Supporting Document 1 (at Approval)

37 Pages·2011·2.1 MB·English

Supporting Document 1 (at Approval)

APPLICATION A1049 – FOOD DERIVED FROM
HERBICIDE-TOLERANT, HIGH OLEIC ACID
SOYBEAN LINE MON87705

SAFETY ASSESSMENT REPORT

SUMMARY AND CONCLUSIONS

Background

Soybean line MON87705 has been genetically modified (GM) to produce soybean oil with
increased levels of oleic acid, a monounsaturated fatty acid, and decreased levels of linoleic
acid, a polyunsaturated fatty acid (PUFA), to improve its suitability for food and industrial
uses. At the same time, a slight reduction in saturated fats was achieved. The changes in
the fatty acid profile are due to the down-regulation of two key enzymes, FATB and FAD2,
involved in fatty acid biosynthesis in soybean seeds. This was achieved through the use of
RNA-based suppression of two endogenous soybean genes, fatb1a and fad2-1a. Soybean
MON87705 also contains the 5-enolpyruvylshikimate-3-phosphate synthase gene derived
from Agrobacterium sp. strain CP4. This gene is referred to as cp4 epsps, and confers
tolerance to the herbicide glyphosate.

The oxidative stability of vegetable oils is greatly influenced by the proportion of
monounsaturated to polyunsaturated fatty acids. Conventional soybean oil typically contains
60-65% PUFAs (mostly in the form of linoleic acid) which are susceptible to oxidation and
degradation at high temperatures. High oleic acid soybean oils, such as that from
MON87705 soybean, are estimated to have oxidative stability around 17 times greater than
conventional soybean oil, providing more stable oil for use in food formulations.

This safety assessment report addresses only food safety and nutritional issues. It does not
address: environmental risks related to the environmental release of GM plants used in food
production; the safety of animal feed or animals fed with feed derived from GM plants used
in food production; the safety of animal feed or animals fed with feed derived from GM
plants; or the safety of food derived from the non-GM (conventional) plant.

History of Use

Soybean (Glycine max L. Merr.) has a long history of safe use as food for humans and feed
for livestock, as well as a range of industrial uses. Soybean oil accounts for over 90% of the
soybean products consumed by humans.

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Soybean MON87705 is intended primarily for use as a broad acre field soybean and not for
vegetable or garden use. After extraction of the nutrient-altered oil from MON87705
soybean, the remaining processed food fractions would likely be blended with those derived
from other commercial soybean varieties before entering the food supply in a range of
processed products.

Molecular Characterisation

Comprehensive molecular characterisation of MON87705 indicated that a functional two-
gene suppression cassette and intact cp4 epsps gene co-integrated into the soybean
genome at a single insertion site. The complete FAD2-1A/FATB1-A suppression cassette,
under the control of a seed specific promoter, encodes an RNA transcript corresponding to
short complementary segments of the soybean fad2-1a and fatb1-a genes. The RNA
transcript forms a double-stranded RNA structure (hairpin) that suppresses endogenous
FATB and FAD2 RNA levels via RNA interference (RNAi) mechanisms, subsequently
leading to the desired fatty acid phenotype.

As demonstrated in other herbicide tolerant GM crops, expression of the cp4 epsps gene in
chloroplasts confers tolerance to glyphosate herbicides at the level of the whole plant.

Some genomic rearrangements were observed at the insertion site, however bioinformatic
analysis did not indicate unintended gene products with homology to known toxins or
allergens.

The genetic elements introduced into MON87705 were demonstrated to be stably integrated
into the soybean genome and follow a Mendelian pattern of inheritance for a single genetic
locus.

Characterisation of Novel Protein

Soybean MON87705 expresses one novel protein, CP4 EPSPS, which is expressed in
leaves, roots, forage and seeds. The average CP4 EPSPS content in seeds of MON87705
soybean is 110 µg/g dry weight.

The safety of CP4 EPSPS as a novel protein conferring tolerance to glyphosate has been
assessed multiple times in different GM crops. Based on numerous studies and use in GM
crops for almost two decades, the CP4 EPSPS protein has been demonstrated to lack any
biologically significant sequence similarity to known protein toxins or allergens, and to be
readily degraded in conditions that simulate normal digestion. The evidence confirms that
the CP4 EPSPS protein is neither toxic nor allergenic in humans.

Compositional Analyses

Detailed compositional analyses of seeds harvested from MON87705 soybean, the isogenic
control and a number of commercial soybean varieties grown at different field sites,
demonstrate that MON87705 contains the same five major fatty acids as found in
conventional soybean, but in different proportions. Oleic acid is significantly increased from
approximately 23% in conventional soybean to around 76% of total fatty acids in MON87705
soybean. The levels of linoleic acid are significantly decreased from 53% total fatty acids to
approximately 10% in MON87705. In addition the saturated fatty acids, palmitic and stearic
acids, together comprise 6% of fatty acids in MON87705 compared with 15% in conventional
soybean. Smaller differences in other fatty acids were within the range established for
commercial reference soybean varieties, or in the published literature.

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Except for the intended changes to four major fatty acids, the composition of MON87705
seed is comparable to that of the conventional soybean control. No significant differences
between MON87705 and conventional soybean were observed from analysis of proximates,
fibre, amino acids, key nutrients and anti-nutrients present in soybean. Comparisons of the
composition of processed soybean fractions: refined, bleached and deodorised soybean oil,
protein isolate and lecithin from MON87705 and conventional soybean varieties, confirmed
the intended changes in the fatty acid profile of MON87705 soybean oil and did not reveal
other differences of any biological or nutritional significance.

Nutritional Impact

Dietary exposure assessments indicated that substitution with high oleic acid soybean oil
derived from MON87705 for conventional soybean oil in food products, would result in
marginally higher intakes of oleic acid and lower intakes of saturated fats, within the range
normally associated with consumption of a variety of edible vegetable oils.

In addition, a 90-day feeding study in rats and a 42-day dietary study in broiler chickens both
demonstrated that a diet containing MON87705 soybean meal is equivalent to a
conventional soybean diet in its nutritional quality and ability to support typical growth and
wellbeing in animals.

Conclusion

No potential public health and safety concerns have been identified in the assessment of
high oleic acid soybean MON87705. On the basis of the data provided by the Applicant, and
information obtained from other sources, food derived from soybean MON87705 is
considered as safe for human consumption as food derived from conventional soybean
varieties.
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TABLE OF CONTENTS
SUMMARY AND CONCLUSIONS ........................................... ERROR! BOOKMARK NOT DEFINED.
1 INTRODUCTION .................................................................................................................... 2
2 HISTORY OF USE ................................................................................................................. 2
2.1 DONOR ORGANISM ................................................................................................................ 2
2.2 HOST ORGANISM ................................................................................................................... 2
3 MOLECULAR CHARACTERISATION ................................................................................ 3
3.1 METHOD USED IN THE GENETIC MODIFICATION ................................................................... 3
3.2 FUNCTION AND REGULATION OF INTRODUCED GENE SEQUENCES .................................... 4
3.3 CHARACTERISATION OF THE GENES IN THE PLANT ............................................................. 7
3.4 OPEN READING FRAME ANALYSIS ....................................................................................... 9
3.5 STABILITY OF GENETIC CHANGES ...................................................................................... 10
3.6 CONCLUSION ABOUT MOLECULAR CHARACTERISATION ................................................... 12
3.7 MODE OF ACTION AND EVIDENCE OF SUPPRESSION OF FATB AND FAD2 GENES ......... 13
4 CHARACTERISATION OF NOVEL PROTEINS .............................................................. 13
4.1 DESCRIPTION AND FUNCTION OF THE NOVEL PROTEIN .................................................... 14
4.2 EQUIVALENCE STUDIES ...................................................................................................... 14
4.3 CP4 EPSPS PROTEIN EXPRESSION ANALYSIS ................................................................. 16
4.4 BIOINFORMATICS REVIEW OF CP4 EPSPS ...................................................................... 18
4.5 SUMMARY AND CONCLUSION FROM CHARACTERISATION OF NOVEL PROTEIN ................ 19
5 COMPOSITIONAL ANALYSES ......................................................................................... 19
5.1 KEY COMPONENTS .............................................................................................................. 20
5.2 STUDY DESIGN .................................................................................................................... 20
5.3 ANALYSES OF KEY COMPONENTS ...................................................................................... 21
5.4 COMPOSITION OF PROCESSED SOYBEAN FRACTIONS ...................................................... 27
5.5 ALLERGENICITY ASSESSMENT ............................................................................................ 28
5.6 CONCLUSION FROM COMPOSITIONAL STUDIES ................................................................. 29
6 NUTRITIONAL IMPACT ...................................................................................................... 29
6.1 DIETARY INTAKE ESTIMATES .............................................................................................. 29
6.2 FEEDING STUDIES ............................................................................................................... 30
6.3 CONCLUSION ....................................................................................................................... 32
REFERENCES AND BIBLIOGRAPHY ...................................................................................... 33

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1 INTRODUCTION

Soybean line MON87705 has been genetically modified (GM) to produce an altered fatty
acid profile and for tolerance to glyphosate. The intended nutritional change, to increase the
level of oleic acid (C18:1) in soybean oil, was brought about by silencing two endogenous
genes involved in fatty acid biosynthesis in soybean plants. The net effect of the modification
also results in decreased levels of linoleic acid (C18:2), and saturated fatty acids palmitic
acid (C16:0) and stearic acid (C18:0), relative to conventional soybean oil. Due to the higher
oleic acid levels, soybean oil from MON87705 is suitable for a greater range of food
applications compared with conventional soybean oil.

The glyphosate tolerance trait introduced into MON87705 plants has been described
previously by FSANZ. Tolerance to glyphosate herbicides is conferred by expression of cp4
epsps, a gene derived from the common soil bacterium, Agrobacterium sp. strain CP4.

2 HISTORY OF USE

2.1 Donor organism

The donor for the only novel gene present in MON87705 soybean (cp4 epsps) is
Agrobacterium tumefaciens, a Gram-negative, non-spore forming, rod-shaped bacterium
commonly found in soil. It is closely related to other soil bacteria involved in nitrogen fixation
by certain plants. As such, Agrobacterium and related nitrogen fixing bacteria are commonly
found in association with plants, especially plant roots. Many strains of Agrobacterium
naturally contain a plasmid (the Ti plasmid) which has the ability to enter plant cells and
insert a portion of the plasmid into plant chromosomes. The result of this is the forced
production by the plant of specialised amino acids (called opines) that the bacterium uses as
a nitrogen source. As such, Agrobacterium is a plant pathogen that causes root deformation
in several crop species. However, techniques have been developed that allow use of
Agrobacterium sp to transform a broad range of plant species without causing adverse
effects in the host plant.

As Agrobacterium species are commonly found in soil and in association with plants, it is
likely that humans have had long exposure to them. They are not known to be pathogenic to
humans or animals and are not commonly allergenic (FAO/WHO, 1991). The cp4 epsps
gene from Agrobacterium sp. strain CP4 has been used widely to confer glyphosate-
tolerance to GM crop plants, and is not associated with any safety concerns.

2.2 Host organism

Soybean (Glycine max (L.) Merr.) is grown as a commercial crop in over 35 countries
worldwide and has a long history of safe use as human food and feed for livestock. The
major producers of soybeans, accounting for 90% of world production, are the United States,
Argentina, Brazil and China. In 2007, soybeans represented 56% of total world oilseed
production, and 32% of those soybeans were produced in the United States (The American
Soybean Association, 2008). In many soybean producing countries, GM soybean (mainly
with a herbicide tolerant trait) accounts for a significant proportion of the total soybean grown
e.g. United States (91%); Argentina (99%); Brazil (63%); South Africa (87%); Uruguay (99%)
(Brookes and Barfoot, 2009).

Australia, while a net importer of soybean, grows conventional crops in latitudes extending
from the tropics (16o S) to temperate regions (37o S), mainly in the eastern states and as a
rotational crop (James and Rose, 2004). The seed is used mainly to produce meal for use in
animal feed (Grey, 2006).
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Food products are derived either from whole or cracked soybeans. Whole soybeans are
used to produce soy sprouts, baked soybeans, roasted soybeans and traditional soy foods
such as miso, tofu, soy milk and soy sauce. Cracked soybeans have the hull (seed coat)
removed and are then rolled into flakes which undergo solvent extraction to remove the oil.
This crude oil is further refined to produce cooking oil, shortening and lecithins as well as
being incorporated into a variety of edible and technical/industrial products. The flakes are
dried and undergo further processing to form products such as meal (for use in livestock, pet
and poultry food), protein concentrate and isolate (for use in both edible and
technical/industrial products), and textured flour. Unprocessed (raw) soybeans are not
suitable for food use, and have only limited feed uses, as they contain toxicants and anti-
nutritional factors, such as lectins and trypsin inhibitors (OECD, 2001). Appropriate heat
processing inactivates these compounds.

3 MOLECULAR CHARACTERISATION

Molecular characterisation is necessary to provide an understanding of the genetic material
introduced into the host genome and helps to frame the subsequent parts of the safety
assessment. The molecular characterisation addresses three main aspects: the
transformation method together with a detailed description of the DNA sequences introduced
to the host genome; a characterisation of the inserted DNA including any rearrangements
that may have occurred as a consequence of the transformation; and the genetic stability of
the inserted DNA and any accompanying expressed traits.

Studies submitted:
A. Skipworth, K.R. Lawray, Q. Tian, J.D. Masucci. (2009). Amended report for MSL0022130:
Molecular analysis of soybean MON87705. Monsanto study report MSL0022384

H. Tu and A. Silvanovich. (2009). Bioinformatics evaluation of DNA sequences flanking the 5’and 3’
junctions of the inserted DNA in MON87705: Assessment of putative polypeptides. Monsanto study
report MSL 0021929

A. Silvanovich and H. Tu. (2009). Additional bioinformatic evaluation of DNA sequences flanking the
5’ junction of the inserted DNA in MON87705: assessment of putative polypeptides. Monsanto study
report MSL 0022346

H. Tu and A. Silvanovich. (2010). Updated bioinformatics evaluation of the DNA sequences flanking
the insertion site in MON87705: BLASTn and BLASTx analyses. Monsanto laboratory project study
number RAR-10-071

H. Tu and A. Silvanovich. (2010). Updated bioinformatics evaluation of the transfer DNA in
MON87705 utilizing the AD_2010, TOX_2-10, and PRT_2010 databases. Monsanto laboratory
project study number RAR-10-070

3.1 Method used in the genetic modification

Conventional soybean cultivar A3525 was transformed with the plasmid PV-GMPQ/HT4404
(see Figure 1), using standard Agrobacterium-mediated transformation methods (Martinell et
al., 2002). The vector consisted of two independent T-DNAs, each flanked by the Left and
Right border sequences from Agrobacterium.
Meristem tissues were excised from the embryos of germinated A3525 seed, and co-
cultured with Agrobacterium tumefaciens, carrying PV-GMPQ/HT4404 (see Figure 1). After
co-culturing, the meristem tissues were placed on glyphosate-containing medium to select
for transformed tissues. The selected tissues were then cultured on medium conducive to
shoot and root development, and rooted plants with normal phenotypic characteristics were
transferred to soil for further assessment.
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Figure 1: Map of plasmid PV-GMPQ/HT4404 showing two independent T-DNAs, labelled T-
DNA I and T-DNA II (see Section 3.2).
These R0 plants were self-pollinated to create the R1 generation. R1 plants were screened
for homozygosity of the inserted cp4 epsps gene. Only those homozygous plants that also
produced the desired fatty acid composition were advanced for development and further
characterisation. MON87705 was selected based on its desired phenotypic and agronomic
characteristics, and molecular profile, as described in Figure 2.
3.2 Function and regulation of introduced gene sequences

Information on the genetic elements present in the transforming plasmid PV-GMPQ/HT4404
is provided in Table 1. The vector contained two independent T-DNAs, as described in the
following sections. T-DNA I included a cp4 epsps expression cassette and a partial gene
suppression cassette. T-DNA II contained another partial gene suppression cassette. The
suppression cassette comprised the sequences and regulatory elements necessary for the
RNA-based suppression of the endogenous FAD2 and FATB genes.

The FATB enzymes, localised in plastids, are acyl-acyl carrier protein (ACP) thioesterases,
which hydrolyse saturated fatty acids from the ACP-fatty acid moiety. In MON87705
soybean, suppression of FATB results in a decrease in transport of saturated fatty acids out
of the plastid, thus increasing their availability for other reactions, specifically conversion to
oleic acid via a desaturation reaction.

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The FAD2 enzymes are delta-12 fatty acid desaturases, catalysing the conversion of oleic
acid (C18:1) to linoleic acid (C18:2). Suppression of FAD2 in MON87705 soybean causes
reduced desaturation of oleic acid to linoleic acid, resulting in more oleic acid being available
for transport from the endoplasmic reticulum to the oil body.

The combined effect of the RNA-based suppression of the two genes in MON87705 is that
soybean oil contains (i) reduced levels of saturated fatty acids (C16:0 palmitic acid and
C18:0 stearic acid), (ii) increased levels of monounsaturated oleic acid, and (iii) decreased
levels of linoleic acid, compared with conventional soybean oil.

Figure 2: Summary of the development of soybean MON87705.

3.2.1 The FAD2-1A/FATB1-A suppression cassettes

The partial suppression sequences were derived from the fad2-1a and fatb1-a genes from
soybean. T-DNA I contains sequences corresponding to the sense strand of a portion of the
soybean FAD2-1A intron, and a portion of the FATB1-A 5’ UTR (untranslated region),
including the plastid targeting sequence.
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Table 1: Description of the genetic elements contained in the vector PV-GMPQ/HT4404
Location in
Genetic element Plasmid Source and Function (Reference)
T-DNA I
DNA region from Agrobacterium tumefaciens containing the left border
Left Border 7657-8098 sequence used for transfer of T-DNA (Barker et al., 1983)

Intervening sequence 8099-8134 Sequence used in DNA cloning
Chimeric promoter consisting of enhancer sequences from the promoter of the
Figwort Mosaic Virus 35S RNA (Richins et al., 1987) combined with the
P-FMV/TsfI 8135-9174 promoter from the TsfI gene of Arabidopsis thaliana that encodes elongation
factor EF-1 alpha (Axelos et al., 1989)

5’ untranslated leader (exon 1) from the TsfI gene of Arabidopsis thaliana -
L-TsfI 9175-9220 encodes elongation factor EF-1 alpha (Axelos et al., 1989)

Intron with flanking exon sequence from the TsfI gene of of Arabidopsis thaliana
I-TsfI 9221-9842 that encodes elongation factor EF-1 alpha (Axelos et al., 1989)

Intervening sequence 9843-9851 Sequence used in DNA cloning
Targeting sequence from the ShkG gene encoding the transit peptide region of
Arabidopsis thaliana EPSPS (Klee et al., 1987) that directs transport of CP4
TS-CTP2 9852-10079
EPSPS to the chloroplast.

Codon modified coding sequence of th3e aroA gene from Agrobacterium sp.
strain CP4, encoding the CP4 EPSPS protein (Barry et al., 1997; Padgette et
CS-cp4 epsps 10080-11447
al., 1996)

Intervening sequence 11448-11505 Sequence used in DNA cloning
3’ untranslated region of the pea RbsS2 gene which functions to direct
T-E9 11506-12148 polyadenylation of the mRNA (Coruzzi et al., 1984)

Intervening sequence 12149-12236 Sequence used in DNA cloning
Promoter and leader from the SphasI gene of Glycine max encoding beta-
P-7Sα’ 12237-13077 conglycinin storage protein (alpha’-bcsp) (Doyle et al., 1986) that directs
transcription in seed

Intervening sequence 13078-11 Sequence used in DNA cloning
Partial sequence from intron #1 of the Glycine max FAD2-1A gene that encodes
FAD2-1AP 12-277 the delta-12 desaturase (Fillatti et al., 2003) which forms part of the suppression
cassette

Partial sequence from the 5’ untranslated region and the plastid targeting
sequence from the Glycine max FATB1-A gene that encodes the palmitoyl acyl
FATB1-AP 278-578 carrier protein thioesterase (Fillatti et al., 2003) which forms part of the
suppression cassette.

Intervening sequence 579-616 Sequence used in DNA cloning
DNA region from Agrobacterium tumefaciens containing the right border
Right Border 617-973 sequence used for transfer of the T-DNA (Zambryski et al., 1982)

T-DNA II
DNA region from Agrobacterium tumefaciens containing the left border
Left Border 5128-5569 sequence used for transfer of the T-DNA (Barker et al., 1983)

3’UTR sequence of the H6 gene from Gossypium barbadense encoding a fibre
T-H6 5668-6103 protein involved in secondary cell wall assembly (John and Keller, 1995)

Intervening sequence 6104-6115 Sequence used in DNA cloning
Partial sequence from intron #1 of the Glycine max FAD2-1A gene that encodes
FAD2-1AP 6116-6381 the delta-12 desaturase (Fillatti et al., 2003) which forms part of the suppression
cassette

Partial sequence from the 5’ untranslated region and the plastid targeting
sequence from the Glycine max FATB1-A gene that encodes the palmitoyl acyl
FATB1-AP 6382-6682 carrier protein thioesterase (Fillatti et al., 2003) which forms part of the
suppression cassette.

Intervening sequence 6683-6693 Sequence used in DNA cloning
DNA region from Agrobacterium tumefaciens containing the right border
Right Border 6694-7024 sequence used for transfer of the T-DNA (Zambryski et al., 1982)

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T-DNA II contains the same partial gene segments, but the sequence encodes the
corresponding antisense strand, followed by the H6 3’ untranslated sequence to terminate
transcription (John and Keller, 1995).

Homologous recombination between the border sequences of adjacent T-DNAs I and II,
leads to the formation in the soybean genome of a single segment of inserted DNA
corresponding to the intact cp4 epsps expression cassette (see below), and a functional
RNAi-mediated fad2-1a/fatb1-a suppression cassette. Transcription of the suppression
cassette leads to production of RNA which forms a double-stranded hairpin structure due to
the presence of the complementary partial gene sequences. This double-stranded RNA
(dsRNA) structure specifically interferes with expression of the endogenous fad2-1a and
fatb1-a genes via RNA interference mechanisms (Siomi and Siomi, 2009), thus reducing the
amount of FAD2-1A and FATB1-A proteins produced in the cell (see Figure 3).

Transcription of these sequences is under the regulation of the seed specific promoter 7Sα’
from soybean itself, which drives expression in immature seeds (Doyle et al., 1986). The use
of a tissue specific promoter limits the phenotype (modification to oil composition) to the
seeds of the plant.

3.2.2 The cp4 epsps expression cassette

MON87705 soybean contains a functional expression cassette comprising the cp4 epsps
gene sequence derived from Agrobacterium sp. strain CP4. The cp4 epsps gene is under
the regulation of the FMV/Tsf1 promoter, a chimeric promoter consisting of enhancer
sequences from the 35S promoter of the Figwort Mosaic Virus (FMV; Richens et al., 1987)
combined with the promoter from the Tsf1 gene of Arabidopsis thaliana, which encodes the
elongation factor EL-1 alpha (Axelos et al., 1989). The cassette also contains sequences
corresponding to the 5’ untranslated region of the Tsf1 gene, and CTP2 (chloroplast transit
peptide) from Arabidopsis which targets the CP4 EPSPS protein to the chloroplast (see
Table 1).

Expression of the CP4 EPSPS protein in MON87705 confers tolerance to glyphosate-
containing herbicides. The CP4 EPSPS protein in MON87705 is the same as that expressed
in several glyphosate-tolerant crops previously assessed and approved by FSANZ, such as
soybean (Applications A338 and A592), corn (Application A416), canola (Application A363),
sugarbeet (Applications A378 and A525) and cotton (Applications A355 and A553).

3.3 Characterisation of the genes in the plant

To fully characterise the changes in the soybean genome, a series of experiments were
conducted to determine whether the T-DNAs are present at more than one insertion site, or
whether incorporation of more than one copy of the T-DNAs within the one site has
occurred. Southern blots and DNA sequence analyses of plant material from the R3
generation of soybean MON87705 (see Figure 4) were performed to (i) characterise the
nature of the insert, (ii) evaluate insert copy number, and (iii) determine the presence or
absence of plasmid backbone sequences. The Southern analysis used multiple probes
(described in Table 2), corresponding to specific segments of the entire transforming plasmid
PV-GMPQ/HT4404. The parent line A3525, was used as a reference control for the
characterisation studies.

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