| Summary of the safety assessment (food safety): |
Dow AgroSciences Industrial Ltda. requested a CTNBio Technical Opinion
related to biosafety of the insect-resistant glufosinate ammonium-tolerant
genetically modified cotton (Gossypium hirsutum), namely WideStrike Cotton, for
the purpose of free registration, use in the environment, human and animal
consumption, marketing and industrial use and any other use and activity related
to this GMO including derivative lineages and cultivars as well as byproducts,
all under the remaining regulations and requirements applicable to any use of
cultivated species of the genus Gossypium effective in Brazil. WideStrike Cotton
was produced by retro-crossing (“gene stacking”, “stacking”), between events
281-24-236/3006-210-23, containing the synthetic gene cry1F that codifies the
synthetic protoxin Cry1F and event 3006-210-23 containing the synthetic gene
cry1Ac that codifies synthetic Protoxin Cr1Ac. Both are insecticide crystallized
proteins also referred as d-endotoxins, obtained from Bacillus thuringiensis var.
aizawai strain PS811 and Bacillus thuringiensis var. kurstaki strain HD73. In
addition to insect resistance given by the action of genes cry1F and cry1Ac,
event WideStrike also displays resistance to the herbicide glufosinate ammonium
due to the presence of two copies of gene pat, which codifies enzyme
phosphinotricin-acetiltransferase (PAT). Gene pat is a synthetic version based on
the natural pat gene of Streptomyces viridochromogenes, a non-pathogenic
bacterium found in the soil. Inclusion of the pat gene enables selection of plants
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from well-succeeded transforms that express proteins Cry1F and Cr1Ac of
Bacillus thuringiensis. The PAT protein fails to grant any pesticide activity, and
there is no known adverse effect to the environment or to man, such as toxicity or
allergenicity. The agronomic purpose of the event is the control of cotton pests:
Heliothis virescens, Helicoperva zea, Spodoptera frugiperda, Alabama argillacea,
Pectinophora gossypiella, Spodoptera exigua, Spodoptera eridiania,
Pseudoplusia includens, and Trichoplusia ni. Events Cry1F 281-24-236 and
Cr1Ac 3006-210-23 were obtained from transformation of Agrobacterium
tumefaciens from cotton cultivar ‘Germain’s Acala GC510’ (Gossypium hirsutum
L.). Each event was later retro-crossed with genotype PSC355 (Phythogen Seed
Company). The first generations (F1) of such crossings in each event was then
retro-crossed for three additional generations for PSC355, to create a BC3F1
lineage of each event. The two transgenic lineages BC3F1 were crossed to obtain
the stacked cotton lineage Cry1F/Cr1Ac that was used in the final product. The
source of the cry1F and cry1Ac is the bacterium Bacillus thuringiensis (Bt
subspecies aizawai and kurstaki, respectively). The d-endotoxins, insecticides Bt
expressed in the cotton plant, are proteolitically cleavaged in the alkaline
intestine of lepidopters, resulting in a form of active insecticide. The active
insecticide protein interacts with a receptor molecule that is present only in the
epithelial cells of the middle intestine of susceptible insects, generating pores in
the cell membranes. The process disturbs the cell equilibrium, causing a dialysis
of intestine cells of insects, leading them to death(53). However, recent studies
suggested that the cause of the insect death may be the presence of pores in the
intestinal epithelium that causes the passage of bacteria present in the middle
intestine to the hemolymph, leading the insect to death by generalized
infection(24). There are no binding sites for the d-endotoxins of Bacillus
thuringiensis at the surface of the intestinal cells of mammals, therefore domestic
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animals and humans are not susceptible to such proteins. A number of
experiments testing Bt proteins showed the lack of toxicity to man and
vertebrates, absence of adverse effects to non-target organisms and to the
environment. Gene stacking events of 281-24-236/3006-210-23 cotton were fieldtested
in 1999, 2000, 2001 and 2002, and are under assessment until now in the
main cotton regions in the United States, Costa Rica, Argentina, Australia,
Mexico, Spain and China. An experiment in Brazil was conducted in the
2005/2006 crop. Data and information related to the agronomic characteristics,
resistance to pests and diseases were collected during such tests. Reports are
that events 281-24-236/3006-210-23 do not exhibit pathogenic properties to
plants and it is unlikely that may harm other insects that are beneficial to
agriculture. There is no evidence or suggestion that proteins Cry1F and Cr1Ac of
Bt increased the potential for the transformed cotton plant to change into an
invading plant, since its phenology, morphology, and other agronomic aspects
remained unaltered. Therefore, according to the results obtained and mentioned
in the Cotton Biosafety report, it is unlikely that events of cotton events 281-24-
236/3006-210-23 change into pest plants for agriculture or become invaders o
natural habitats; cross with wild relatives or create hybrid descendants that may
change into pests or invading plants, despite genetic compatibility among the
species. It is also unlikely that they change into a plant pest; have adverse effects
on non-target species, including man, or that they have any effect on biodiversity.
Summarizing, the focus of this application are the transformation events 281-24-
236 and 3006-210-23, which are combined by retro-crossing (conventional
improvement). Cotton cultivars containing Cry1F and Cr1Ac will be marketed with
stacked events 281-24-236/3006-210-23. The release of stacked product 281-24-
236/3006-210-23 together with the practices of insect resistance management
shall reduce the selection pressure towards development of insecticide resistance
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and help maintaining an effective control of Lepidoptera. For the foregoing,
commercial release of WideStrike Cotton is not potentially harmful to either
human or animal health, and is not an event of significant degradation for the
environment. According to Article 1 of Law nº 11,460, of March 21, 2007,
“research and cultivation of genetically modified organisms may not be conducted
in indigenous lands and areas of conservation units”. Under Article 14 of Law no.
11,105/2005, CTNBio found that the request complies with the applicable rules
and legislation securing the biosafety of environment, agriculture, human and
animal health. According to Annex I of Regulating Resolution no. 5, of March 12,
2008, the applicant shall have a term of thirty (30) days from the publication
date of this Technical Opinion to adjust its proposal to the post-commercial
release monitoring plan.
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CTNBio TECHNICAL OPINION
I. GMO Identification
GMO name: WideStrike Cotton, Event 281-24-236/3006-210-
23 .
Applicant: Dow AgroScience Industrial Ltda.
Species: Gossypium hirsutum L.
Inserted characteristics: Tolerance to insects [genes cry1Ac and cry1F]
and to herbicide glufosinate ammonium [gene
pat]
Method of insertion: Transformation mediated by Agrobacterium
Prospective use: Production of fibers for the textile industry and
kernels for human and animal consumption of the
GMO and derivatives.
II. General Information
Cotton belongs to genus Gossypium, Tribe Gossipiae, Family Malvaceae, order
Malvales (62,136). The genus is divided into four sub-genera (Gossypium, Sturtia,
Houzingenia and Karpas), which, in turn, are divided into nine sections and a
number of subsections(61).
Original centers of G. hirsutum are Mexico and Guatemala, while those of G.
barbadiense are in Peru and Bolivia(173). Allelotetraploid species exhibit in their
genome a combination of two distinct diploid species(136).
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Two types of cotton plants are predominantly cultivated in Brazil: conventional
cotton and a caterpillar-resistant genetically modified cotton plant. These plants
are responsible for practically all the cotton produced in the country. Besides,
three other cotton plants featuring genetic or ecologic special characteristics are
cultivated: the naturally colored, the organic and the agroecologic cotton plant.
The colored cotton is almost exclusively concentrated in the State of Paraíba,
being the area planted, in 2007, of about 300 hectares. Certified organic cotton is
planted in the States of Paraná and Paraíba, and the area cultivated in 2007 was
250 hectares. Tillage of agroecologic cotton plants were conducted by 235
farmers in the semiarid bioma of four States of the Northeastern region, producing
42 tons(116).
Chains of special, conventional and transgenic cotton have satisfactorily lived
together, without problems of coexistence being reported. The area planted with
cotton in Brazil in the past 2007/2008 harvest reached about one million and one
hundred thousand hectares, of which over 85% concentrated in the Cerrado
bioma, especially in the states of Mato Grosso, Bahia, Goiás and Mato Grosso do
Sul. Other cultures are present in other states of the country, mainly in the
semiarid of the Northeastern region, Paraná, Minas Gerais and São Paulo(94).
Cotton plants are unique in their utilitarian aspects, including weaving fibers and
oil- and protein-bearing seeds used in human and animal food. Cotton plant
species were developed since ancient times both in the old and the new world.
Cotton (Gossypium spp.) is a plant domesticated by man since 3000 BC and
cultivated in all continents. Its main use is in the production of fibers and food,
especially for animals.
Cotton plant (Gossypium hirsutum L.) is one or the four species cultivated for
cotton fiber in the world(147), economically exploited in a wide tropical strip and in
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some subtropical regions. Culture of cotton in Brazil ranks among the ten main
crops in Brazil and is sixth in cultivated area.
Cotton species commercially cultivated in Brazil are Gossypium hirsutum and, in
a lesser area, G. barbadense. G. hirsutum is more adaptable, more productive
and is prevalent in the world. Its fiber is used in the production of textiles, other
non-textile products and is the source of industrial cellulose for different products.
G. barbadiense is important for the quality and length of its fiber and is used in the
production of fine fabrics.
Among the main cotton pests in Brazil, one may mention cotton leafworm
(Alabama argillacea), cotton budworm (Heliothis virescens), pink bollworm
(Pectinophora gossypiella), fall armyworm (Spodoptera frugiperda), cotton aphid
(Aphis gossypii), cotton bug (Horcias nobilellus), and boll weevil (Anthonomus
grandis). Control of such pests has been mainly conducted with the use of
insecticides. In Brazil, over 10 tons of insecticide are consumed each year in
cotton fields only, causing a US$ 190 million increase in production costs. The
excessive use of non-specific insecticides leads to negative environmental
impacts, such as severe reduction of beneficial organisms and potential upsurge
of pests resistant to conventional insecticides.
WideStrike Cotton’s purpose is to obtain insect-pest resistant plants through
introduction of two gens from bacterium B. thuringiensis (Bt), namely gene cry1Ac
(Bt species kurstaki) and gene cry1F (Bt subspecies aizawa) that synthesize
endotoxins, mainly against Lepidoptera. In addition, gene pat was also introduced
(enzyme phosphinothricin acetyltransferase) from bacterium Streptomyces
viridochromogenes. The enzyme produced is used as a marker and imparts
resistance to glufosinate ammonium.
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The use of Bt technology in Brazil may contribute towards the reduction of the use
of such insecticides and, consequently, diminish the impacts from the use of such
pesticides to the environment, and human and animal health. Besides, adoption
of technologies that reduce spraying of chemical products in crops may favor the
appearance of secondary benefits. Such benefits are reduction in the use of
inputs to produce agricultural pesticides, conservation of fuels used to produce,
distribute and apply the pesticides and elimination of the need for use and
discarding of pesticide packaging.
III. Description of GMO and Proteins Expressed
WideStrike Cotton was developed by retro-crossing (“stacking”) between events
281-24-236, containing synthetic gene cry1F that codifies protein Cry1F and
event 3006-210-23, containing synthetic gene cry1Ac that codifies protein
Cr1Ac. Both are crystallized insecticide proteins, also referred as d-endotoxins,
obtained from Bacillus thuringiensis var. aizawai strain PS811 and Bacillus
thuringiensis var. kurstaki strain HD73, respectively. In addition to insectresistance
for the action of genes cry1F and cry1Ac, event WideStrike is also
glufosinate ammonium-resistant due to the presence of two copies of the gene
pat, which codifies enzyme phosphinothricin acetyltransferase (PAT). Gene pat is
a synthetic version based on the natural pat gene of Streptomyces
viridochromogenes.
Events Cry1F 281-24-236 and Cr1Ac 3006-210-23 were obtained through
transformation by Agrobacterium tumefaciens of the cotton cultivar ‘Germain’s
Acala GC510’ (Gossypium hirsutum L.). Each event was later retro-crossed with
genotype PSC355 (Phytogen Seed Company). The first generations (F1) of such
crossings in each event were retro-crossed for three additional generations for
CSC355 in order to generate a BC3F1 lineage of each event. The two transgenic
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lineages BC3F1 were crossed to obtain the cotton stacked lineage Cry1F/Cr1Ac
that was finally used in the commercial product.
The transformation was made by Agrobacterium tumefaciens using binary vectors
pAGM281 and pMYC3006, the maps and other gene elements of which are
described by the applicant in the proceedings. Genetic elements of region T-DNA
of plasmids pAGM281 and pMYC3006 are described below.
Plasmid pAGM281
Genetic Element Size (kpp)1 Location (bp) Details
(40CS)Dmas 2’ 0.61 7028-7636
(complementary)
Promoter of manopine synthase of Agrobacterium
tumefaciens strain LBA 4404 pTi15955
Cry1F (synpro) 3.45 3571-7017
(complementary)
Plant optimized synthetic, full length version of
Cry1F of B.t. var. aizawai.
ORF25 poliA 0.73 2818-3544 Bidirectional terminator of Agrobacterium
tumefaciens strain LBA 4404.
pat 0.55 2259-2810 Plant optimized synthetic gene of glufosinate
ammonium resistance, based on a sequence of
phosphinotricin-acetiltransferase from S.
viridochromogenes.
Ubi Zm1 1.99 260-2252 Zea mays promoter plus Zea mays exon 1
(enhanced not translated) and intron 1.
Plasmid pMYC3006
Genetic Element Size (kpp)1 Location (bp) Details
Ubi Zm1 1.99 6080-8072
(complementary)
Zea mays promoter plus Zea mays exon 1
(enhanced not translated) and intron 1.
Cry1Ac (synpro) 3.47 2587-6057
(complementary)
Plant optimized full length synthetic version of
Cr1Ac1 of B.t. var. kurstaki.
ORF25 poliA 0.73 1835-2561 Bidirectional terminator of Agrobacterium
tumefaciens pTi5955.
pat 0.55 1276-1827 Plant optimized synthetic gene of glufosinate
ammonium resistance, based on a gene
sequence of phosphinotricin-acetiltransferase
from Streptomyces viridochromogenes.
(40CS)Dmas 2’ 0.61 643-1251 Promoter of manopine synthase of pTi15955
(Barker et al., 1983, Plant Mol. Biol. 2, 335-350),
including four copies of octopine synthase (OCS)
of pTiAch5.
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For both lineages, the transformation took place using segments of cotton
cotyledons isolated from plants with 7-10 days of in vitro germination. The
segments were cultivated with disarmed A. tumefaciens (strain LBA4404)
containing the above described plasmids. After the transformation, the segments
were transferred to a medium of callus induction with herbicide glufosinate
ammonium. In the middle of the callus formation the antibiotic carbenicillin was
also used with the purpose to destroy any remaining Agrobacterium.
The source of genes cry1F and cry1Ac is bacterium Bacillus thuringiensis (B.t.
subspecies aizawai and kurstaki, respectively). Insecticide d-endotoxins
expressed in the cotton plant are proteolitically cleavaged in the alkaline intestine
of Lepidoptera insects, resulting in an active insecticide form. The active
insecticide protein interacts with a receptor molecule, present only in the epithelial
cells of the medium intestine of susceptible insects, generating pores in the cell
membranes. This process causes a disturbance in the cell equilibrium, promoting
dialysis of the intestine cells of insects, leading them to death(53). Recent studies,
however, identified that the cause of the insect death is that the presence of pores
in their intestinal epithelium promotes the passage of medium intestine bacteria to
the hemolymph leading the insect to death by generalized infection(24). There are
not binding sites for the Bacillus thuringiensis d-endotoxins at the surface of
mammal’s intestinal cells and therefore domestic animals and man are not
susceptible to such proteins.
A detailed molecular analysis of WideStrike cotton was submitted by applicant,
discussing the results of the insertion number characterization by Southern Blot
analysis; identification of terminations 5’ and 3’ and border regions of the DNA
inserted by cloning and PCR; DNA sequencing; and assessment of whether other
transcriptions of potential mRNA resulting from the insertion sequences are or not
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present.
The initial Southern Blot analysis indicated that the commercial WideStrike
contains a single copy of the T-DNA from the binary vector pAGM281 jointly with
the T-DNA of the binary vector pMYC3006. Insert pMYC3006 contains an intact
copy of insect resistance, cry1Ac, jointly with one intact copy of the marker for
selection of molecular weight, gene pat, while insertion of pAGM281 contains one
intact copy of the insect resistant gene, cry1F and one intact copy of the pat gene.
Besides, one copy of promoter UbiZm1 and a truncated copy (231 bp) of the pat
gene were identified adjacent to the 3’ T-DNA terminal border. Southern blot
analyses also suggested the genotypic stability of insects in different generations
of the stacked event or of lineages, individually, besides verifying the absence of
antibiotic-resistant genes or sequences of the vector backbone in cotton 281-24-
236/3006-210-23. The results are in line with the expectations, since the
transformation method using A. tumefaciens, even with the unplanned, though
acceptable, presence of a truncated copy of the gene pat close to the insert
Cry1F / pat(108, 109, 194).
IV. Aspects Related to Human and Animal Health
Transgenic cotton resistant to lepidopters order insects event 281-24-236/3006-
210-23 contains DNA sequences derived from the following organisms: Bacillus
thuringiensis, Agrobacterium tumefaciens, Streptmyces viridochromogenes, and
Zea mays. None of such donor organisms in known as a source of toxins for
mammals or as being allergenic to man.
Proteins Cr1F/Cr1Ac are microbial d-endotoxins produced by Bacillus
thuringiensis (Bt). The toxins act at the intestine of larvae of different caterpillars
of the order Lepidoptera, where they have receptors. This bind prevents the
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insects from feeding. Man and animals do not have such receptors and, therefore,
in principle, are not subject to the effects caused by such bind.
Protein Cry1F is expressed in all the plant parts, except nectar, bran and oil.
Protein Cr1Ac is expressed in all the plant parts, except nectar, husk, bran and
oil. Protein PAT is expressed in very low levels for event 281-24-236 and for
cotton 281-24-236/3006-210-23, however this protein was seldom detected in
tissues of event 3006-210-23.
Analyses carried out in ashes, total fat, humidity, protein, carbohydrates, calories,
total fiber, fiber in acid detergent, fiber in neutral detergent in the modified cotton
showed results similar to those of the control cotton, varying within the values of
the literature. An analysis of fatty acids in the oil of products processed from
cottonseed showed that there is no difference between conventional and
transgenic cottons. Regarding anti-nutritional factors, the contents of gossypol
and cyclopropenoid fatty acids were essentially the same between the control and
the modified cotton.
Protein PAT is degraded by gastric juice of animals and by similar to human
artificial gastric juice, losing its physicochemical characteristics. Therefore, it is
not expected that the protein may be fully absorbed being therefore unlikely that it
may produce adverse toxic effects. As mentioned above, PAT protein is
expressed in very low levels in event 281-24-236 and cotton 281-24-236/3006-
210-23; this protein was seldom detected in event 3006-210-23.
References to acute toxicity are described in the sites of the United States
Environment Protection Agency,
(http://www.epa.gov/fedrgstr/EPA-PEST/1997/April/Day-11/p9373.htm),
and at the European Commission Health and Consumer Protection Directorate341/
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General,
(http://ec.europa.eu/food/fs/sc/oldcomm7/out02en.htm)
indicating lack of toxicity of PAT protein.
Regarding proteins Cry1F and Cr1Ac, according to the United States
Environment Protection Agency (http://www.epa.gov) one may state with
reasonable certainty that there are not risks to humans caused by exposure to
such proteins.
The above considerations are also in the report of Cotton Biosafety, event 281-
24-236/3006-210-23, submitted by Dow AgroSciences. Toxicity and allergenicity
data of this same event were submitted to and analyzed by the United States
Environment Protection Agency that considered both proteins Cry1F and Cr1Ac,
pesticides, innocuous to Human and Animal Health.
It shall be mentioned that recently, in Brazil, EMBRAPA released a larvicide to
combat dengue using Bacillus thuringiensis (FSP 04.06.2008) to be added to
water for human consumption. The release notice contained the phrase “This
product is totally health and environment safe. In case a child drinks a little water
containing the larvicide, this is not a problem”. It is worth stressing that the greater
concentration of the Cry proteins is in the cotton leaves and that in the
cottonseed, the part potentially consumed by man and animals, the concentration
is smaller. In cotton bran none of the inserted protein (Cr1Ac, Cry1F and PAT)
was detected. In case they are consumed by animals (bran and kernel) and by
man (cottonseed oil) the proteins will be rapidly digested by the stomach, before
being available for absorption. Cotton bran is preferably destined to ruminants
because of the presence of gossypol, which is toxic for non-ruminants: not an
impediment for its use provided inclusion in the diet remains at levels lower than
10%. For ruminants, cotton bran is used in larger quantities, but a large part is
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degraded at the rumen and the remaining is digested by the true stomach, the
abomasum. For humans, the main ingestion is through cottonseed oil, that has
extremely low levels of proteins (generally below detection threshold) and in case
the inserted proteins (Cry1F, Cr1Ac and PAT) are contained in the oil, their
quantities will be even smaller. This fact, coupled with the rapid stomach digestion
of these proteins and thermal lability above 75ºC makes any risk to human health
extremely unlikely. Another fact contributing towards reducing the likelihood of
toxicity, both for man and animals, is the specificity of the Cry proteins that, in
order to act, bind to receptors that are present only in target-insects.
The PAT protein, which grants resistance to glufosinate ammonium, was obtained
from Streptomyces viridochromogenes, present in the soil, recognized as nonpathogenic
to man and animals. Tests conducted with inclusion of cotton bran in
poultry rations failed to show any adverse effect in the weight gain and mortality
rate. It is worth stressing that the feed conversion rate of modified bran was better
than the rates of controls, that is to say, the birds consumed a smaller amount of
ration to gain the same weight. Another important fact is that the test begun with
newly-born birds, sensitive to any kind of adversity and, even so, the presence of
modified cotton in the diet did not have any negative effect.
In an acute toxicity test, conducted with CD1 mice, a dose of 2000 mg/kg of
microbially produced Cry1F and Cr1Ac was fed, with no record of severe
pathologic lesion and with animals gaining weight.
For being proteins, the risks of allergenic effects were also assessed. Allergens
originated from food are normally resistant to heat, acid and proteases, may be
glycosylated and present in high concentrations. The proteins essayed are
promptly digested by gastric juice, are not glycosylated, and heating leads to loss
of bioactivity. Experiments conducted in animals fail do suggest any allergenic
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potential.
Both B. thuringiensis and Streptomyces viridochromogenes did not have records
of being allergy-triggering factors, and as both are present in the environment,
mainly in the soil, they may be ingested as food contaminants, mainly vegetables,
without causing adverse effects (no records in the literature). Genes introduced in
modified organisms do not codify known allergens and fail to share
immunologically significant sequences. The sequence of amino acids of proteins
Cry1F, Cr1Ac and PAT were compared with two data banks using the software
provided by Genetics Computer Group and no allergenic sequence was identified.
The above facts, together with the small quantity of modified proteins present in
the diet, rapid gastric digestion (one minute for proteins Cry1F and Cr1Ac and
less than 30 seconds for protein PAT) and their thermolability make the risk of
triggering an allergic reaction practically inexistent.
Safety assessment of food derived from genetically modified organisms is entirely
based on the concept of substantial equivalence. The doctrine emerged and has
been basically discussed by the international community in the context of safety
assessment of new foods. According to the doctrine, if a genetically modified
product maintains the same characteristics, composition, nutritional values and
utility of another non-modified product, there is no motive to segregate it from
the remaining so-called conventional ones for the reason that Molecular Biology
tools were used, since they are in fact the same product, obtained by different
production methods.
According to the substantial equivalence doctrine, a food may only cease being
an equivalent of another when a scientific assessment finds a characteristics such
as composition, nutritional value, nutritional effect or utility that differentiates the
product from a corresponding already existing food.
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In general, the assessment needed for approving the marketing of such products
includes analysis of the transformation vectors, molecular structure of the newly
inserted gene, intentional and non-intentional effects associated to its expression,
chemical composition in macro- and micronutrients and toxic compounds and
products secondary to metabolism. Besides, biological essays (both nutritional
and toxicologic ones), chemical analyses, in silico essays (bioinformatics) and
biological and biochemical essays shall be conducted to check any allergenic
potential of the heterolog protein.
Consumption of alimentary cotton products by man is very limited. Therefore,
there will be an insignificant exposure to Cry1F, Cr1Ac and PAT proteins in the
human diet. The predominant food product derived from cotton is the seed oil,
where the protein content is null. On the other hand, animals may consume cotton
seed, bran, husk and byproducts as part of their feeding. However, an
investigation of the respective products of heterolog expression showed that the
recombinant proteins displayed the expected molecular and catalytic
characteristic.
One fact in favor of protein PAT safety is that transacetylases (the category of
PAT) are very common in nature (found in microbes, plants and animals) and are
recognized as being non-toxic and non-allergenic. Besides, up to the present,
there was no record of potential glycosylation sites or peptide, a sign that they
could result in transportation to the endoplasmatic reticulum, place where
potential glycosylation could occur, were recorded. PAT protein rapidly degrades
in high temperatures. Data about its behavior in simulated digestive fluids and
acute oral toxicity caused EPA to issue a final notice in 1977 exempting PAT of
the need to establish a tolerance level for all unprocessed commodities when
used as an inert protection incorporated to the plant.
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Regarding Bt proteins, decades of experiments testing the proteins showed their
absence of toxicity to man and vertebrate animals and absence of adverse effects
to non-target organisms and the environment. Besides, commercial formulations
of B. thuringiensis containing such proteins have been used in Brazil and other
countries to control some agricultural pests for over 40 years. Since this
bacterium is a soil microorganism, the exposure of living organisms and
environment to this bacterium or any element extracted thereof is an event that
occurs abundantly in nature. Besides, proteins Cry1F and Cr1Ac have very
specific action and operate, only through ingestion, in some species of the
Lepidoptera order.
Further favoring the safety of such recombinant proteins, data were submitted
from the complete characterization of each protein. The more significant data are:
absence of homology of the proteins with known toxins and allergenics;
pronounced thermolability; rapid digestion under simulated gastric conditions;
absence of glycosylation; absence of acute toxicity in rodents; and absence of
adverse effects in poultry fed with rations containing the recombinant proteins.
Besides, all possible interactions among proteins Cry1F, Cr1Ac and PAT were
assessed to estimate possible unforeseeable interactions that may cause adverse
effects to man and animals. All results supported the initial evidences that the
stacked cotton safe from the alimentary viewpoint.
Data submitted by applicant proved that the heterolog proteins are present in very
low quantity and in different tissues, an excellent sign of the recombinant proteins
safety for humans, since they are clearly different from those typically presented
as allergenic proteins. In general, allergenic proteins are reserve proteins of plant
organs or tissues, pathogeny- or stress-related proteins, and, in addition
allergenic proteins are abundant in the plant and present in high levels over the
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plant tissues.
Experiments additionally proved that genetically modified cotton plants fail to
exhibit morphologic, phenologic or architectural alterations and that there was no
effect of the gene insertion to the quality of fibers. Except for the tolerance to
target-insects along the crop, the genetically modified cotton plants displayed
equivalence in all the analyzed phenotypic and agronomic characteristics against
the standard displayed by the non-transformed parental lineage and other
varieties used in commercial farming. Examination of documents submitted
supports the conclusion that cultivation of cotton event 281-24-236/3006-210-23
will not cause changes in soil and its ecologic and functional relations different
from the ones caused by conventional cotton varieties.
Besides, studies conducted did not show changes in main natural components
and antinutrients found in cotton. Safety of alimentary products from the
transgenic cotton was determined by equivalence in composition of macro- and
micronutrients in salubrity studies with animals. The conclusion was that the
product, as a component of animal fodder and the recombinant proteins
expressed in the plant tissues proved to be safe and displaying a nutritional value
equivalent for human and animal consumption. Quality and composition analyses
of seeds from cotton event 281-24-236/3006-210-23 showed that the properties
of the genetically modified cotton and its processed fractions were comparable to
the properties of conventional cotton.
For the foregoing, based on the data submitted by applicant and independent
papers consulted in the literature and considering the internationally accepted
criteria in the process of risk analyses of genetically modified raw-materials,
based on the concept of substantial equivalence, a conclusion emerges that
cotton event 281-24-236/3006-210-23 is as safe for human and animal
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consumption as its conventional equivalent.
V. Environmental Aspects
Modern agriculture is an activity responsible for significant negative
environmental impacts(9, 42, 79, 155, 193) and, therefore, the risk assessment of any
GM event shall be conducted in relation to that impact inherent to conventional
agriculture(13, 46, 138). Therefore, the analysis conducted by CTNBio intended to
assess whether the impact caused by WideStrike Cotton is significantly higher
than the one caused by conventional cotton varieties considering the practices
associated to each system.
All species of the Gossypium genus posses perfect flowers. Fecundation takes
place promptly after anthesis, and either self-fecundation, crossed pollination or
both are possible. The cotton plant pollen is relatively large, ranging from 81 to
143 micra, viscous (making the pollen grains to adhere to each other), spherical
in format, covered by a large amount of spicules and in practice is not transported
by wind(47). In the fields, its viability extends to late afternoon, but may last for up
to 24 hours if stored at temperatures from 2ºC to 3ºC(27). In order for crossed
fecundation to happen, presence of pollinating insects is necessary, mainly those
insects of the Hymenopterae family(29, 149, 150, 164). The crossing rate recorded
between cotton plant cultures is relatively low, displaying figures that enable
classifying cotton as a partially autogamous species or a species of a mixed
reproduction system.
Some authors suggest that gene flow from GM plants to wild genotypes may
result in biodiversity reduction. However, reduction of genetic variability results
from gene introgression, a process far more complex than simple hybridization(46,
49, 80, 175). In order for introgression to happen, hybridization is first necessary and
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later a series of retro-crossings so that a gene be incorporated in a permanent
way into a new genome(80, 81).
Ecotoxicity studies with Cry1F and Cr1Ac were conducted in soil invertebrates
and the results showed that protein Cry1F or Cr1Ac derived by microbial way,
either pure or in combination with protein Cr1Ac or Cry1F failed to display toxicity
to earthworms (Eisenia foetida). A laboratory study was also conducted to
determine the chronic effects of the protein Cry1F to survival and reproduction of
the collembolan soil invertebrate (Folsimia candida), which plays an important
role in soil ecosystems due to the fact that it feeds on decomposed plant material,
using Cry1F derived by microbial way added to beer yeast, the staple collembolan
diet(192). The tested concentration was 709 mg of Cry1F protein per kg of diet, or
702 mg of Cry1F protein per kg of diet in combination with protein Cr1Ac. There
was no noticeable effect with the exposure to protein Cry1F in the diet. A
laboratory study was conducted with protein Cr1Ac to determine the chronic
effects of this protein by microbial way added to beer yeast, the staple
collembolan diet(192). Protein Cr1Ac failed to cause significant effects to survival
and reproduction of adults.
The effects of proteins Cry were tested in water organisms and no adverse effects
were recorded for the water invertebrate Daphnia magna. Acute toxicity from
protein Cry1F for rainbow trout (Onchorynchus mykis) was determined for fish
exposed for eight days to a commercial type of trout pelletized diet containing
10% of bran prepared with cotton expressing proteins Cry1F and Cr1Ac(128). This
produced a diet containing an initial dose of 0.209 gram of Cry1F per gram of
food in combination with protein Cr1Ac. The control diet consisted of the same
commercial fish diet prepared with non transgenic cotton bran. No mortality of fish
of sub-lethal effects were reported for both the fish feeding on control diet and fish
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feeding on the GMO diet. Acute toxicity from the diet with protein Cr1Ac to the
rainbow trout (Onchorynchus mykiss) was determined for individuals exposed for
eight days to a commercial-type trout pelletized diet containing 10% of bran
prepared with cotton expressing proteins Cry1F and Cr1Ac(128). This produced a
diet containing an initial dose of 0.118 gram of Cr1Ac per gram of food in
combination with protein Cr1Ac. The control diet consisted of the same
commercial diet prepared with non-transgenic cotton bran. No mortality of fish or
sub-lethal effect was recorded for both, the control diet and the diet containing the
GMO.
Regarding non-target arthropods, no effects were recorded in average survival of
bees exposed to 2 mg of pollen of event expressing Cry1F , or 1.98 mg/ml of
protein Cry1F in combination with protein Cr1Ac(124). CL50 in a diet for green
lacewing (Chryosperia carnea) exposed to protein Cry1F, pure or in combination
with protein Cr1Ac, was investigated in a series of studies with the microbial
protein administered in a diet of moth eggs(185). There was no effect of Cry1F at
5.2 mg/g in combination with Cr1Ac. The test with Cry1F alone also failed to show
any effect. Toxicity for green lacewing is not held as ecologically relevant for risk
assessment of cotton event 281-24-236, since the exposure, as case it happens,
shall be indirect and results of field censuses failed to reveal any impact of protein
Cry1F(125). Parasite hymenoptera (Nasonia vitripennis) were exposed to a single
boundary concentration of protein Cry1F, pure and in combination with protein
Cr1Ac, in sugared water for 10 days. There were no significant difference in
mortality between the two groups of treatment and a control negative for sugared
water on day 9, which was the last day of observation before mortality increased
beyond the ceiling established in the protocol for negative control(20%). The
same result was obtained with protein Cr1Ac, pure and in combination with
protein Cry1F. Adult ladybugs (Hipodamia convergens) were not affected when
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exposed to protein Cry1F or Cr1Ac expressed by microbial way, pure or in
combination with protein Cr1Ac or Cry1F.
Non-target organisms may be either directly or indirectly exposed to protein
Cry1F expressed in 281-24-236. Exposure estimates for organisms feeding
directly on cotton tissues expressing protein Cry1F are based on the upper limit of
expression in the specific plant tissue to which the non-target organism may be
exposed through direct ingestion. Estimates of upper exposure limit (UEL)
represent 90% of the upper limit of expression recorded(200). Indirect exposures
represent unadvised exposures to protein Cry1F through the soil, pollen on
tissues of the host plant, and multitrophic interactions. Such exposures are known
as Estimated Environmental Concentrations (EEC) and are calculated in a
conservative way using the higher estimates for the parameters used in the
calculation.
Direct feeding on plants of parts or plants constitute the primary ways of exposure
of organisms to protein Cry1F expressed in event 281-24-236 and to protein
Cr1Ac expressed in event 3006-210-23. The plant parts subject to feeding are
mainly the leaves, roots, stalks and, possibly, nectar. Organisms feeding directly
on cotton as a primary source of food within agroecosystems are characterized as
plant pests and are not analyzed here. Organisms incidentally exposed to
residues of plants and organisms consuming cotton plants of parts or cotton
plants as an occasional or supplementary source of food are held as non-target
organisms that deserve consideration in this exposure assessment. Secondary
exposure to protein residues through tritrophic interactions may occur for
predators or parasites of organisms that feed on plants. Residues occurring in the
soil or water matrices may be a way of additional secondary exposure to protein
Cry1F and Cr1Ac. Absence of effects in ecotoxicity tests for non-target organisms
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show large safety margins related to environment concentrations of foreseen
exposures, in a conservative manner, and such observations are supported by
field monitoring of the species abundance(125). Exposure ways postulated in here
are therefore relevant for exposure and risk characterization for the rate of
Lepidoptera potentially susceptible.
Conditions favorable to degradation of proteins Cry1F and Cr1Ac in the soil were
described in studies where proteins Cry1F and Cr1Ac, either derived from plants
or derived by microbial way were mixed to the soil, incubated under standard
laboratory conditions, and then sampled for bioassay in different time intervals(84).
Bioessays were performed in insects to measure protein degradation, as loss of
biological activity, by applying mixtures of water-agar in soil samples put in
artificial diets and leaving the neonate caterpillars of tobacco budworm (Heliothis
virescens) to feed on this prepared medium. Based on the bio-essay results
(GI50) for soil added of Cry1F, derived by microbial way, the half-life was 1.3
days under laboratory conditions, suggesting a high rate of decomposition in the
soil. This fast decomposition was verified in liofilized cotton tissue containing
protein Cry1F in combination with protein Cr1Ac, where the half life of bioactivity
was below one day. Bioessays with truncated protein Cry1F also displayed a half
live in soil of less than one day(85). Microbial protein Cr1Ac failed to decompose
when applied to microorganisms in non viable soil(84), but the rapid decomposition
took place when the leaf tissue of the liofilized cotton 281-24-236/3006-210-23
containing proteins Cry1F and Cr1Ac was applied in viable soil(84). These results
are consistent with degradation of protein Cr1Ac in soil mediated by
microorganisms. Based on results of the bioassay (GI50) in soil to which liofilized
cotton tissue was added, the half life of crystallized insecticides Cry1F/Cr1Ac was
1.3 day under laboratory conditions, suggesting a high decomposition rate in soil.
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A soil representative of cotton agroecosystem was examined in a laboratory
study. The protein decomposed very fast and was consistent with what has been
seen for other Bt proteins in a lineage of soils(86, 87). The study submitted
demonstrates the ability of soil microbes to rapidly degrade such Bt proteins. In
addition, field studies including multiple locations and a lineage of soils were
planned to focus the potential accumulation of proteins Cry1F and Cr1Ac
expressed in cotton 281-24-236/3006-210-23. A report published in one of such
multiple field studies found non accumulation(83) and served as a model for the
study conducted with cotton 3006-210-23/281-24-236.
Bt cotton, when compared with conventional cotton with insecticides, failed to
affect arthropod populations collected in soil pitfalls, yellow tray and yellow
adhesive card. The main arthropods collected in such pitfalls were the
phytophagous Euxesta sp., Chaetopsis sp., Liriomyza sp., Aphis sp. and thrips of
the Thripidae family; predators Dolichopodidae sp., parasitoids Aphididae,
Aphidius sp. and Encyrtidae; and decomposers Phora sp and Sarcophagidae. Bt
cotton failed to affect occurrence of predator arthropods in plants, among which
one may mention bugs Orius sp., Nabis sp. Geocoris sp. and Zelus sp; ladybugs
Cycloneda sanguine, Scymnus sp., Hyppodamia convergens and Stetorus sp.;
spiders Chiracantum sp., Oxyopes sp., Phidippus sp., Misumenopsis sp., and
Latrodectus, sp.; crysopid Ceraeochrysa sp., Doru luteipes and the sylphid
Toxomerus dispar. Hymenoptera parasitoids captured in yellow trays were not
abundant in any of the treatments, totaling 3% of collected insects. The
conclusions were that:
- Bt cotton failed to cause a negative impact on arthropod populations in
the soil;
- Bt cotton, when compared with conventional cotton with insecticides,
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failed to affect the arthropod population collected in soil pitfalls, yellow
tray and adhesive card;
- Bt cotton failed to harm the occurrence of predator arthropods in plants.
Cotton is basically a culture of self-fecundation, with some crossed pollination by
bumble bees, Melisode bees and honeybees; Lepidoptera insects are not
pollinators of cultivated cotton(129). Consequently, environmental exposure of a
lepidopter sensitive to cotton pollen would be indirect, through contamination of
their source of food by noxious species. Indirect exposure to cotton pollen was
considered to be non significant.
Incidental exposure of a non-target butterfly or moth, in the larval stage sensitive
to Cry1F may occur if the pollen of 281-24-236 cotton is present in host plants
and is consumed. In a study conducted in the United States, indirect exposure of
a larva of a hypothetical non-target lepidopter sensitive to cotton pollen was
insignificant, as shown in the case of the Monarch butterfly feeding on Sapium
Glandulatum, as an example of a non-target lepidopter occurring in host plants
inside or close to cotton fields. The likelihood of exposure is remote due to the
non-significant pollen flow of cotton; there is, therefore, an insignificant risk for
non-target butterflies and moths present in cotton fields of event 281-24-236. The
Monarch butterfly was simply used as a model species for the purpose of this
assessment, since it would be minimally exposed to pollen of event 281-24-236,
since its migration in spring through cotton cultivation regions takes place before
flowering time. Incidental exposure of a non-target butterfly or moth, during the
larval stage sensitive to Cr1Ac may occur if the pollen of cotton 3006-210-23 is
present in host plants and is consumed. Indirect exposure of a non-target
lepidopter larva sensitive to cotton pollen is insignificant. The likelihood of
exposure is remote due to the insignificant dispersion of cotton pollen. The
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Monarch butterfly was used as a model species for the purpose of this
assessment.
The consequences of cultivating cotton expressing proteins Cry1F and Cr1Ac on
beneficial insects was studied by Wolt (2002)(206). Over 300 species of beneficial
insects are known inhabitants of cotton fields. Common arthropods, predators and
parasites in cotton fields represent orders that are not sensitive to Cry1 proteins.
Besides, such organisms are predominantly predators and parasites, and only in
few examples they consume plant products (pollen and nectar) and, in these
cases, consumption is made by adults, in a stage of life when the species is no
longer sensitive. Direct risks to beneficial insects, therefore, of exposure to protein
Cry1F expressed in cotton event 281-24-236 and to protein Cr1Ac expressed in
cotton event 3006-210-23 are negligible for practical purposes. Indirect risk to
proteins Cry1F and Cr1Ac through tritrophic feeding with host/prey insects is also
non-significant, due to the low levels of exposure foreseen in comparison with
levels of effects shown in the tests conducted. Security margins shown in risk
assessments are very conservative, since they are based on a level of expression
of the proteins in plants, while secondary exposure would be significantly reduced
in tritrophic feeding.
Based on the above analysis, there are no ecologically relevant concerns with
cultivation of cotton 281-24-236/3006-210-23 expressing proteins Cry1F and
Cr1Ac. Selectivity, exposure ways and concentrations restrict any potential risk
posed to lepidopter insects directly exposed to residues of event 281-24-
236/3006-210-23. For this rate, the likelihood of an adverse environmental
consequence is insignificant, as shown by the toxicity test of non-target
organisms, which indicate concentrations with no effect in doses well above the
concentrations of the environmental exposure foreseen in a conservative manner.
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Abundance in fields corroborates the absence of risk at the typical rates of cotton
agroecosystems. Experience with commercial farming in other countries
corroborates such experimental results. No adverse environmental effect
associated with this product was detected.
VI. Restrictions to the use of GMO and derivatives
Technical opinions related to agronomic performance concluded that there is
equivalence between transgenic and conventional plants. Therefore, the data
suggest that transgenic cotton plants are not fundamentally different from the
genotypes of non-transformed cotton plants, except for the resistance to certain
insects. In addition, there is no evidence of adverse reactions to the use of
WideStrike Cotton event 281-24-236/3006-210-23. For the foregoing, there are no
restrictions to the use of this cotton or its derivatives, either as human or animal
food.
As established by Article 11 of Law no. 11,460, of March 21, 2007 “research and
cultivation of genetically modified organisms may not be conducted in indigenous
lands and areas of conservation units.”
VII. Considerations on particulars of different regions of the country
(contribution to supervision agencies)
WideStrike cotton event 281-24-236/3006-210-23 technology was shown to be
usable under all agricultural practices commonly used in different regions under
different conditions, considering availability of inputs and labor, among other
inputs used in the culture of cotton.
There are not creole varieties of cotton plants and the chains of special cotton
plants, both conventional and transgenic, have lived together in a satisfactory
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fashion, without any record of coexistence problems.
VIII. Conclusion
Whereas:***
1. Event 281-24-236/3006-210-23 Cotton is as safe for human and animal
consumption as the conventional cotton;
2. All possible interactions among proteins Cry1F, Cr1Ac and PAT were
assessed in order to estimate any possible unforeseen interactions that
my cause adverse effects to man and animals, and all results supported
the initial evidences that stacked cotton is safe from the alimentary point
of view;
3. Event 281-24-236/3006-210-23 Cotton contributes to reduce the use of
insecticides and, consequently, reduces the impacts of such pesticides in
the environment, human and animal health;
4. The use of technology such as insect-resistant Bt cotton may have a
positive impact to population preservation of non-target organisms and
beneficial insects, making integrated management of crop pests easier;
5. Cultivation of event 281-24-236/3006-210-23 cotton will not cause
changes in the soil and its functional and ecologic relations different from
the changes caused by conventional corn varieties;
6. Proteins Cry1F and Cr1Ac have very specific action and act, through
ingestion only, in some species of the Lepidoptera order;
7. Protein PAT was extensively discussed by CTNBio during analysis
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related to commercial release of Liberty Link corn, Bt corn and Liberty
Link cotton, where the conclusion was that such protein has no known
toxic and allergenic potential and that is rapidly digested by enzymes
(proteases) in the digestive system of animals, eliminating most of this
protein potential to be allergenic when consumed;
8. Assessment of proteins Cry1F and PAT expressed in event 281-24-236
and proteins Cr1Ac and PAT expressed in event 3006-210-23 failed to
identify any allergenicity potential in food products;
9. The event produces a protein that is toxic to some cotton pest insects
only, and does not have properties allergenic to mammals;
10. The source organism of proteins Cry1F and Cr1Ac, Bacillus thuringiensis,
and PAT, Streptomyces viridochromogenes are not recognized as
allergenic;
11. There are no ecologically relevant concerns with cultivation of 281-24-
236/3006-210-23 cotton expressing proteins Cry1F and Cr1Ac, since no
adverse environmental effect associated to this product was recorded;
12. Event 281-24-236/3006-210-23 Cotton is nutritionally equivalent to
conventional cotton;
13. Nutritional and productive equivalence of WideStrike Cotton was
evidenced in different research works conducted in the United States and
herein enclosed;
14. Nutritional quality of cotton remained unchanged related to ashes, total
fat, humidity, protein, carbohydrate, calorie, total fiber, fiber in acid
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detergent, fiber in neutral detergent, amino acids in seeds and bran, fatty
acids in seed and seed oil, tocopherols in seed oil fraction, antinutrients in
leaf and floral bud and seed, oil and bran;
15. Crossing of WideStrike Cotton event 281-24-236/3006-210-23 with wild
kindred cotton and generation of hybrid offspring that may change into
pest plants harmful to agriculture or plants invaders of natural habitats is
unlikely;
16. Finally, given the detailed data submitted by applicant, the results
obtained in control and safety essays on this genetically modified
organism, the elements credited to authors of scientific works mentioned
and inexistence of facts contrary to nutritional, toxicological and allergenic
safety, no matter how much they have been scrutinized;
CTNBio finds that this activity is not a potential cause of significant degradation to
the environment nor harmful to human and animal health. Restrictions to the use
of such GMO and its derivatives are conditions to the provisions of CTNBio Ruling
Resolution nº 03 and Ruling Resolution nº 04.
According to Annex I of Ruling Resolution 5, of March 12, 2008, applicant shall
make adjustments to its proposed post-commercial release monitoring plan within
thirty (30) days from publication of this Technical Opinion.
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