TAC Sub-Committee

Recommendations and
Findings
Genuity VT Double PRO (MON 89034 x NK603)
Hybrid DK 6767 (DK 919)
Executive summary

Monsanto Company, USA has developed the insect-protected and herbicide-tolerant combined trait maize
product MON 89034 × NK603 (Genuity VT Double PRO®) through conventional breeding techniques by
crossing two individual biotechnology-derived products: insect-protected maize (MON 89034) and
herbicide-tolerant Roundup Ready® 2 maize (NK603). MON 89034 × NK603 offers benefits to Pakistani
farmers for the control of economically important lepidopteran pests and weeds that occur in maize fields.

National Biosafety Committee approved to import seed and do different types of field trials during 2009
and 2010. Monsanto conducted the following studies to do the safety assessments of the technology.

• Field Trial for Agronomic Phenotypic Evaluation, and production of crop tissues for Gene
Expression Studies done in autumn 2009.

• Field Trial for Roundup® herbicide efficacy and crop safety done in autumn 2009

• Field Trial for insect Bio- efficacy done in autumn 2009

• Field trial for Agronomic Phenotypic evaluation done in autumn 2010

• Field Trial for Roundup herbicide efficacy and crop safety done in autumn 2010

• Large Scale Field trial to monitor the insect Bio-efficacy, Roundup® herbicide crop safety and
economic value done in autumn 2010

As maize has two major growing seasons (autumn and spring), Monsanto conducted the studies in both
the seasons; however, the hybrids were such which suit to the respective season only.

The present case of commercialization relates to only autumn season hybrid’ DK6767 (DK919).

Conclusion

An extensive set of data collected in 2009 and 2010 in Pakistan (on Phenotypic, agronomic and
environmental interaction characteristics) suggests that MON 89034 × NK603 does not possess weedy
characteristics, increased susceptibility or tolerance to specific abiotic stress, diseases, or arthropods, or
characteristics that would confer a plant pest risk or significant environmental impact compared to
conventional maize. The insect protection trait in MON 89034 × NK603 provides an effective option for
control of the major lepidopteran pests (C. partellus, H. armigera, and S. litura) compared to the
conventional control treated with different insecticide regimes targeting these insects. Field trials support
a conclusion that Roundup herbicide sprayed over the top of glyphosate-tolerant MON 89034 x NK603 at
all application rates provides an effective control of weeds with no evidence of any damage to plants

Based on the information presented by Monsanto in the submission, it is concluded that MON 89034 ×
NK603 maize is unlikely to present any biosafety risks compared to conventional maize. Therefore,

®
Genuity VT Double PRO and Roundup Ready are registered trademarks of Monsanto Technology LLC.
Technical Advisory Committee recommend the approval of MON 89034 × NK603 to the National
Biosafety Committee for commercial release.

PART -1 SUMMARY OF FOOD, FEED AND ENVIRONMENTAL SAFETY

Product Description

In Pakistan, maize is the third most important cereal crop after wheat and rice. Currently in Pakistan,
maize is grown on over 950,000 hectares and has a production of 3.5 million metric tons and productivity
of 3670 kg/ha in 2009 (http//www.fao.org). Maize Stem borer (Chilo partellus) and American bollworm
(Helicoverpa armigera) are the major insect pests in Pakistan, which have the potential to reduce yield
considerably; similarly, weeds in maize can also reduce yields drastically. The pest problems have arisen
due to large-scale cultivation of maize as a sole crop.

A transgenic maize hybrid that expresses both lepidopteran insect resistance and herbicide tolerance will
provide maize farmers a package of solutions to safeguard yields from lepidopteran insect infestations
and weed competition in a cost-efficient and environmentally friendly way. MON 89034 × NK603 is an
ideal fit for Pakistan, as all acres planted with hybrid maize are treated with insecticide and a majority of
acres also have perennial weed infestations. The maize selective herbicides which are available in
Pakistan have limited activity on these weeds that are easily be controlled by Roundup® herbicide.

MON 89034 was developed as a second generation insect-resistant maize product to provide enhanced
benefits for the control of lepidopteran insect pests. In addition to the wider spectrum of insect control,
the combination of the Cry1A.105 and Cry2Ab2 insecticidal proteins in the same plant provides a dual
mode of action which will delay the development of insect resistance.

Herbicide-tolerant Roundup Ready® 2 maize (NK603) contains two cp4 epsps genes that encode two
near-identical glyphosate-tolerant EPSPS proteins (CP4 EPSPS) that confer tolerance to glyphosate, the
active ingredient in Roundup® agricultural herbicides. The epsps (5-enolpyruvylshikimate-3-phosphate
synthase) coding sequences from Agrobacterium sp. strain CP4 were transferred into the genome of
conventional maize using an Agrobacterium tumefaciens-mediated system.

The biosafety data on MON 89034 × NK603 to support the commercial release includes:

• A detailed molecular characterization of the inserted DNA, where the results confirm the
insertion of functional cry1A.105, cry2Ab2 and cp4 epsps genes within the maize genome;

• An extensive set of biochemical evaluations that characterize the Cry1A.105, Cry2Ab2 and CP4
EPSPS proteins, including in plant expression;

• An assessment of toxicity and allergenicity potential that demonstrates that the Cry1A.105,
Cry2Ab2 and CP4 EPSPS proteins are not likely to be a toxins or allergens;

® Roundup is a registered trademark of Monsanto Company

 • A compositional and nutritional assessment which confirms that MON 89034 × NK603 maize
grain and forage are compositionally and nutritionally equivalent to, and as safe as, those of
conventional maize.

• An extensive evaluation of phenotypic and agronomic characteristics and environmental

interactions that demonstrate that the cultivation of MON 89034 x NK603 is not likely to result in
adverse environmental impacts, compared to the cultivation of conventional maize; and,

• An assessment on the potential impact to non-target organisms (NTOs) concludes that MON
89034 × NK603 is unlikely to have adverse effects on these organisms under normal agricultural
practices.

Molecular Characterization of Inserted DNA

MON 89034 × NK603 maize hybrids are derived from two genetically modified parental inbred lines of
MON 89034 and NK603 through traditional breeding methods. Southern blot analysis of the combined
trait product MON 89034 × NK603 showed that the molecular fingerprints of MON 89034 × NK603
were consistent with those for the individual traits, MON 89034 and NK603.

The integrated DNA in MON 89034 and NK603 were characterized by Southern blot analyses. The data
indicate that both MON 89034 and NK603 contain one copy of the insert at a single integration locus and
all expression elements are present. These data also demonstrated that both MON89034 and NK603 do
not contain detectable backbone or selectable marker sequence from plasmid PVZMIR245 and PV-
ZMGT32L respectively.

Expression of the Inserted Proteins

The expression levels of Cry1A.105, Cry2Ab2, and CP4 EPSPS proteins were determined by validated
enzyme-linked immunosorbent assays (ELISA) in maize tissues of MON 89034 × NK603 collected from
plants grown at three field sites in Pakistan during 2009. ELISA analyses were conducted on leaf, root,
whole plant, forage, forage root, pollen, silk and grain. Protein levels for each tissue type were calculated
on a microgram (µ g) per gram (g) fresh weight (fwt) basis. Moisture content was then measured for
each tissue type from each site and protein levels were converted and reported on a dry weight (dwt)
basis.

Protein Level in MON 89034 × NK603 maize

Mean (μg/g dwt) Mean (μg/g dwt)
Samp Samp
Cry1A.1 Cry2A CP4 EPS Cry1A.1 Cry2A CP4 EPS
le le
05 b2 PS 05 b2 PS
Type Type
Forag
Leaf 120 100 230 23 35 57
e Root
Root 68 68 110 Pollen 8.9 0.75 410
Whole
88 47 190 Silk 17 150 120
Plant
Forag
36 59 94 Grain 3.9 2 8.1
e
Safety Assessment of the Cry1A.105, Cry2Ab2 and CP4 EPSPS Proteins

The safety assessment of the Cry1A.105, Cry2Ab2 and CP4 EPSPS proteins was based on their detailed
biochemical characterization, history of safe consumption, and their toxic and allergenic potential. Since,
the expression levels of plant-produced proteins are relatively low, it was necessary to produce the
proteins in a high-expressing, recombinant microorganism in order to obtain sufficient quantities of the
proteins for safety studies.

The modes and sites of biological activity are different for the Cry and CP4 EPSPS proteins and there is
no known or conceivable mechanism of interaction between the proteins in MON 89034 × NK603 which
could lead to adverse environmental or health effects in animals or humans.

History of Safe Consumption and Acute Toxicity of Cry1A.105 and Cry2Ab2

The Cry1A.105 and Cry2Ab2 proteins belong to a family of Bt Cry proteins. Commercial sprays of
sporulated Bt have a long history of safe use for pest control in agriculture. The potential toxicity of the
Cry1A.105 and Cry2Ab2 proteins was tested by acute oral gavage exposure in mice, and by conducting
searches for amino acid sequence homology of the proteins with those of known toxins. The acute oral
LD50 of the Cry1A.105 protein in mice is greater than 2072 mg/kg body weight and since no signs of
toxicity were observed, this level was also considered to be the no observed effect level (NOEL) for this
protein. In acute toxicity study with Cry2Ab2 protein, no mortality or other evidence of toxicity was
observed at the maximum attainable dose. Therefore, the acute oral LD50 of the Cry2Ab2 protein in mice
is greater than 2,198 mg/kg body weight, which was designated as the NOEL for this protein. The results
of bioinformatic searches revealed no biologically relevant sequence similarities between Cry1A.105 and
Cry2Ab2 proteins and toxins.

History of Safe Consumption and Acute Toxicity of CP4 EPSPS

There is no prior reason to expect any toxicity to be associated with the CP4 EPSPS proteins. CP4 EPSPS
is functionally similar to the EPSPS proteins typically present in foods and feeds derived from plant and
microbial sources, based on the reaction catalyzed. The potential toxicity of the CP4 EPSPS was tested
by acute oral gavage exposure in mice, and by conducting searches for amino acid sequence homology of
the protein with those of known toxins. No adverse effects were observed when CP4 EPSPS was
administered at levels up to 572 mg/kg of body weight. Similarly, no adverse effects were observed when
CP4 EPSPS L214P was administered to mice at levels up to 817 mg/kg of body weight.The results of the
searches revealed no biologically relevant sequence similarities between CP4 EPSPS and known toxins.

Assessment of Allergenic Potential of the Cry1A.105, Cry2Ab2, and CP4 EPSPS Proteins

Potential allergenicity was assessed based on knowledge of the source organism of the protein, an
assessment of structural similarities to known allergens, in vitro digestibility in simulated digestive fluids,
and abundance of the proteins in maize grain.

The Cry1A.105 protein is present at approximately 0.003% of the total protein found in the grain of MON
89034 × NK603. Similarly, the Cry2Ab2 protein is present at approximately 0.002% of the total protein
found in the grain of MON 89034 × NK603. The low levels of the Cry1A.105 and Cry2Ab2 proteins in
MON 89034 × NK603, combined with their rapid in vitro digestibility in gastric fluids and lack of
homology to any known allergens suggests that they are unlikely to be allergens.

The CP4 EPSPS and CP4 EPSPS L214P proteins together are present at approximately 0.01 % of the
total protein found in the grain of MON 89034 × NK603. The low levels of the CP4 EPSPS proteins in
NK603, combined with the rapid digestibility of these proteins in the in vitro digestibility test, establishes
an extremely low probability of the CP4 EPSPS proteins that they are likely to be allergens.

Food and Feed Safety Assessment

The fundamental principle for the food, feed and environmental safety assessment of products derived
from modern biotechnology is the concept of substantial equivalence in which the composition of the
transgenic plant, in this case MON 89034 × NK603 maize, is compared to appropriate conventional
controls that have an established history of safe use.

Composition and Nutrition of Grain

The composition of forage and grain produced by MON 89034 × NK603 was compared to conventional
control maize with similar genetic background, as well as with other commercially available maize
hybrids. Analyses of grain samples included measurements of proximates (protein, fat, ash, moisture),
acid detergent fibre (ADF), neutral detergent fibre (NDF), amino acids, fatty acids, vitamin E, minerals
(calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc), and the
antinutritional components, phytic acid and trypsin inhibitor.

The statistical analyses showed that all of the 366 comparisons made between the MON 89034 × NK603
and the control maize were either: a) not statistically significantly different, b) significantly different
(p<0.05) but the composition values for MON 89034 × NK603 were within the calculated 99% tolerance
interval for the population of conventional reference hybrids used in this study, or c) significantly
different but the composition values for the MON 89034 × NK603 were within the range of values
reported in the International Life Sciences Institute (ILSI) Crop Composition Database and, therefore, not
considered as being biologically meaningful.

Nutritional Equivalence

Broiler feeding studies are the model of choice for comparing the wholesomeness of genetically modified
grain to conventional grain, because the rapid weight gains of broilers makes them sensitive to small
nutrient or antinutrient changes in the diet. A study was conducted to compare bird performance (feed
intake, body weight, and feed conversion), carcass yield, and meat quality of the birds fed diets
containing MON 89034 × NK603, control (similar genetic background to the test maize), and
conventional reference maize. It was concluded that the diets containing MON 89034 × NK603 were
nutritionally equivalent to diets containing the control or conventional reference maize grain.
Two studies on the oral administration of MON 89034 and NK603 maize grains in repeat dose manner to
rats for 90 days have shown no adverse effects on the growth or health of the animals. Therefore, the
consumption of MON 89034 × NK603 maize, produced by traditional breeding of MON 89034 × NK603
is considered safe, and unlikely to produce any adverse effects to humans or animals.

Overall Assessment for Food and Feed Safety

The compositional and nutritional analyses lead to the conclusion that MON 89034 × NK603 forage
and grain are not different in nutritional and anti-nutritional composition compared to maize hybrids
currently grown, marketed, and consumed and are substantially equivalent to, and as safe and
nutritious as the parental and commercially available conventional maize hybrids. The history of safe
use of the introduced proteins and their low potential for toxicity and allergenicity support a
conclusion on safety of MON89034 × NK603 for human and animal consumption.

Environmental Safety Assessment

An extensive set of data collected in Pakistan over two years (2009 and 2010) were used to assess
whether the introduced traits altered the plant pest potential of MON 89034 × NK603 compared to
conventional maize. Phenotypic, agronomic, and environmental interaction characteristics of MON
89034 × NK603 were evaluated and compared to those of the conventional control and considered within
the variation among commercial reference hybrids. These assessments included plant growth and
development characteristics; observations of abiotic stress response; disease, bird, and arthropod damage;
shoot fly damage and arthropod abundance. Results from the phenotypic, agronomic, and environmental
interactions assessment indicate that MON 89034 × NK603 does not possess weedy characteristics,
increased susceptibility or tolerance to specific abiotic stress, diseases, or arthropods, or characteristics
that would confer a plant pest risk or significant environmental impact compared to conventional maize.

Weediness Potential

No competitive advantage has been conferred to MON 89034 × NK603, other than that conferred by
tolerance to glyphosate herbicide and resistance to lepidopteran insects. These introduced traits will not,
in themselves, render maize weedy or invasive of natural habitats since none of the reproductive or
growth characteristics were modified in MON 89034 × NK603.

Secondary and Non-Target Adverse Effects

No environmentally adverse effects were detected in MON 89034 × NK603 fields during the two years (2
seasons) of trials. MON 89034 × NK603 and the individual events, MON 89034 and NK603, have been
approved for environmental release in many countries and have also undergone environmental safety
evaluation in several importing countries in case there is an unintended planting of these events.

Outcrossing

Gene exchange between MON 89034 × NK603 and other cultivated maize varieties will be similar to that
which occurs naturally between cultivated maize varieties at the present time. In geographies where few
wild plant species closely-related to maize are found, the likelihood of gene flow to these wild species is
remote due to spatial and temporal flowering differences. Cultivated maize, Zea mays L. subsp. mays, is
sexually compatible with other members of the genus Zea, and to a much lesser degree with members of
the genus Tripsacum.

Overall assessment for Environment Safety

Based on the data and information presented in the submissions, it is concluded that MON 89034 ×
NK603 maize is unlikely to: a) have an increased weediness potential b) pose a risk to non-target
organisms, c) outcross with wild relatives of maize, and d) have an adverse environmental impact
compared to conventional maize. The successful adoption of MON 89034 × NK603 maize is expected
to increase economic benefits to Pakistani farmers by protection of maize yields and decreased
chemical insecticide usage.
PART – 2 SUMMARY OF FIELD TRIALS

Agronomic/Phenotypic Evaluation- 2009

The Multi Location Research Trials (MLRTs) were conducted to evaluate phenotypic characteristics and
ecological interactions of MON 89034 × NK603 compared to the conventional maize control. Data were
collected from field trials conducted in autumn season 2009 at three sites within maize production regions
of Punjab, Pakistan. MON 89034 × NK603 maize (test hybrid DK 919), the conventional maize control
hybrid and three different commercially-released reference maize hybrids were evaluated at each site (a
total of 6 hybrids across the sites). The experimental design at each site was a randomized complete
block with four replications. MON 89034 × NK603 was compared to the conventional maize control for
early stand count, seedling vigor, plant growth stage, days to 50% pollen shed, days to 50% silk, stay
green, ear height, plant height, ear tip fill, grain fill, number of stalk lodged plants, number of root lodged
plants, final stand count, grain moisture, test weight, yield, plant response to abiotic stressors, disease,
bird, arthropod, and shoot fly damage, and arthropod abundance. The observations of plant response to
abiotic stressors, disease damage, and arthropod damage were performed four times during the growing
season at all sites. Shoot fly damage was assessed three times during the growing season at all sites.
Beneficial arthropod abundance was assessed both from sticky trap collections (five times) and visual
observations (five times) during the growing season at all sites. MON 89034 × NK603 was compared to
the conventional maize control across all sites (combined site analysis) and within each individual site
(individual site analysis). The minimum and maximum mean values (reference range) were established
from the maize reference hybrids to provide phenotypic characteristic, plant response to abiotic stress,
disease damage, arthropod damage, and beneficial arthropod abundance values representative of the range
of natural variation within commercial maize hybrids.

Phenotypic Characteristics of MON 89034 × NK603

MON 89034 × NK603 and the conventional maize control were within the same range for all 23 growth
stage observations among the sites. Thus, there were no developmental differences observed between
MON 89034 × NK603 and the conventional maize control.

In the combined site analysis, no statistical difference were detected between MON 89034 × NK603 and
the conventional maize control for seedling vigor, days to 50% pollen shed, days to 50% silk, plant
height, uniformity of plant height, stay green, grain fill, ear tip fill rating, number of stalk lodged plants,
number of root lodged plants, grain moisture contents, test weight, and yield. Early stand count (92.17 vs.
135.00 plants/plot) was statistically lower for MON 89034 × NK603 compared to the conventional maize
control and this was related to the difference in planting rate for MON 89034 × NK603 (1 seed/hill)
compared to the conventional maize control and reference substances (2 seeds/hill). However, all plots
were thinned down to final target plant density (~41 plants/8 m row). Ear count (78.67 vs.72.00 ears/plot)
and final stand count (81.17 vs. 73.75 plants/plot) were statistically higher for MON 89034 × NK603
compared to conventional maize control. However, the mean values for ear count and final stand count
were within the respective ranges of commercial reference hybrids. Thus, the differences detected in ear
count and final stand count was considered not biologically meaningful in the context of increased
weediness of MON 89034 × NK603 compared to conventional maize.
Ecological Interaction Evaluations of MON 89034 × NK603

Abiotic Stressors Disease Damage and Arthropod Damage

In an assessment of abiotic stress response, disease damage, and arthropod damage, no differences were
observed between MON 89034 × NK603 and the conventional maize control for 101 of 108 comparisons
(including 36 abiotic stressors, 36 disease damage, and 36 arthropod damage comparisons) among all
observations across all sites. The seven observed differences were in the arthropod damage category.
The insect-protection trait in MON 89034 × NK603 was developed to control certain lepidopteran pests,
therefore it was expected that where feeding pressure was sufficient to cause measurable damage, MON
89034 × NK603 would have less damage from lepidopteran pests compared to the conventional maize
control. The results showed that MON 89034 × NK603 had less damage caused by lepidopteran pests (C.
partellus, H. armigera, and Spodoptera spp.) than the conventional maize control.

Beneficial Arthropod Abundance

In the combined site analysis of beneficial arthropod abundance data from sticky trap samples, no
statistical differences were detected between MON 89034 × NK603 and the conventional maize control
for any of the 14 comparisons, including, Chrysoperla spp., Coccinellidae, spiders, dragon fly, preying
mantis, and syrphid fly collected across all locations. Lack of variability in the data precluded statistical
comparisons between MON 89034 × NK603 and conventional maize control for 11 additional
comparisons; however, the mean values for MON 89034 × NK603 and the conventional maize control
were similar, indicating no biological differences.

In the combined site analysis of beneficial arthropod abundance data from visual observations, no
statistical differences were detected between MON 89034 × NK603 and the conventional maize control
for 29 out of 30 comparisons observed across all sites. A single difference was detected where the
abundance of Chrysoperla spp. at Collection 2 was statistically higher for MON 89034 × NK603
compared to conventional maize control (0.25 vs. 0.00 per plot). However, the mean abundance values
for Chrysoperla spp. were within the range of the commercial reference hybrids and no differences were
detected at other four collection times. Thus, the difference detected in abundance of Chrysoperla spp.
was considered not biologically meaningful in terms of an adverse environmental impact of
MON 89034 × NK603 compared to conventional maize.

Bird Damage Assessment

In the combined site analysis of bird damage data, no statistical differences were detected for bird damage
between MON 89034 × NK603 and the conventional maize control.

Shoot fly Damage Assessment

No shoot fly damage was present in all three observations from MON 89034 × NK603, the conventional
control, or the commercial reference hybrids at any of the three sites. P-values could not be generated
due to lack of variability in the data.

Based on the assessed characteristics, the results of this study demonstrate there were no unexpected
changes in the phenotype or ecological interactions that are indicative of increased plant weed
potential or adverse environmental impact for MON 89034 × NK603 compared to the conventional
maize control.
Bio-Efficacy Assessment – 2009

The Multi Location Research Trials (MLRTs) were conducted to assess the bio-efficacy of MON 89034 ×
NK603 maize hybrid against target arthropod pests in field trials during autumn 2009. Data were
collected from field trials conducted in autumn season 2009 at three sites within maize production regions
of Punjab, Pakistan. The experimental design at each site was a randomized complete block with six
treatments and four replications. MON 89034 × NK603 (Treatment 1), the conventional control
(Treatment 2) and the conventional control with four insecticide management regimes targeting
lepidopteran insect pests (Treatments 3, 4, 5 and 6), were evaluated at each site. The efficacy of the
insect-protection trait in MON 89034 × NK603 was assessed by comparing MON 89034 × NK603 to the
appropriate control for target pest damage. Plant phenotypic data were also collected to demonstrate that
the growth and development of the maize crop was as expected and to give an indication of the impact, if
any, of the target pest infestations.

Phenotypic Data

The overall assessment of phenotypic data indicated that the growth and development of MON 89034 ×
NK603 maize grown in the study area was as expected and MON 89034 × NK603 plants were in general
similar in all treatments. A statistically significant difference was detected between MON 89034 ×
NK603 and the untreated conventional control for yield. MON 89034 x NK603 had higher yield (8.25-
vs. 6.89- mt/ha). Thus, the data support that where pressure from the target pests are sufficient to cause
significant measureable damage, as demonstrated by pressure from target pests, MON 89034 × NK603
effectively provides feeding protection from the pests.

Pest Arthropod Damage Assessment

The insect protection trait in MON 89034 × NK603 was developed to control major lepidopteran pests
(C. partellus, H. armigera, and S. litura), therefore it was expected that where feeding pressure was
sufficient to cause measureable damage, MON 89034 × NK603 would exhibit reduced feeding damage
from these species compared to the control.

In an assessment of Agrotis ipsilon, no damage was observed in any of the treatments. Lack of infestation
from A. ipsilon prevented evaluation of damage from this species at all sites.

In an assessment of S. litura, MON 89034 × NK603 had less damage compared to the untreated
conventional control, and the conventional control treated with four management regimes. However,
damage from S. litura was statistically significantly lower on MON 89034 × NK603 compared to other
treatments in some instances in individual-site analyses.

In an assessment of C. partellus, percent damaged plants was statistically significantly lower for MON
89034 × NK603 compared to the untreated conventional control and the conventional control treated with
four management regimes. MON 89034 × NK603 had less leaf damage from C. partellus compared to all
treatments at 35 and 45 days after crop emergence. MON 89034 × NK603 had lower number of dead
hearts compared to all treatments at 35 and 45 days after crop emergence. C. partellus damage was
statistically significantly lower for MON 89034 × NK603 compared to all other treatments as indicated by
fewer entry/exit holes and fewer stalk galleries. In addition, stalk gallery length was clearly lower
compared to the untreated conventional control and the conventional control treated with four
management regimes.
In an assessment of H. armigera, severity of ear damage caused by H. armigera was statistically
significantly lower for MON 89034 × NK603 compared to the untreated conventional control and the
conventional control treated with four management regimes. In addition, percent damaged ears due to
feeding damage from H. armigera was lower compared to the untreated conventional control and the
conventional control treated with four management regimes.

Based on the assessed characteristics, the results from the 2009 field trials in Pakistan, support the
conclusion that when pressure from the target insect pests are sufficient to cause significant
measureable damage, MON 89034 × NK603 provides effective protection from feeding damage. As
expected, the insect protection trait in MON 89034 × NK603 provides an effective option for control of
the major lepidopteran pests (C. partellus, H. armigera, and S. litura) compared to the conventional
control treated with four management regimes targeting these insects.

Roundup Herbicide Efficacy for Weed Control and Crop safety - 2009

The Multi Location Research Trials (MLRTs) were conducted to evaluate the efficacy of Roundup ®
herbicide for weed control in MON 89034 x NK603 maize. Data were collected from field trials
conducted in autumn season 2009 at three sites within maize production regions of Punjab, Pakistan.
MON 89034 × NK603 maize (DK919) was used as starting seed for this study. The experimental design
at each site was a randomized complete block with 10 treatments (T1-T10) and four replications.
Roundup® IPA salt was applied as two sequential applications at the V2-V4 and V5-V6 crop growth
stages (T1-T4) and as a single application for treatments T5 and T6 at the V2-V4 crop stage. Treatments
(rates) were: T1 (788 + 788 ae/ha), T2 (1212 + 1212 ae/ha), T3 (788 + 1212 ae/ha), T4 (1212 +788
ae/ha), T5 (788 ae/ha), and T6 (1212 ae/ha). Doses selected for MON89034 × NK603 were the
maximum proposed in its registration application in Pakistan to be used in association with NK603 and
any stack containing NK603 (or any event expressing the CP4 EPSPS protein which confers resistance
against glyphosate). Treatments 7, 8, and 9 included three common weed management regimes currently
used in conventional maize in Pakistan. Treatment 10 served as control and was not subjected to any
weed control practice. Weed assessments, crop safety (herbicide tolerance), and phenotypic data were
collected at each site.

To assess herbicide efficacy, weed species were identified and counted, and the percentage of weed
covered ground was estimated from 1 m2 area in each plot prior to herbicide application and at 7, 14, and
21 days after herbicide treatment. Visual observation of percent weed control was also conducted using
0-10 rating scale in each plot prior to herbicide application and at 7, 14, 21, 30 days after herbicide
treatment. Herbicide tolerance (crop safety) was evaluated by the following characteristics: chlorosis,
necrosis, and herbicide induced damage ratings prior to herbicide application and at 7, 14, and 21 days
after herbicide treatment. Plant phenotypic data were also collected to demonstrate that the growth and
development of the maize crop was as expected and to provide additional evidence of herbicide tolerance.
For phenotypic and herbicide tolerance data, Roundup treatments T1, T2, T3, T4, T5, and T6 were
compared to a treatment without any weed control (T10) and treatments managed with three common
weed management regimes (T7, T8, and T9) currently used in conventional maize in an individual-site
analysis (within each site) and combined-site analysis (across all sites). Weed count, percent weed
covered area, and percent weed control were statistically analyzed only within individual observations
and sites due to the variation in temporal and geographical distribution of the weed flora.
The overall assessment of phenotypic data indicated that the growth and development of MON89034 ×
NK603 maize grown in the study area was as expected and MON89034 × NK603 plants were in general
similar in all treatments.

From the data collected, it is apparent that all herbicide treatments including hand weeding at all sites
effectively controlled weeds when compared with unweeded control. Roundup herbicide at all
application rates provided effective control in controlling both grasses as well as broad-leaf weeds.
Visual assessments indicated a statistically significantly greater percent weed control by Roundup
herbicide treatments (T1 – T6) compared to Atrazine and Premextra Gold herbicide treatments (T7 and
T8, respectively). No chlorosis, necrosis or herbicide-induced damage was observed on
MON 89034 × NK603 maize plants at any application rate of Roundup herbicide.

Based on the assessed characteristics, the results, support a conclusion that Roundup herbicide
sprayed over the top of glyphosate-tolerant MON 89034 x NK603 at all application rates provides an
effective control of weeds with no evidence of any damage to plants.

Agronomic/Phenotypic Evaluation- 2010

The layout and data collection parameters were the same as that of 2009 trials. The results are
discussed below.

Phenotypic Characteristics of MON 89034 × NK603

MON 89034 × NK603 and the conventional maize control were within the same range of plant growth
stages for all 24 growth stage observations among the sites. Thus, there were no developmental
differences observed between MON 89034 × NK603 and the conventional maize control.

In the combined-site analysis, no statistical differences were detected between MON 89034 × NK603 and
the conventional maize control for seedling vigor, days to 50% pollen shed, days to 50% silking, stay
green, husk cover, plant height, uniformity of plant height, grain filling in cob, tip filling in cob, stalk
lodged plants, root lodged plants, grain moisture, test weight, dropped ear, and yield. Four statistically
significant differences were detected in the combined-site analysis where MON 89034 × NK603 had
higher early stand count (74.75 vs 65.33 plants per plot), ear height (110.82 vs. 105.55 cm), ear count
(70.42 vs 59.08 ears per plot) and final stand count (70.67 vs 61.33 plants per plot) compared to the
conventional maize control. The mean values for early stand count, ear height, ear count and final stand
count were within the respective ranges of commercial reference hybrids. Thus, the differences detected
in ealrly stand count, ear height, ear count and final stand count were considered not biologically
meaningful in the context of increased weediness of MON 89034 × NK603 compared to conventional
maize.

Ecological Interaction Evaluations of MON 89034 × NK603

Abiotic Stressors Disease Damage and Arthropod Damage

In an assessment of abiotic stress response, disease damage, and arthropod damage, no differences were
observed between MON 89034 × NK603 and the conventional maize control for any of the 108
comparisons (including 32 abiotic stressors, 40 disease damage, and 36 arthropod damage comparisons)
among all observations across all sites.
Arthropod Abundance

In the combined-site analysis of arthropod abundance from sticky trap samples, no statistically significant
differences were detected between MON 89034 × NK603 and the conventional control for 14 out of 16
comparisons, including Chrysoperla spp., Coccinellidae, jassid, and spiders collected across all sites. The
abundance of Coccinellidae during Collection 1 and jassid during Collection 3 was lower for MON 89034
× NK603 compared to conventional maize control (1.25 vs. 3.58 per plot and 18.17 vs. 29.67 per plot,
respectively). The mean abundance values for Coccinellidae during Collection 1 and jassid during
Collection 3 from MON 89034 × NK603 were within the respective ranges of the commercial reference
hybrids. Thus, the differences detected in Coccinellidae and jassid abundance were considered not
biologically meaningful in terms of an adverse environmental impact of MON 89034 × NK603 compared
to conventional maize

In the combined-site analysis of arthropod abundance from visual observations, no statistical differences
were detected between MON 89034 × NK603 and the conventional maize control for 15 out of 16
comparisons including, Chrysoperla spp., Coccinellidae, jassid, and spiders observed across all sites. A
single difference was detected where the abundance of spider during Observation 3 was statistically lower
for MON 89034 × NK603 compared to conventional maize control (0.08 vs. 0.50 per plot). However, the
mean abundance value for spiders from MON 89034 × NK603 was within the range of commercial
reference maize hybrids. In addition, this difference was not consistently detected across observation
times. Thus, this difference was not indicative of a consistent plant response associated with the trait and
is unlikely to be biologically meaningful in terms of an adverse environmental impact of MON 89034 ×
NK603 compared to conventional maize.

Bird Damage Assessment

No bird damage was present in MON 89034 × NK603, the conventional control, or the commercial
reference hybrids at any of the three sites. P-values could not be generated due to lack of variability in
the data.

Shoot fly Damage Assessment

No shoot fly damage was present in all three observations from MON 89034 × NK603, the conventional
control, or the commercial reference hybrids at any of the three sites. P-values could not be generated
due to lack of variability in the data.

Based on the assessed characteristics, the results of this study demonstrate there were no unexpected
changes in the phenotype or ecological interactions that are indicative of increased plant weed
potential or adverse environmental impact for MON 89034 × NK603 compared to the conventional
maize control.

Roundup Herbicide Efficacy for Weed Control and Crop Safety- 2010

The layout and data collection parameters were the same as that of 2009 trials. The results are
discussed below.
The overall assessment of phenotypic data indicated that the growth and development of MON 89034 ×
NK603 maize grown in the study area was as expected and MON 89034 × NK603 plants were in general
similar in all treatments.
From the data collected, it is apparent that all herbicide treatments including hand weeding at all sites
effectively controlled weeds when compared with unweeded control. Roundup herbicide at all
application rates provides an effective control in controlling both grasses as well as broad-leaf weeds.
Visual assessments indicated a statistically significantly greater weed control by Roundup herbicide
treatments (T1 – T6) compared to Atrazine and Premextra Gold herbicide treatments (T7 and T8,
respectively). No chlorosis, necrosis or herbicide-induced damage was observed on
MON 89034 × NK603 maize plants at any application rate of Roundup herbicide

Based on the assessed characteristics, these results support a conclusion that Roundup herbicide
sprayed over the top of glyphosate-tolerant MON 89034 x NK603 at all application rates provides an
effective control of weeds with no evidence of any damage to plants.

Assessments in Large Scale Trials (LSTs)- 2010

The Large Scale Trials were planted in autumn 2010 with the purpose to 1) assess the bio-efficacy of
MON89034 x NK603 against lepidopteran insect pests; 2) evaluate the efficacy of Roundup herbicide for
weed control in MON 89034 x NK603; and 3) determine the benefits of MON 89034 x NK603 compared
to the conventional maize managed under local practices.

Data were collected from LSTs conducted in the autumn season of 2010 at five sites within maize
production regions of Punjab, Pakistan. There were three non-replicated plots at each site: MON 89034 ×
NK603 treated with Roundup herbicide (Treatment 1), the conventional maize control treated with pre-
emergent herbicide and insecticides targeting lepidopteran insect pests (Treatment 2) and the
conventional maize control without any insecticide or herbicide application (Treatment 3).

Data on the following phenotypic characteristics were collected: seedling vigor, number of stalk lodged
plants, number of root lodged plants, final stand count, number of harvested ears, grain moisture, and
yield. The number of harvested ears and yield for MON 89034 × NK603 treated with Roundup herbicide
were compared to the conventional control treated with herbicide + insecticide and untreated conventional
control to evaluate the impact, if any, of the target weed and insect pest infestations. The efficacy of the
insect-protection trait in MON 89034 × NK603 was assessed by comparing MON 89034 × NK603 to the
appropriate control treatment for target pest damage. Pest arthropod damage was assessed once for H.
zea, two times for C. partellus and three times for S. litura under natural infestation pressure during the
growing season at each site. To assess herbicide efficacy, percent of weed control and percent weed
covered area was observed in each plot prior to herbicide application and at 21 days after herbicide
treatment. Herbicide tolerance (crop safety) was evaluated using following characteristics: chlorosis,
necrosis, and herbicide induced damage ratings prior to herbicide application and at 7, 14, and 21 days
after herbicide treatment. All pest arthropod damage, weed control, and herbicide tolerance assessments
were made from each of the 4 predetermined quadrants in each non-replicated plot. Results are described
across site (combined-site) and at each site (individual-site).

Phenotypic Assessments

The overall assessment of phenotypic data indicated that the growth and development of MON 89034 ×
NK603 maize grown in the study area was as expected and maize plants were in general similar in all
treatments. Combined-site data shows that MON 89034 × NK603 treated with Roundup herbicide had
clearly higher number of harvested ears and yield compared to the conventional control treated with
herbicide + insecticide (84.50 vs. 74.60 ears per plot and 7.60 vs. 6.83 tonnes/ha, respectively) and
untreated conventional control (84.5 vs. 71.80 ears per plot and 7.60 vs. 5.64 tonnes/ha, respectively).
Thus, the data support the conclusion that where pressure from the target weeds or insect pests are
sufficient to cause significant measureable damage, MON 89034 × NK603 treated with Roundup
herbicide effectively provides superior weed control, feeding protection from the insect pests, excellent
crop safety and maximum yield potential.

Pest Arthropod Damage Assessment

The insect protection trait in MON 89034 × NK603 was developed to control major lepidopteran pests (S.
litura, H. armigera, and C. partellus), therefore it was expected that where feeding pressure was
sufficient to cause measureable damage, MON 89034 × NK603 would have less damage from these
species compared to the conventional control.

In an assessment of combined-site data, S. litura, H. armigera, and C. partellus damage was lower for
MON 89034 × NK603 compared to the conventional control treated with insecticide and untreated
conventional control.

Weed Control and herbicide Tolerance Assessment

From the data collected, it is clear that Roundup herbicide application in MON 89034 × NK603 plots
provided effective and better weed control compared to Primextra gold herbicide application in
conventional control and untreated conventional control. Roundup herbicide application (2.5 L/ha)
provides an excellent control both for grasses and broad-leaf weeds at all sites. No chlorosis, necrosis or
herbicide-induced damage was observed on MON 89034 × NK603 maize plants where application of
Roundup herbicide was made.

Value creation / Economic Benefits

Value creation defined commercially in a prospect farmer has tangible and intangible value associated to
a new product. Considering only the tangible part of value creation following elements were considered
critical to estimate the economic benefits from MON 89034 × NK603 technology:

1. The constant cost across the treatments includes land rent, land preparation, labor, fertilizer,
irrigation, seed cost1 etc.
2. Variable cost across treatments includes pesticide application.

The results of this analysis show that MON 89034 × NK603 treated with Roundup® herbicide has the
potential to provide significant economic benefits for growers compared to both conventional control
treatments. These estimates show that MON 89034 × NK603 can provide an economic benefit of
approximately Rs. 8,910 over the conventional control treated with herbicide + insecticides and an
economic benefit of approximately Rs. 17,018 over the untreated conventional control, respectively2. In
addition to the provision of a potential monetary benefit, it is important to note that the other benefits
associated with MON 89034 × NK603 are significant, the environmental benefits of reduced pesticide use
as well as reduced pesticide exposure and labor cost. Overall, these benefits contribute to greater
flexibility in farmer’s management practice and profitability.

1
Monsanto has not determined the price of MON 89034× NK603 seed.
2
These economic benefit calculations are based on pesticide and produce rates in January 2011.
The overall results from the large scale field trials in Pakistan, support a conclusion that where
pressure from the target weeds or insect pests are sufficient to cause significant measureable damage,
MON 89034 × NK603 treated with Roundup herbicide effectively provides superior weed control,
feeding protection from the insect pests, excellent crop safety, and maximum yield potential.
Furthermore, this study demonstrates that MON 89034 × NK603 will provide a net economic benefit to
the grower compared to conventional maize.

Overall Conclusion of Field trials

An extensive set of data collected over two years (2009 and 2010) in Pakistan were used to assess
whether the introduced traits altered the plant pest potential of MON 89034 × NK603 compared to
conventional maize. Phenotypic, agronomic, and environmental interaction characteristics of MON
89034 × NK603 were evaluated and compared to those of the conventional control and considered within
the variation among commercial reference hybrids. These assessments included plant growth and
development characteristics; observations of abiotic stress response, disease, bird, and arthropod
damage; and arthropod abundance. Results from the phenotypic, agronomic, and environmental
interactions assessment indicate that MON 89034 × NK603 does not possess weedy characteristics,
increased susceptibility or tolerance to specific abiotic stress, diseases, or arthropods, or characteristics
that would confer a plant pest risk or significant environmental impact compared to conventional maize.
The insect protection trait in MON 89034 × NK603 provides an effective option for control of the major
lepidopteran pests (C. partellus, H. armigera, and S. litura) compared to the conventional control treated
with different insecticide regimes targeting these insects. Field trials support a conclusion that Roundup
herbicide sprayed over the top of glyphosate-tolerant MON 89034 x NK603 at all application rates
provides an effective control of weeds with no evidence of any damage to plants