific legislation was enacted in the United States
protect the site, increasing the cost for the field trial drastically. that provided guidance or directives relating to the regulation of
Nevertheless, the field was attacked twice in 2009 and again in genetically engineered plants. Rather, it was concluded in the 1980s
2011. Fortunately, the destroyers were only able to tear some of the that existing statutes provided sufficient general authority for the
plants out of the soil, and in some cases it was possible to identify implementation of genetically engineered crop regulations. The
the plants so they could be replaced into the soil and the trial could White House Office of Science and Technology Policy (OSTP)
be evaluated, at least partially. established the Biotechnology Science Coordinating Committee
(BSCC, 1984), with representatives from 18 agencies including the
Risk Assessment and Regulation of Molecular Current Pharmaceutical Design, 2013, Vol. 19, No. 00 9
National Institutes of Health (NIH), the United States Department promoters in genetic engineering, the plants produced from these
of Agriculture (USDA), the US Environmental Protection Agency agents are considered plant pests.
(EPA), the US Food and Drug Administration (FDA), and the US A GM crop developer must obtain APHIS authorization
National Science Foundation [71, 72]. During the Reagan admini-
through either the permit process or notification process before a
stration in 1986, the Coordinated Framework for Regulation of genetically engineered crop can be field tested or released into the
Biotechnology was published in the US Federal Register (51 Fed. environment. Movement and planting restrictions are imposed by
Reg. 23302; June 26, 1986). This document included policy state-
permits while notification can be used in lieu of a permit when the
ments from the agencies involved as well as a proposed USDA plant is not considered a noxious weed [77]. Notifications, i.e., an
regulation governing the introduction of organisms and products expedited permitting process, are used primarily for field trial ap-
that are plant pests that were developed through genetic engineering
proval as well as importation and transport within the US of certain
[73]. Of significance and in contrast to European policies, an under- genetically engineered plants. To be eligible for a notification, spe-
lying principle of the US Coordinated Framework is that genetically cific criteria as well as designated performance and characteristics
engineered organisms are not inherently risky and regulatory re-
standards must be met. For example, among other things, the GM
quirements should focus on the product instead of the process used plant cannot be considered a noxious weed under USDA regula-
to derive the organism. Additionally, the existing agencies and laws tions or a weed in the area of release, the function of the genetic
were deemed sufficient to regulate this new technology and the
material must be known and its expression must not cause plant
agencies agreed to cooperate in the process. The resulting Coordi- disease, the modification should not contain human or animal
nated Framework allowed (1) USDA to monitor planting and field pathogenic sequence, and the crop cannot express a plant-made
testing as well as to regulate genetically engineered organisms that
pharmaceutical or a plant-made industrial product [78]. Permits are
might be plant pests, (2) FDA to ensure the safety of most domestic used when the GE plant does not meet the requirements for notifi-
and imported foods or feed, including genetically engineered foods, cation.
and to enforce pesticide tolerances established for food or feed by
In 2003, the USDA amended its regulations to require that in-
the EPA, and (3) the EPA to establish pesticide tolerances for food
troductions of plants genetically engineered to produce industrial
and feed and regulate genetically engineered organisms that contain
compounds be conducted only under permit, i.e., not eligible for
pesticidal substances and the genetic material necessary for incor-
notification (68 FR 46434-46436, Docket No. 03-038-1, August 6,
poration of pesticidal substances, new uses of existing pesticides,
2003). As with the notifications, developers must provide APHIS
and novel micro-organisms [71, 74, 75]. Although these divisions
scientists with details about the nature of the GE organism. The
of responsibility may be clear in theory, in practice the lines are
main consideration for field trials is safety and prevention of unin-
often blurred as into whose purview a genetically engineered plant
tended release in order to prevent spread of a potential plant pest to
falls. The US policy decision to use existing laws, in effect, led to
the environment.
three different agencies with significant and often duplicative roles
in the regulation of GM crops. Indeed, in most cases, different regu- APHIS regulation 7 CFR Part 340.6 permits a crop developer to
latory dossiers and data sets must be submitted to at least two US petition APHIS to evaluate submitted data to determine if a particu-
agencies, although these data sets are not mutually exclusive. lar article presents a plant pest risk, and if not, should no longer be
Twenty-six years after the implementation of the US Coordinated regulated [71, 77]. Once a developer has sufficient evidence that its
Framework (i.e., in early 2012), the USDA, EPA, and FDA entered GM product is no more risky than the equivalent non-GM parent,
into a memorandum of understanding (77 Federal Register 5012, the developer may petition APHIS for a determination of non-
FRL–9328–7, MOU 225-11-0001) to permit information sharing regulated status to introduce the organism into US agriculture and
between the three agencies. This may improve the overall coordina- commerce without APHIS oversight – i.e., to become “deregu-
tion of regulation in the US although that remains to be seen. lated”. Petitions must contain extensive data on details about plant
biology, genetics, the nature and origin of the genetic material,
5.1.1. Regulatory Considerations of the US Department of Agri-
expression of the gene product, disease and pest susceptibilities,
culture – Animal Plant Health Inspection Service (APHIS)
field test reports and effects on other plants. In addition, data on the
The primary regulatory arm of the USDA that is relevant for
impact of the GM plant on other organisms including the potential
plants and crops is the Animal and Plant Health Inspection Service for gene flow must be determined. Petitions are reviewed by APHIS
(APHIS). The focus of APHIS is to protect US agriculture, food, with a focus on the potential for the GM plant to become a weed or
and natural resources and it is in this context that it regulates GM
plant pest [77, 78]. During the process of evaluation, APHIS at a
crops [76], specifically genetically engineered organisms capable of minimum prepares an Environmental Assessment (EA) and a “De-
posing risks to plant or animal health. termination of Non-Regulated Status” as well as taking into consid-
The US Plant Protection Act (Title IV Public Law 106-224, 114 eration comments from the public.
Statute 438, 7 U.S.C. §7701-7772) requires the USDA to prevent
While this review is not focused on the legal aspects of regula-
the introduction and dissemination of plant pests and weeds into the
tions, it warrants mentioning that APHIS has worked through sev-
United States and provides specific authority to prohibit or restrict eral lawsuits filed by the Center for Food Safety (CFS) and the
the importation, exportation, and interstate movement of plants,
International Center for Technology Assessment (ICTA) [70].
plant products, certain biological control organisms, noxious weeds,
These organizations alleged, in the early 2000’s, that APHIS did
and plant pests [69]. APHIS regulations under 7 CFR §340.1 stipu- not adequately evaluate the environmental risks of pharmaceutical-
late that any organism which has been altered or produced through
producing GM crops using the directives of the National Environ-
genetic engineering is considered a regulated article if the donor
mental Policy Act (NEPA) of 1969. An environmental assessment
organism, recipient organism, or vector or vector agent meets the prior to issuing permits in some instances would have prevented
definition of a plant pest, or is an unclassified organism and/or an
litigation over the field-testing of these crops. Current litigation
organism whose classification is unknown, or any product which
involves the decisions by APHIS to de-regulate glyphosate-tolerant
contains such an organism. Additionally, any other organism or (GT) alfalfa and sugar beet. These complex cases allege that APHIS
product altered or produced through genetic engineering which the
failed to consider outcrossing of GT alfalfa to organic crops or GT
Administrator determines is a plant pest or has reason to believe is a
sugar beet to seed production sugar beet crops [70]. In each case,
plant pest is also regulated [77]. APHIS defines plant pests broadly the de-regulation decision was accompanied by a “Finding of No
to include any organisms that can damage or cause injury to plants
Significant Impact” or FONSI. In response to these cases, the
or plant parts. Since many plant pathogens are used as vectors or
USDA prepared Final Environmental Impact Statements (FEIS) for
GT alfalfa and sugar beet. USDA published the FEIS in December
10 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 Sparrow et al.
2010 for the GT alfalfa lines J101 and J163 (APHIS-2007-0044- pesticides or plant incorporated protectants (PIPs). PIPs, such as the
12532). These lines were determined to be unlikely to pose plant Bacillus thuringiensis (Bt) toxin in corn, are considered pesticides
pest risks and thus were no longer considered regulated articles. In under FIFRA because they are introduced into plants with the inten-
the FEIS published by the USDA in May 2012, the USDA found tion of preventing, destroying, repelling, or mitigating a pest (FI-
that GT sugar beets (H7-1) are not expected to become more inva- FRA §2). Therefore, Plant-Made Pharpaceuticals are not regulated
sive in natural environments or have any different effect on critical by EPA. In 2001, EPA published a rule that established the regula-
habitat than conventional sugar beets which do not establish or tory framework for PIPs and included (1) an exemption from FI-
persist in the environment (APHIS-2010-0047-4658). FRA requirements for those PIPs that are derived entirely through
5.1.1.1. APHIS and New Breeding Technologies conventional breeding between sexually compatible plants and (2)
an exemption from the requirement of the Federal Food, Drug, and
In recent years, APHIS has taken steps to refine and limit its
Cosmetic Act (FFDCA) (21 U.S.C. § 301 et seq.) for the establish-
regulatory authority to only plants that are plant pests, were created
ment of a residue tolerance level for pesticides [71].
with plant pests, or used a plant pest in the method to create the
plant. This has resulted in the exemption from APHIS regulation A developer of a crop that contains a pesticidal property that
was not developed entirely through conventional breeding must
several new breeding technologies.
submit an application to EPA to register a PIP. The EPA evaluates
Cibus Global (www.Cibus.com) received a green light from
each submission to register a pesticidal substance produced by a a
APHIS in 2004 when it learned that its herbicide tolerant canola
GM plant on a case-by-case basis, and requires data related to the
developed with oligonucleotide-directed mutagenesis was not sub-
product characterization which are similar to the data required by
ject to regulation. In the March 2004 letter, APHIS indicated that it
other agencies regarding the description of the GM plant and the
had no authority to regulate products created by mutagenesis tech-
details of its modification: i.e., organic source of the pesticidal gene
niques.
construct (promoter, terminator, enhancer, marker genes and other
In May 2006, APHIS exempted from regulation a maize Zinc inserted DNA). The dossier should include information on the biol-
Finger Nuclease (ZFN) - deletion technology used by Dow Agro- ogy of the plant, the stability of the genetically engineered DNA,
Science (May 26, 2010 letter). The ZFN-12 maize plants with in- and the anti-nutritional substances produced by the plant, among
duced deletions due to the use of zinc finger nuclease technology others.
were not considered to be articles regulated by APHIS as ZFN-12
Additionally, EPA requires data on mammalian toxicity, the
was not a plant pest and did not pose a plant pest risk.
effects on non-target organisms, environmental metabolism and an
APHIS clarified the scope of its regulatory authority in mid- insect resistance management program for plants specifically ex-
2011 when it informed the public that a genetically engineered, pressing Bt products. Since the EPA focuses on the pesticidal prop-
herbicide tolerant Kentucky bluegrass developed by Scotts did not erties, developers should include a full biochemical and amino acid
meet the definition of a regulated article (i.e., a plant pest) and thus description of the pesticidal protein. Since most PIPs are proteins,
was exempt from regulation by APHIS (APHIS-2011-0080). This the issue of mammalian allergenicity is highly important. Acute
conclusion was based on the fact that the Kentucky bluegrass itself digestibility assays and amino acid sequence homology are neces-
is not a plant pest and that no organisms or the method used to cre- sary to demonstrate absence of allergenicity concerns. EPA is also
ate the variety were plant pests. In a July 2011 Factsheet regarding concerned with gene flow issues as they may result in expanded
the action, APHIS made clear that it would exempt from regulation exposure to novel pesticidal substances as well as the environ-
other GE organisms that are not a plant pest, are not made using mental fate of PIP substances and the effect on non-target organ-
plant pests, and when there is no reason to believe it is a plant pest. isms [71].
While each of these decisions was made relative to a specific In addition to the requirement for a PIP to be registered before
case, they indicate that APHIS will not regulate new breeding tech- use unless it is exempted (as noted above), FIFRA section 7 re-
nologies in and of themselves. Rather, it appears that APHIS will quires that the producers of pesticides register the facilities where
continue to focus its regulatory requirements on plants that are a the pesticide is produced and report the amount produced. In con-
plant pest or that pose pest risk, without regard to the technology trast to APHIS requirements, experimental use permits are required,
used to create the plant. generally, for larger field trials (i.e., over 10 acres). In an effort to
5.1.2. Regulatory Considerations of the Environmental Protection delay the onset of resistance to pesticides, EPA requires manage-
Agency (EPA) ment strategies after commercialization. Farmers cultivating GM
crops with PIPs are required to follow the appropriate insect resis-
The main role of the US Environmental Protection Agency
tance management (IRM) practices that are approved by EPA usu-
(EPA) is to ensure that US citizens are protected from significant
ally including refuge areas.
risks to health and that the environment is protected from invasive
species. EPA regulations are designed to make US communities 5.1.2.1. EPA and New Breeding Technologies
and ecosystems diverse, sustainable, and economically productive In March 2011, the EPA issued a Notice of Proposed Rule-
[76]. The regulatory activities of the EPA are governed by the re- Making (NPRM) that would specifically exempt from registration
quirements of specific environmental laws. These laws direct EPA and the need for experimental use permits cisgenic PIPs (EPA-HQ-
to regulate many kinds of pollution, including pesticides, with a OPP-2009-0499; FRL-8873-5). Additionally, the proposed rule
focus on how they affect different aspects of the environment. Envi- would contain proposed revisions to the data requirements for regis-
ronment is generally defined in these statutes as including human tration of PIPs or for an experimental use permit. In the NPRM, the
health, plant and animal life, water quality, and air quality [79]. The EPA notified the public that it had sent the draft rule to the USDA
courts, the scientific community, and the regulatory community and the FDA. The EPA has yet to finalize the proposed rule and
recognize that EPA should consider other factors along with science submit to the Federal Register for comments.
when it makes decisions about risk management [80].
5.1.3. Regulatory Considerations of the US Food and Drug Ad-
Under the Federal Insecticide, Fungicide, and Rodenticide Act ministration (FDA)
(FIFRA) (7 U.S.C. §135 et seq., 1972), the EPA must determine if a
The Food and Drug Administration (FDA) is an agency within
new pesticide is able to perform its intended function without un-
the US Department of Health and Human Services and is the pri-
reasonable adverse effects on human health and the environment
mary agency with responsibility for ensuring the safety of food and
(FIFRA §3) before it can be registered for use. Within the Coordi-
feed, except for meat, poultry, and egg products which are regulated
nated Framework, these pesticides include genetically engineered
by the USDA. The FDA primarily assures the safety, effectiveness,