Introduction {#pbi12551-sec-0001}
============

Genetically modified (GM) crops have been widely adopted since commercial release more than 20 years ago, with a steady growth each year in biotech hectarage planted globally (James, [2014](#pbi12551-bib-0028){ref-type="ref"}). The benefits of GM crops were recently documented by Klumper and Qaim ([2014](#pbi12551-bib-0029){ref-type="ref"}) and include an overall increase in yield of almost 22%, a 36.9% reduction in pesticide use and an increase in farmer profit slightly over 68%. GM crops are increasingly being exploited globally, benefiting 18 million farmers in 28 countries in 2014 (James, [2014](#pbi12551-bib-0028){ref-type="ref"}).

What is a GM stack? {#pbi12551-sec-0002}
-------------------

Crop productivity has undergone continuous improvement by conventional plant breeding since crop plants were originally domesticated for agricultural purposes. For example, over 100 genes conferring beneficial traits from wild relatives have been incorporated into the genomes of 19 of the world\'s major crops by conventional breeding, creating new varieties with disease resistance, abiotic stress resistance and improved quality (Hajjar and Hodgkin, [2007](#pbi12551-bib-0024){ref-type="ref"}).

Conventional breeding techniques can also be used to combine traits from two or more GM events. A GM event is defined as the insertion of DNA into the plant genome as a result of a single transformation process (Pilacinski *et al*., [2011](#pbi12551-bib-0037){ref-type="ref"}). Individual events may contain one or more transgenes, and these events are sometimes referred to as molecular stacks (Que *et al*., [2010](#pbi12551-bib-0038){ref-type="ref"}). Alternatively, using conventional breeding to combine GM events does not involve insertion of new recombinant DNA sequence into the genome and does not modify the existing genomic DNA, so stacked GM events are, by definition, not a new event (Pilacinski *et al*., [2011](#pbi12551-bib-0037){ref-type="ref"}). The data presented in this study include analysis of stacked GM events created by conventional breeding of single events developed by Syngenta and will be referred to as stacked GM events or stacks.

Why are stacked events important? {#pbi12551-sec-0003}
---------------------------------

The ability to offer multiple benefits in a single product have resulted in many GM events being combined by conventional breeding techniques, offering combinations of insect resistance and herbicide tolerance genes. These types of stacks are also referred to as 'pyramids', characterized by multiple traits that confer resistance to a specific pest or pest complex. For example, Syngenta\'s Agrisure Viptera^®^ 3111 stack provides control of 14 insect pests and tolerance to both glyphosate and glufosinate herbicides.[1](#pbi12551-note-1001){ref-type="fn"} GM events combining different modes of action for protection against specific insect(s) pests are a valuable strategy for management of insect resistance.

How are single GM events assessed for risk? {#pbi12551-sec-0004}
-------------------------------------------

The principles underpinning risk assessments of GM events have been well described by the Food and Agricultural Organization/World Health Organization (FAO/WHO [1996](#pbi12551-bib-0021){ref-type="ref"}, [2000](#pbi12551-bib-0022){ref-type="ref"}), the Organization for Economic Cooperation and Development (OECD [2010](#pbi12551-bib-0035){ref-type="ref"}), the Codex Alimentarius ([2003](#pbi12551-bib-0006){ref-type="ref"}, [2009](#pbi12551-bib-0007){ref-type="ref"}) and by national regulatory agencies (EC [2013](#pbi12551-bib-0008){ref-type="ref"}, EFSA [2011a](#pbi12551-bib-0016){ref-type="ref"}, EPA [2014](#pbi12551-bib-0020){ref-type="ref"}, FDA [1992](#pbi12551-bib-0023){ref-type="ref"}, Health Canada [2006](#pbi12551-bib-0026){ref-type="ref"}). Most commercial GM crops express transgenes, which encode proteins that confer the desired traits, for example pest control or herbicide tolerance. Many of these transgenes are derived from other plants or microorganisms ubiquitous and/or abundant in the environment and food and feed chain. Regardless of the nature and origin of the transgenes, newly developed GM events undergo comprehensive safety assessments prior to commercial release. The history of safe use of the conventional crop, which has been consumed for decades, can be used as a baseline for the safety assessment of new GM plant varieties (Kok and Kuiper, [2003](#pbi12551-bib-0030){ref-type="ref"}).

Data generated for a risk assessment of a new GM single event intended for food and feed typically includes (i) molecular characterization of the genetic insertion, (ii) bioinformatic assessment of the inserted sequences for similarity to known allergens or toxins, (iii) protein toxicity studies, (iv) analysis of the physicochemical properties of the introduced proteins as they pertain to stability and activity, (v) quantitation of the proteins expressed by the transgenes *in planta*, (vi) nutritional equivalence assessed by compositional analysis and animal feeding studies, and (vii) an estimation of human dietary exposure. Although individual regulatory agencies may have different specific requirements, these data meet the requirements of most global regulatory agencies in regions that import food and feed commodities.

The molecular characterization of a GM event includes verifying the sequence of the inserted DNA and the host genome flanking sequences and confirmation of the copy number of the inserted sequences. Correct position of the promoter and terminator sequences with respect to the coding sequence(s) confirms that only the intended transcripts(s) and protein(s) are encoded by the transgene(s). The flanking sequence is used to assess the possible disruption of endogenous genes in the host plant and the potential generation of new open reading frames.

Proteins are an essential component of both human and animal diets, and most are safe to consume. But all known food allergens are proteins and there are also rare examples of proteins that are toxic to animals, including humans. The potential for proteins expressed by the transgenes to be allergenic or toxic is assessed using comparative bioinformatic data to identify similarities in sequence to known allergens or toxins and by characterization of physicochemical properties consistent with proteinaceous allergens and toxins.

Protein expression studies are conducted to generate a profile of transgenic protein expression across the life cycle of the crop produced under field conditions. These data can be used to estimate potential human and livestock exposure to the transgenic protein present in food and feed products and for determining appropriate dose levels for protein toxicity studies. Protein toxicity studies often use rats or mice to evaluate the effects of direct exposure to transgenic proteins administered via gavage, based on the recommended limit dose (OECD [2001](#pbi12551-bib-0032){ref-type="ref"}) or the theoretical exposure level (OECD [2008](#pbi12551-bib-0034){ref-type="ref"}).

The major objective of compositional equivalence evaluations of new GM crop varieties is to assess whether the new GM event is comparable in nutrient and antinutrient content to the conventional crop that already has an established history of safe consumption.

In addition to compositional equivalence studies, some regulatory agencies require animal feeding studies with diets prepared from the raw agricultural commodity originating from the GM crop to further assess nutritional value and wholesomeness.

How are stacked events assessed for safety? {#pbi12551-sec-0005}
-------------------------------------------

When a GM stack is created by conventional breeding, the inserted DNA from each individual event is transferred from parent to progeny similarly to that for endogenous genes. There is no additional recombinant DNA sequences introduced when stacking GM events by conventional breeding. For the purposes of this discussion on food and feed risk assessment of GM stacks, it is assumed that the single events that comprise the stack have been individually and comprehensively evaluated for food and feed safety, as described above, and that the novel proteins expressed in the GM stack have been shown to pose negligible risk to human and animal health.

Currently, there is no global consensus for the regulation of previously approved GM events combined by conventional breeding (Pilacinski *et al*., [2011](#pbi12551-bib-0037){ref-type="ref"}). Consequently, some regulatory agencies require no additional safety data, while others require a repeat of studies already conducted for the safety assessments of the individual events. Still others have chosen to regulate only the GM stacks, which are inclusive of the greatest number of events in the stacks under consideration ('higher‐order stacks', Pilacinski *et al*., [2011](#pbi12551-bib-0037){ref-type="ref"}; Ramjoue, [2008](#pbi12551-bib-0039){ref-type="ref"}). Countries that have adopted policies whereby approval of a higher‐order stack may cover lower‐order stacks containing the same events include Argentina, Paraguay, Uruguay, Philippines, the European Union and Brazil (e.g. EC [2013](#pbi12551-bib-0008){ref-type="ref"}, EFSA [2011a](#pbi12551-bib-0016){ref-type="ref"}, Philippines [2013](#pbi12551-bib-0036){ref-type="ref"}).

For countries that require additional data for food and feed approval of stacked GM events, these requirements may include confirmation of copy number and intactness of the inserted sequence contributed by each of the single events, verification that expression of the introduced genes is consistent with that of the single events, and nutritional equivalence studies. Recently, a new requirement to confirm transgenic insert stability by resequencing of the individual single events in stacked GM events has been implemented for import approval in EU member countries (EC [2013](#pbi12551-bib-0008){ref-type="ref"}).

Compositional equivalence, protein expression and molecular characterization (insert stability and intactness) data generated for food and feed safety assessments of stacked GM events and the component single events are described for stacks containing two, three and four Syngenta events in six stacked event combinations (Bt11 × GA21, Bt11 × MIR604, MIR604 × GA21, Bt11 × MIR604 × GA21, Bt11 × MIR162 × GA21 and Bt11 × MIR604 × MIR162 × GA21). Data from individual studies were compiled to create a compendium representing the range of variation in composition and expression of single events and stacks across many years of agricultural production in different environments and in different germplasm. As expected, both environment and germplasm had an impact on composition and expression, but there were no indications that stacking the events had either an additive or synergestic effect. These data, together with molecular studies demonstrating insert stability, provide compelling evidence that when there is no likely interaction between the single event, extrapolation of single event risk assessment data to the stacked GM events is warranted.

An understanding of the modes of action of the individual transgenes, the products they produce and the transgenic protein expression profile can be used with a problem formulation approach to identify potential interactions between the transgenes and transgene products contained in the combined events, on a case‐by‐case basis. When there is a likelihood of interaction, additional studies can be designed to address anticipated interaction(s) and to either confirm or update the existing safety assessments.

Results {#pbi12551-sec-0006}
=======

Compositional analysis {#pbi12551-sec-0007}
----------------------

For each nutritional component, the mean levels for the single events and the stacks were very similar (Table [1](#pbi12551-tbl-0001){ref-type="table-wrap"}). The compositional analysis data for Bt11 was limited, so no comparison with that single event was possible for many of the analytes measured in the stacks. The range of values for each of the six stacked event hybrids were compared to the compiled prediction intervals derived from data of each of the corresponding single events contained in each stack. For example, the range of protein levels in the Bt11 × MIR162 × GA21 stack (10.0--12.7% dry weight) was compared to the range of the prediction intervals for Bt11, MIR162 and GA21 (6.87%--13.5%). Most values of the stacks were within the collective prediction interval ranges for the corresponding singles. When a low or high value for a stack was not within the prediction intervals for the singles, it was often due to only one or a few of the stack values falling outside of the prediction intervals.

###### 

Levels of nutritional and antinutritional components in grain from single event and stacked event maize

  Event or stack                                                      Bt11                                        GA21                                        MIR604           MIR162                                      Bt11 MIR162 MIR604 GA21   Bt11 MIR162 GA21   Bt11 MIR604 GA21                            Bt11 MIR604                                 Bt11 GA21    MIR604 GA21
  ------------------------------------------------------------------- ------------------------------------------- ------------------------------------------- ---------------- ------------------------------------------- ------------------------- ------------------ ------------------------------------------- ------------------------------------------- ------------ -------------------------------------------
  *N* =                                                               21[a](#pbi12551-note-0002){ref-type="fn"}   53[b](#pbi12551-note-0003){ref-type="fn"}   52               34[c](#pbi12551-note-0004){ref-type="fn"}   18                        18                 36[d](#pbi12551-note-0005){ref-type="fn"}   18[e](#pbi12551-note-0006){ref-type="fn"}   36           18[f](#pbi12551-note-0007){ref-type="fn"}
  Protein (%)                                                                                                                                                                                                                                                                                                                                                                
  Mean                                                                8.59                                        10.8                                        10.8             9.85                                        10.7                      10.9               10.3                                        9.88                                        10.5         11.1
  SD                                                                  0.8055                                      1.336                                       1.111            0.8663                                      0.8052                    0.7492             1.279                                       0.8326                                      1.446        0.9609
  Min                                                                 7.30                                        6.7                                         7.96             7.50                                        9.40                      10.0               7.39                                        7.50                                        7.78         9.3
  Max                                                                 10.6                                        13.7                                        13.3             11.2                                        12.5                      12.7               13.3                                        10.8                                        13.0         12.5
  Prediction interval                                                 6.87--10.3                                  8.06--13.5                                  8.50--13.0       8.06--11.6                                                                                                                                                                                    
  Fat (%)                                                                                                                                                                                                                                                                                                                                                                    
  Mean                                                                3.2                                         4.0                                         3.9              3.7                                         4.5                       4.2                4.3                                         3.8                                         4.4          4.6
  SD                                                                  0.433                                       0.540                                       0.420            0.340                                       0.304                     0.423              0.436                                       0.335                                       0.538        0.422
  Min                                                                 2.5                                         2.9                                         2.9              3.3                                         4.0                       3.2                3.3                                         3.3                                         3.2          3.9
  Max                                                                 4.1                                         4.9                                         4.7              4.6                                         **5.2**                   5.0                5.1                                         4.3                                         **5.4**      **5.3**
  Prediction interval                                                 2.3--4.1                                    2.9--5.1                                    3.1--4.8         3.0--4.4                                                                                                                                                                                      
  Carbohydrates (%)                                                                                                                                                                                                                                                                                                                                                          
  Mean                                                                --                                          83.8                                        83.8             84.9                                        83.3                      83.4               83.9                                        84.9                                        83.5         82.6
  SD                                                                  --                                          1.576                                       1.260            0.9564                                      0.9775                    0.9192             1.477                                       0.9694                                      1.673        1.113
  Min                                                                 --                                          81.0                                        80.5             83.2                                        81.2                      80.7               80.7                                        83.7                                        80.9         81.0
  Max                                                                                                             87.8                                        86.6             87.1                                        85.3                      84.3               86.8                                        **87.0**                                    86.3         84.8
  Prediction interval                                                 --                                          80.6--87.0                                  81.2--86.4       83.0--86.9                                                                                                                                                                                    
  Acid detergent fibre (%)                                                                                                                                                                                                                                                                                                                                                   
  Mean                                                                --                                          3.7                                         4.6              4.4                                         3.6                       3.5                3.6                                         4.8                                         5.0          5.8
  SD                                                                  --                                          0.577                                       1.16             1.02                                        0.493                     0.631              0.372                                       0.910                                       1.44         0.756
  Min                                                                 --                                          2.6                                         2.7              3.2                                         2.7                       2.7                *2.4*                                       3.2                                         3.3          4.9
  Max                                                                                                             5.4                                         7.7              7.0                                         4.5                       5.5                4.4                                         6.1                                         **8.4**      7.5
  Prediction interval                                                 --                                          2.6--4.9                                    2.3--7.0         2.3--6.5                                                                                                                                                                                      
  Neutral detergent fibre (%)                                                                                                                                                                                                                                                                                                                                                
  Mean                                                                --                                          11.0                                        12.0             11.7                                        9.70                      11.2               10.2                                        11.5                                        11.4         13.0
  SD                                                                  --                                          1.761                                       1.861            1.148                                       0.9773                    2.061              1.043                                       0.7710                                      0.9503       1.189
  Min                                                                 --                                          7.50                                        9.38             10.0                                        7.70                      8.80               *7.20*                                      9.90                                        9.62         10.9
  Max                                                                                                             14.4                                        15.5             15.1                                        11.9                      **18.1**           12.7                                        13.2                                        13.4         15.5
  Prediction interval                                                 --                                          7.47--14.6                                  8.19--15.7       9.34--14.1                                                                                                                                                                                    
  Starch (%)                                                                                                                                                                                                                                                                                                                                                                 
  Mean                                                                72.7                                        61.3                                        60.9             66.3                                        62.0                      66.3               67.8                                        65.0                                        66.0         59.6
  SD                                                                  0.9532                                      5.147                                       7.275            4.302                                       6.224                     6.186              4.919                                       2.089                                       5.355        2.906
  Min                                                                 70.8                                        46.8                                        51.4             54.8                                        48.0                      58.5               53.8                                        61.5                                        55.3         55.1
  Max                                                                 74.3                                        71.2                                        73.4             73.1                                        69.5                      **78.4**           75.6                                        69.2                                        **74.9**     64.7
  Prediction interval                                                 70.6--74.7                                  50.9--71.8                                  46.1--75.6       57.4--75.2                                                                                                                                                                                    
  Calcium (ppm)                                                                                                                                                                                                                                                                                                                                                              
  Mean                                                                --                                          51.1                                        47.9             46.4                                        47.7                      44.4               50.1                                        39.7                                        54.0         45.2
  SD                                                                  --                                          8.248                                       12.15            12.99                                       9.358                     4.278              8.738                                       4.467                                       10.00        4.090
  Min                                                                 --                                          37.3                                        30.5             29.4                                        34.4                      38.7               38.7                                        31.7                                        39.2         37.5
  Max                                                                                                             74.4                                        85.4             93.7                                        **76.2**                  56.6               71.9                                        48.0                                        **85.2**     49.8
  Prediction interval                                                 --                                          34.4--67.9                                  23.2--72.5       19.5--73.2                                                                                                                                                                                    
  Copper (ppm)                                                                                                                                                                                                                                                                                                                                                               
  Mean                                                                --                                          1.31                                        1.76             1.71                                        1.41                      1.58               1.48                                        1.27                                        1.59         1.29
  SD                                                                  --                                          0.1672                                      0.5628           0.4760                                      0.1813                    0.1063             0.3616                                      0.3969                                      0.2436       0.09364
  Min                                                                 --                                          0.980                                       1.00             0.960                                       1.01                      1.40               1.05                                        0.950                                       1.21         1.17
  Max                                                                                                             1.81                                        4.30             2.90                                        1.72                      1.80               2.65                                        2.70                                        **2.20**     1.45
  Prediction interval                                                 --                                          0.971--1.65                                 0.623--2.90      0.732--2.70                                                                                                                                                                                   
  Iron (ppm)                                                                                                                                                                                                                                                                                                                                                                 
  Mean                                                                --                                          22.4                                        26.3             21.6                                        22.1                      28.3               22.3                                        20.4                                        22.6         23.1
  SD                                                                  --                                          4.149                                       4.758            2.926                                       1.708                     1.739              3.568                                       1.971                                       3.904        2.440
  Min                                                                 --                                          15.7                                        17.4             17.3                                        18.6                      24.3               15.5                                        17.5                                        16.3         18.9
  Max                                                                                                             42.3                                        44.5             33.4                                        25.5                      **32.2**           **36.8**                                    24.0                                        **38.0**     27.2
  Prediction interval                                                 --                                          14.0--30.8                                  16.6--35.9       15.5--27.6                                                                                                                                                                                    
  Magnesium (ppm)                                                                                                                                                                                                                                                                                                                                                            
  Mean                                                                --                                          1233                                        1220             1290                                        1283                      1323               1232                                        1252                                        1351         1503
  SD                                                                  --                                          117.97                                      125.00           102.62                                      94.980                    96.881             126.60                                      117.39                                      195.75       85.392
  Min                                                                 --                                          896                                         919              1090                                        1060                      1150               *966*                                       1080                                        1030         1380
  Max                                                                                                             1510                                        1480             1530                                        1420                      **1540**           **1520**                                    **1490**                                    **1790**     **1660**
  Prediction interval                                                 --                                          994--1472                                   967--1473        1078--1502                                                                                                                                                                                    
  Manganese (ppm)                                                                                                                                                                                                                                                                                                                                                            
  Mean                                                                --                                          6.68                                        5.63             6.38                                        6.30                      6.61               5.64                                        6.20                                        6.55         7.40
  SD                                                                  --                                          1.048                                       1.002            1.972                                       0.9441                    0.8353             0.9827                                      1.093                                       1.607        1.413
  Min                                                                 --                                          3.86                                        3.35             4.14                                        4.69                      5.02               3.81                                        4.39                                        *4.48*       4.98
  Max                                                                                                             8.87                                        7.93             16.2                                        8.22                      8.13               7.57                                        **8.53**                                    **9.88**     **9.62**
  Prediction interval                                                 --                                          4.56--8.81                                  3.60--7.66       2.31--10.5                                                                                                                                                                                    
  Phosphorus (ppm)                                                                                                                                                                                                                                                                                                                                                           
  Mean                                                                --                                          3295                                        3092             3034                                        3459                      3543               3116                                        3127                                        3383         3993
  SD                                                                  --                                          405.24                                      501.26           261.05                                      318.63                    256.49             523.95                                      185.88                                      741.71       269.09
  Min                                                                 --                                          2180                                        1660             2430                                        2610                      2940               *1740*                                      2790                                        *1880*       3560
  Max                                                                                                             4170                                        3710             3550                                        3850                      4000               3980                                        3470                                        **4760**     **4400**
  Prediction interval                                                 --                                          2474--4115                                  2076--4108       2496--3573                                                                                                                                                                                    
  Potassium (ppm)                                                                                                                                                                                                                                                                                                                                                            
  Mean                                                                --                                          3743                                        3879             3694                                        3988                      3994               3967                                        3373                                        4182         4399
  SD                                                                  --                                          380.36                                      360.00           474.25                                      215.39                    236.47             305.74                                      162.62                                      340.87       149.96
  Min                                                                 --                                          2920                                        3060             3160                                        3570                      3570               3350                                        *3010*                                      3650         4120
  Max                                                                                                             4720                                        4650             4930                                        4340                      4400               **4670**                                    3610                                        **5020**     **4650**
  Prediction interval                                                 --                                          2973--4514                                  3150--4609       2715--4672                                                                                                                                                                                    
  Selenium (ppm)                                                                                                                                                                                                                                                                                                                                                             
  Mean                                                                --                                          0.178                                       0.188            0.292                                       0.206                     0.145              0.175                                       0.216                                       0.206        0.271
  SD                                                                  --                                          0.09044                                     0.08153          0.1862                                      0.1248                    0.1029             0.1157                                      0.1367                                      0.1509       0.1857
  Min                                                                 --                                          \<LOQ                                       \<LOQ            \<LOQ                                       0.0718                    \<LOQ              \<LOQ                                       0.0569                                      \<LOQ        \<LOQ
  Max                                                                                                             0.337                                       0.360            0.655                                       0.492                     0.399              **0.501**                                   **0.444**                                   **0.625**    **0.607**
  Predictioninterval                                                  --                                          \<LOQ--0.368                                \<LOQ--0.363     \<LOQ--0.683                                                                                                                                                                                  
  Zinc (ppm)                                                                                                                                                                                                                                                                                                                                                                 
  Mean                                                                --                                          24.1                                        23.2             20.9                                        25.1                      25.6               22.3                                        21.5                                        22.3         22.1
  SD                                                                  --                                          3.163                                       3.702            2.354                                       3.084                     3.598              3.868                                       1.899                                       3.150        1.708
  Min                                                                 --                                          17.9                                        16.9             15.9                                        20.3                      19.9               16.1                                        18.2                                        *17.2*       19.1
  Max                                                                                                             31.0                                        31.2             24.4                                        **31.8**                  **32.9**           29.7                                        25.4                                        28.5         25.4
  Predictioninterval                                                  --                                          17.7--30.5                                  15.7--30.7       16.0--25.8                                                                                                                                                                                    
  β--Carotene (mg/100 g)                                                                                                                                                                                                                                                                                                                                                     
  Mean                                                                --                                          0.112                                       0.123            0.217                                       0.0950                    0.113              0.0992                                      0.344                                       0.170        0.222
  SD                                                                  --                                          0.04016                                     0.02362          0.07010                                     0.01792                   0.01164            0.01882                                     0.02576                                     0.06540      0.01144
  Min                                                                 --                                          0.0576                                      0.0841           0.120                                       0.0710                    0.0912             0.0613                                      0.311                                       0.0692       0.201
  Max                                                                                                             0.208                                       0.195            0.316                                       0.123                     0.140              0.130                                       **0.409**                                   **0.257**    0.241
  Prediction interval                                                 --                                          0.0307--0.193                               0.0723--0.362    0.0755--0.171                                                                                                                                                                                 
  Thiamine HCl (mg/100 g)                                                                                                                                                                                                                                                                                                                                                    
  Mean                                                                --                                          0.38                                        0.45             0.42                                        0.43                      0.46               0.43                                        0.41                                        0.42         0.42
  SD                                                                  --                                          0.0643                                      0.0553           0.0388                                      0.0355                    0.0354             0.0306                                      0.0255                                      0.0320       0.0321
  Min                                                                 --                                          0.24                                        0.37             0.35                                        0.34                      0.40               0.37                                        0.36                                        0.35         0.35
  Max                                                                                                             0.48                                        0.59             0.49                                        0.49                      0.53               0.49                                        0.47                                        0.48         0.48
  Prediction interval                                                 --                                          0.251--0.511                                0.337--0.497     0.342--0.566                                                                                                                                                                                  
  Riboflavin (mg/100 g)                                                                                                                                                                                                                                                                                                                                                      
  Mean                                                                --                                          0.193                                       0.157            0.183                                       0.225                     0.203              0.229                                       0.164                                       0.215        0.223
  SD                                                                  --                                          0.03331                                     0.03430          0.03571                                     0.03629                   0.03784            0.03793                                     0.02942                                     0.04888      0.04291
  Min                                                                 --                                          0.137                                       0.111            0.112                                       0.168                     0.157              0.162                                       0.111                                       *0.107*      0.144
  Max                                                                                                             0.339                                       0.245            0.257                                       **0.292**                 **0.307**          **0.365**                                   0.228                                       **0.330**    **0.291**
  Prediction interval                                                 --                                          0.126--0.261                                0.109--0.256     0.0875--0.227                                                                                                                                                                                 
  Niacin (mg/100 g)                                                                                                                                                                                                                                                                                                                                                          
  Mean                                                                --                                          2.65                                        2.81             2.54                                        2.25                      2.86               2.64                                        2.28                                        3.19         3.29
  SD                                                                  --                                          0.7651                                      0.4087           0.2896                                      0.3358                    0.3604             0.5290                                      0.1661                                      0.4157       0.6157
  Min                                                                 --                                          1.55                                        2.18             2.11                                        1.45                      2.13               1.58                                        2.02                                        2.23         2.53
  Max                                                                                                             4.25                                        3.79             3.31                                        2.85                      3.45               3.67                                        2.74                                        3.99         **4.54**
  Prediction interval                                                 --                                          1.10--4.20                                  1.94--3.14       1.98--3.64                                                                                                                                                                                    
  Pyridoxine HCl (mg/100 g)                                                                                                                                                                                                                                                                                                                                                  
  Mean                                                                --                                          0.682                                       0.432            0.529                                       0.623                     0.649              0.679                                       0.522                                       0.718        0.692
  SD                                                                  --                                          0.09275                                     0.2557           0.07769                                     0.06852                   0.08177            0.1039                                      0.05942                                     0.09809      0.05853
  Min                                                                 --                                          0.508                                       0.218            0.385                                       0.480                     0.554              0.503                                       0.436                                       0.518        0.586
  Max                                                                                                             0.952                                       1.11             0.694                                       0.718                     0.831              **0.927**                                   0.636                                       **0.902**    0.848
  Prediction interval                                                 --                                          0.494--0.870                                0.369--0.690     \<LOQ--0.950                                                                                                                                                                                  
  Folic acid (mg/100 g)                                                                                                                                                                                                                                                                                                                                                      
  Mean                                                                --                                          0.0277                                      0.0484           0.0343                                      0.0288                    0.0297             0.0328                                      0.0258                                      0.0400       0.0455
  SD                                                                  --                                          0.004676                                    0.01549          0.01056                                     0.00715                   0.009504           0.00872                                     0.006184                                    0.01548      0.01975
  Min                                                                 --                                          0.0178                                      0.0232           0.0210                                      0.0209                    0.0209             0.0206                                      0.0179                                      0.0217       0.0273
  Max                                                                                                             0.0403                                      0.0813           0.0624                                      0.0437                    0.0525             **0.0626**                                  0.0456                                      **0.0797**   **0.0821**
  Prediction interval                                                 --                                          0.0182--0.0372                              0.0125--0.0561   0.0170--0.0798                                                                                                                                                                                
  α--Tocopherol (mg/100 g)                                                                                                                                                                                                                                                                                                                                                   
  Mean                                                                --                                          0.800                                       0.993            1.02                                        0.814                     0.801              1.19                                        1.27                                        1.26         1.03
  SD                                                                  --                                          0.1946                                      0.2536           0.2809                                      0.1471                    0.1791             0.4741                                      0.1339                                      0.3360       0.1499
  Min                                                                 --                                          0.436                                       \<LOQ            0.619                                       \<LOQ                     0.504              \<LOQ                                       1.06                                        0.850        0.838
  Max                                                                                                             1.27                                        1.86             1.54                                        1.07                      1.11               **2.18**                                    1.59                                        **2.06**     1.32
  Prediction interval                                                 --                                          0.406--1.19                                 \<LOQ--1.60      0.472--1.51                                                                                                                                                                                   
  16:0 Palmitic (% of total)                                                                                                                                                                                                                                                                                                                                                 
  Mean                                                                11.6                                        13.2                                        14.0             14.0                                        14.0                      14.0               14.3                                        12.5                                        15.0         15.4
  SD                                                                  0.9110                                      0.726                                       0.8947           1.403                                       0.4133                    0.2578             0.6363                                      0.3123                                      0.5231       0.3634
  Min                                                                 10.9                                        11.2                                        12.5             12.2                                        12.9                      13.6               13.0                                        11.9                                        13.6         15.0
  Max                                                                 13.5                                        14.5                                        15.9             16.6                                        14.5                      14.7               15.5                                        12.9                                        **15.7**     16.1
  Prediction interval                                                 9.39--13.8                                  11.8--14.7                                  11.1--16.9       12.2--15.8                                                                                                                                                                                    
  18:0 Stearic (% of total)                                                                                                                                                                                                                                                                                                                                                  
  Mean                                                                2.32                                        2.36                                        2.08             1.75                                        1.71                      1.68               2.03                                        1.93                                        2.16         2.19
  SD                                                                  0.2328                                      0.2248                                      0.2764           0.1507                                      0.1219                    0.09912            0.4006                                      0.1671                                      0.2526       0.07253
  Min                                                                 2.02                                        1.89                                        1.72             1.44                                        *1.47*                    1.47               1.51                                        1.68                                        1.78         2.08
  Max                                                                 2.70                                        2.90                                        2.77             1.99                                        1.88                      1.82               **3.20**                                    2.26                                        2.79         2.30
  Prediction interval                                                 1.75--2.89                                  1.91--2.82                                  1.44--2.06       1.52--2.64                                                                                                                                                                                    
  Oleic (% of total)                                                                                                                                                                                                                                                                                                                                                         
  Mean                                                                27.2                                        25.4                                        25.7             23.8                                        26.0                      25.8               28.4                                        25.4                                        27.4         24.7
  SD                                                                  1.277                                       2.595                                       2.502            2.151                                       1.972                     2.108              2.487                                       0.9934                                      2.163        0.4615
  Min                                                                 26.3                                        21.1                                        21.6             19.2                                        22.3                      21.5               22.9                                        23.6                                        25.2         23.3
  Max                                                                 30.3                                        29.1                                        30.6             26.6                                        28.9                      28.8               **33.2**                                    26.5                                        **32.3**     25.3
  Prediction interval                                                 24.1--30.3                                  20.2--30.7                                  19.4--28.3       20.6--30.8                                                                                                                                                                                    
  18:2 Linoleic (% of total)                                                                                                                                                                                                                                                                                                                                                 
  Mean                                                                57.7                                        56.4                                        56.3             57.6                                        55.8                      55.9               52.6                                        57.3                                        52.7         55.0
  SD                                                                  1.759                                       2.928                                       3.633            1.226                                       2.051                     2.041              3.155                                       0.9154                                      2.422        0.5780
  Min                                                                 53.2                                        52.1                                        48.8             55.9                                        52.5                      52.6               *46.4*                                      56.3                                        *46.5*       54.1
  Max                                                                 59.1                                        62.9                                        60.7             59.7                                        59.4                      59.7               58.4                                        59.3                                        55.9         56.5
  Prediction interval                                                 53.4--62.0                                  50.4--62.3                                  55.1--60.1       48.9--63.6                                                                                                                                                                                    
  18:3 Linolenic (% of total)                                                                                                                                                                                                                                                                                                                                                
  Mean                                                                1.32                                        1.58                                        1.62             1.87                                        1.59                      1.67               1.70                                        1.71                                        1.70         1.64
  SD                                                                  0.2805                                      0.08545                                     0.1297           0.1323                                      0.1289                    0.1273             0.1160                                      0.05098                                     0.08048      0.06526
  Min                                                                 0.914                                       1.31                                        1.42             1.66                                        1.46                      1.54               1.54                                        1.64                                        1.58         1.53
  Max                                                                 1.69                                        1.79                                        1.87             2.26                                        1.95                      *2.06*             2.06                                        1.82                                        1.85         1.76
  Prediction interval                                                 0.635--2.00                                 1.41--1.75                                  1.59--2.14       1.35--1.88                                                                                                                                                                                    
  Ferulic acid (ppm)                                                                                                                                                                                                                                                                                                                                                         
  Mean                                                                --                                          1993                                        1937             2663                                        1846                      1866               2005                                        2336                                        2177         2285
  SD                                                                  --                                          242.38                                      252.44           157.72                                      160.78                    169.91             259.01                                      155.95                                      229.02       148.53
  Min                                                                 --                                          1310                                        1340             2380                                        1560                      1560               1530                                        *2010*                                      1540         1990
  Max                                                                                                             2440                                        2510             2990                                        2280                      2120               2510                                        2590                                        **2620**     2520
  Prediction interval                                                 --                                          1502--2484                                  2337--2989       1425--2449                                                                                                                                                                                    
  p--Coumaric acid (ppm)                                                                                                                                                                                                                                                                                                                                                     
  Mean                                                                --                                          189                                         170              224                                         136                       123                201                                         124                                         239          214
  SD                                                                  --                                          27.51                                       81.31            58.06                                       20.39                     15.16              70.85                                       12.13                                       52.22        14.75
  Min                                                                 --                                          140                                         63.3             148                                         87.3                      *96.1*             82.7                                        104                                         178          193
  Max                                                                                                             259                                         371              354                                         167                       157                **338**                                     139                                         **363**      246
  Prediction interval                                                 --                                          133--244                                    5.05--335        104--344                                                                                                                                                                                      
  Inositol (ppm)                                                                                                                                                                                                                                                                                                                                                             
  Mean                                                                --                                          2896                                        2728             2642                                        2742                      2678               2664                                        2859                                        3024         3766
  SD                                                                  --                                          651.93                                      490.63           479.75                                      420.43                    441.2              432.94                                      402.21                                      751.4        382.32
  Min                                                                 --                                          1940                                        1580             1600                                        2170                      1660               1830                                        2190                                        1720         3320
  Max                                                                                                             5540                                        4030             3530                                        3710                      3240               3520                                        3400                                        **4900**     **4570**
  Prediction interval                                                 --                                          1576--4217                                  1652--3632       1733--3722                                                                                                                                                                                    
  Phytic acid (%)                                                                                                                                                                                                                                                                                                                                                            
  Mean                                                                --                                          0.795                                       0.714            0.744                                       0.861                     0.759              0.860                                       0.714                                       0.932        0.940
  SD                                                                  --                                          0.1230                                      0.2597           0.08178                                     0.1089                    0.1805             0.1753                                      0.0767                                      0.1562       0.1105
  Min                                                                 --                                          0.491                                       \<LOQ            0.556                                       0.631                     0.208              0.512                                       0.546                                       0.600        0.750
  Max                                                                                                             1.02                                        1.21             0.880                                       1.14                      0.969              **1.15**                                    0.837                                       **1.19**     **1.20**
  Prediction interval                                                 --                                          0.546--1.04                                 \<LOQ--0.913     0.187--1.24                                                                                                                                                                                   
  Trypsin inhibitor (TIU[g](#pbi12551-note-0008){ref-type="fn"}/mg)                                                                                                                                                                                                                                                                                                          
  Mean                                                                --                                          2.80                                        2.81             3.46                                        1.96                      1.79               2.59                                        2.90                                        3.10         3.11
  SD                                                                  --                                          1.253                                       0.4411           0.9429                                      0.2796                    0.3214             0.696                                       0.4931                                      0.297        0.2416
  Min                                                                 --                                          \<LOQ                                       2.06             2.27                                        1.50                      1.34               1.42                                        2.18                                        2.51         2.62
  Max                                                                                                             5.41                                        3.83             5.03                                        2.55                      2.38               3.99                                        3.63                                        3.69         3.54
  Prediction interval                                                 --                                          0.259--5.34                                 1.51--5.41       1.91--3.70                                                                                                                                                                                    

All values are on a dry weight basis. Each event and stack was grown in separate field trials. *N *= total number of observations, but the mean and standard deviation (SD) do not include observations below the level of quantitation (\<LOQ). Bold values indicates one or more values above the combined prediction interval for all of the single events within the stack; Italic values indicates one or more values below the combined prediction interval.

For starch, *N *=* *18 and for each fatty acid, *N *=* *9;

For starch *N *=* *5;

For iron *N *=* *33;

For copper, *N *=* *35;

For selenium, *N *=* *16;

For selenium, *N *=* *12.

TIU = trypsin inhibitor unit.

John Wiley & Sons, Ltd

About half of the stack ranges with values outside of the prediction intervals occurred among the minerals, which are not metabolized by the plant but are instead absorbed from the surrounding environment and thus are heavily influenced by such environmental parameters as soil type, soil cation exchange capacity and fertilization regimes.

Across all nutritional components, the Bt11 × GA21 stack had the greatest number of values outside of the prediction intervals. The Bt11 and the GA21 events were both present in four other stacks, yet the occurrence of values outside of the prediction intervals was inconsistent compared to the Bt11 × GA21 stack (see highlighted range values, Table 3). If two events in a stack were interacting with one another to cause changes in composition, then that interaction should occur whenever those specific events were combined in a stack. However, examination of the means and ranges of other stacks containing these events showed no such pattern.

All values within the data set fell within the ranges of the International Life Sciences Crop Composition Database (ILSI [2014](#pbi12551-bib-0027){ref-type="ref"}), which reports nutritional component levels for conventional crops.

The data for the single events and stacks were generated from plants grown in multiple environments (years, location and germplasm). The observed variability in grain composition can be attributed to these differences. It is well documented that both growing environment and germplasm have a strong influence on the composition of maize (Harrigan *et al*., [2007](#pbi12551-bib-0025){ref-type="ref"}; Reynolds *et al*., [2005](#pbi12551-bib-0041){ref-type="ref"}; Skogerson *et al*., [2010](#pbi12551-bib-0042){ref-type="ref"}; Venkatesh *et al*., [2014](#pbi12551-bib-0045){ref-type="ref"}). These results are consistent with the expectation that these events have little effect on composition, whether as single events or stacked with one another, and are a good representation of the variability in composition inherent in maize.

Protein expression {#pbi12551-sec-0008}
------------------

The ranges of concentration for each of the transgenic proteins in maize grain for all stacks were within the range of those for the corresponding single events (Figures [1](#pbi12551-fig-0001){ref-type="fig"}, [2](#pbi12551-fig-0002){ref-type="fig"}, [3](#pbi12551-fig-0003){ref-type="fig"}, [4](#pbi12551-fig-0004){ref-type="fig"}, [5](#pbi12551-fig-0005){ref-type="fig"}) except for Cry1Ab in two Bt11 × MIR604 × GA21 samples, which were very slightly lower than the range in Bt11 (Figure [1](#pbi12551-fig-0001){ref-type="fig"}). As expected, PMI concentrations in Bt11 × MIR162 × MIR604 × GA21 maize grain were higher than those in MIR162 or MIR604 due to the expression of two *pmi* genes, each contributed by MIR162 and MIR604 (Figure [5](#pbi12551-fig-0005){ref-type="fig"}). The concentrations of Cry1Ab in Bt11 × MIR162 × GA21 and Vip3Aa20 in Bt11 × MIR162× MIR604 × GA21 were generally higher than in the other stacks (Figures [1](#pbi12551-fig-0001){ref-type="fig"} and [4](#pbi12551-fig-0004){ref-type="fig"}) but were still within the ranges of expression for the single events (Bt11 and MIR162) grown in the same trials and in the same germplasm. Concentrations of PAT in event Bt11 and stacks containing Bt11 are not included because PAT concentrations were typically less than the limit of quantitation in grain. While these results do not indicate a difference in expression levels due to stacking of GM events, they do illustrate how environment and germplasm can affect protein expression levels.

![Cry1Ab concentrations in grain of event Bt11 (dark grey) and stacks containing event Bt11. *N *=* *170 (Bt11), 32 (Bt11 × GA21), 5 (Bt11 × MIR604), 29 (Bt11 × MIR604 × GA21), 10 (Bt11 × MIR162 × GA21) and 9 (Bt11 × MIR162 × MIR604 × GA21); Number of environments (combination of germplasm, year grown and location) = 11 (Bt11), 4 (Bt11 × GA21), 1 (Bt11 × MIR604), 3 (Bt11 × MIR604 × GA21), 1 (Bt11 × MIR162 × GA21), 1 (Bt11 × MIR162 × MIR604 × GA21); × = individual datapoints for Cry1Ab in event Bt11 and Bt11 × MIR162 × GA21 grown together in one field trial.](PBI-14-1899-g001){#pbi12551-fig-0001}

![mCry3A concentrations in grain of event MIR604 (dark grey) and stacks containing event MIR604. *N *=* *101 (MIR604), 5 (MIR604 × GA21), 5 (Bt11 × MIR604), 19 (Bt11 × MIR604 × GA21) and 8 (Bt11 × MIR162 × MIR604 × GA21); Number of environments (combination of germplasm, year grown and location) = 7 (MIR604), 1 (MIR604 × GA21), 1 (Bt11 × MIR604), 3 (Bt11 × MIR604 × GA21) and 1 (Bt11 × MIR162 × MIR604 × GA21).](PBI-14-1899-g002){#pbi12551-fig-0002}

![mEPSPS concentrations in grain of event GA21(dark grey) and stacks containing event GA21. *N *=* *208 (GA21), 5 (MIR604 × GA21), 30 (Bt11 × GA21), 29 (Bt11 × MIR604 × GA21), 10 (Bt11 × MIR162 × GA21) and 8 (Bt11 × MIR162 × MIR604 × GA21); Number of environments (combination of germplasm, year grown and location) = 12 (GA21), 1 (MIR604 × GA21), 4 (Bt11 × GA21), 3 (Bt11 × MIR604 × GA21), 1 (Bt11 × MIR162 × GA21) and 1 (Bt11 × MIR162 × MIR604 × GA21).](PBI-14-1899-g003){#pbi12551-fig-0003}

![Vip3Aa20 concentrations in event MIR162 (dark grey) and stacks containing event MIR162. *N *=* *52 (MIR162), 10 (Bt11 × MIR162 × GA21) and 9 (Bt11 × MIR162 × MIR604 × GA21); Number of environments (combination of germplasm, year grown and location) = 6 (MIR162), 1 (Bt11 × MIR162 × GA21), 1 (Bt11 × MIR162 × MIR604 × GA21); × = individual datapoints for Vip3Aa20 in MIR162 maize and Bt11 × MIR162 × MIR604 × GA21 maize grown together in one field trial.](PBI-14-1899-g004){#pbi12551-fig-0004}

![Phosphomannose isomerase concentrations in event MIR604, event MIR162 (both dark grey) and stacks containing events MIR604 and MIR162. *N *=* *101 (MIR604), 52 (MIR162), 5 (MIR604 × GA21), 5 (Bt11 × MIR604), 29 (Bt11 × MIR604 × GA21), 10 (Bt11 × MIR162 × GA21) and 17 (Bt11 × MIR162 × MIR604 × GA21); Number of environments (combination of germplasm, year grown and location) = 7 (MIR604), 6 (MIR162), 1 (MIR604 × GA21), 1 (Bt11 × MIR604), 3 (Bt11 × MIR604 × GA21), 1 (Bt11 × MIR162 × GA21) and 1 (Bt11 × MIR162 × MIR604 × GA21).](PBI-14-1899-g005){#pbi12551-fig-0005}

Molecular characterization {#pbi12551-sec-0009}
--------------------------

The Southern analyses had the expected hybridization patterns of the *cry1Ab* and *pat* genes from Bt11, the *vip3Aa20* and *pmi* genes from MIR162, the *mcry3A* and *pmi* genes from MIR604 and the *mepsps* gene from GA21 contained in the stacks. An example Southern blot analysis for *pmi* is shown in Figure [6](#pbi12551-fig-0006){ref-type="fig"}. These results demonstrated that inheritance and integrity of the transgenic inserts were preserved during conventional breeding for production of the stacks Bt11 × GA21, Bt11 × MIR604, MIR604 × GA21, Bt11 × MIR604 × GA21, Bt11 × MIR162× GA21 and Bt11 × MIR162 × MIR604 × GA21 maize.

![Southern blot analysis confirming presence of *pmi* in event MIR162, event MIR604 and in Bt11 × MIR162 × MIR604 × GA21.](PBI-14-1899-g006){#pbi12551-fig-0006}

Discussion {#pbi12551-sec-0010}
==========

The World Health Organization (WHO [1995](#pbi12551-bib-0047){ref-type="ref"}) has stated that when two plants, which are substantially equivalent to conventional varieties are crossed by conventional breeding, the combined event product is expected to be substantially equivalent to the individual events. The composition, protein expression and molecular characterization data reported herein for several stacked GM event combinations and the component single events supported the position of the World Health Organization. Additionally, these results were consistent with the conclusions of EFSA following review of applications submitted for import approval of these single events and stacked GM event combinations (EFSA [2005](#pbi12551-bib-0009){ref-type="ref"}, [2009a](#pbi12551-bib-0010){ref-type="ref"},[b](#pbi12551-bib-0011){ref-type="ref"},[c](#pbi12551-bib-0012){ref-type="ref"}, [2010a](#pbi12551-bib-0013){ref-type="ref"},[b](#pbi12551-bib-0014){ref-type="ref"},[c](#pbi12551-bib-0015){ref-type="ref"}, [2011b](#pbi12551-bib-0017){ref-type="ref"}, [2012](#pbi12551-bib-0018){ref-type="ref"}, EFSA, GMO Panel, [2015](#pbi12551-bib-0019){ref-type="ref"}).

More than 20 different GM crops with stacked events have been evaluated by EFSA and other regulatory agencies and in all cases EFSA concluded that there were no compositional, agronomic or phenotypic changes that would raise safety concerns (Kok *et al*., [2014](#pbi12551-bib-0031){ref-type="ref"}).

In considering the safety of stacked GM events for food and feed use, Steiner *et al*. ([2013](#pbi12551-bib-0043){ref-type="ref"}) posed two questions: (i) Is genomic instability increased in stacked GM events, and (ii) Can potential interactions between the transgenes and their products impact safety? An overview of genome plasticity and the mechanisms known to contribute to changes in plant genomes are well described by Weber *et al*. ([2012](#pbi12551-bib-0046){ref-type="ref"}). These molecular mechanisms naturally occur in plants, and as conventional breeding to combine GM events does not introduce additional recombinant DNA into the genome or modify the existing genomic DNA (Pilacinski *et al*., [2011](#pbi12551-bib-0037){ref-type="ref"}), there is no biological reason for the frequency or nature of genomic instability to differ in stacked GM events. If genomic instability does occur that affects the GM sequences contributed by the individual events, the likely outcome would be a reversion to the parental phenotype. The molecular characterization data for the GM stacks described herein confirmed stable inheritance and intactness of the inserted DNA, and supports the view that combining events through conventional breeding does not increase genetic instability.

Taken together, these data provide a strong rationale for revision of the current practices for food and feed safety assessments of stacked GM events through problem formulation focused on potential interactions resulting from the specific combination of GM events (Kok *et al*., [2014](#pbi12551-bib-0031){ref-type="ref"}; Steiner *et al*., [2013](#pbi12551-bib-0043){ref-type="ref"}; Weber *et al*., [2012](#pbi12551-bib-0046){ref-type="ref"}). Problem formulation approaches to risk assessment maximize the possibility of detecting effects that indicate potential risk (Raybould, [2006](#pbi12551-bib-0040){ref-type="ref"}). The identification and assessment of potential interactions between stacked GM events can be undertaken on a case‐by‐case basis, taking into consideration any metabolic pathways that could be influenced by the expression of the transgenes in the single events, and whether such interactions could result in changes in the plant that would impact food and feed safety. Based on each transgene\'s mode of action, it is possible to make predictions concerning potential interactions in the resulting GM stack. An interaction in the plant is not expected when the transgene products are not part of, or do not interact with, common metabolic pathways. Combinations of insect resistance traits fall into this category, as most insecticidal proteins have no known metabolic activity *in planta*. Herbicide tolerance is typically conferred by an herbicide‐insensitive version of the targeted enzyme or by an enzyme that metabolizes the herbicide, rendering it inactive, so it is also unlikely that a plant interaction could occur between proteins engineered for insect resistance and proteins expressed for herbicide tolerance. There are proteins that are known to act synergistically, and the potential for synergism between proteins combined by stacking of GM events should be evaluated. Although the scope of this manuscript is limited to food and feed safety, problem formulation can and should be applied to potential synergism of insect and herbicide resistance genes in assessments related to environmental safety. A diagram representing a decision‐making process for stacked GM event safety assessment data is shown in Figure [7](#pbi12551-fig-0007){ref-type="fig"}.

![Decision process for generation of risk assessment data for stacked events.](PBI-14-1899-g007){#pbi12551-fig-0007}

Conventional breeding has a history of safe use for combining genetic information for the improvement of crop plant varieties (CLI [2011](#pbi12551-bib-0004){ref-type="ref"}). As described herein, all new GM events undergo extensive characterization and risk assessment prior to their commercial release. Because there is no evidence of increased genome instability when GM events are stacked through conventional breeding, if there is also no plausible interaction of the transgenes or their products, then stacked GM events can be considered as safe as their component single events (CLI [2015](#pbi12551-bib-0005){ref-type="ref"}).

Experimental procedures {#pbi12551-sec-0011}
=======================

The events and stacked GM events described herein consisted of the maize events Bt11, MIR162, MIR604 and GA21, and several stacks created by combining these events through conventional breeding. Maize plants derived from event Bt11 express a truncated Cry1Ab protein for control of certain lepidopteran pests and a phosphinothricin acetyltransferase (PAT) protein that confers tolerance to glufosinate herbicide. Maize event MIR162 expresses a Vip3Aa20 protein for control of lepidopteran insect pests and a phosphomannose isomerase (PMI) protein that was used as a selectable marker. Event MIR604 expresses a modified Cry3A (mCry3A) protein for control of corn root worm and phosphomannose isomerase (PMI). Maize event GA21 expresses a modified 5‐enol pyruvylshikimate‐3‐phosphate synthase (mEPSPS) protein that confers tolerance to glyphosate herbicide. Stacked GM events were produced by combining the individual events Bt11, MIR162, MIR604 and GA21 through conventional breeding. The single events and stacks evaluated in this study were in several different genetic backgrounds.

Compositional analysis {#pbi12551-sec-0012}
----------------------

The single events and stacked GM events were grown in field trials to generate grain samples for compositional analysis. The field trials were conducted between 1995 and 2009 in the United States\' Corn Belt, and in Europe, in agricultural regions suitable for the specific hybrids being grown. Each single event or stack was grown in separate field trials at multiple locations (Table [2](#pbi12551-tbl-0002){ref-type="table-wrap"}). Some were grown in the same location and year, but in a different trial and in different genetic backgrounds. The field trials at each location were randomized in a complete block experimental design with three or four replicate plots at each location, except for the Bt11 trials, which consisted of a single plot at multiple locations. Developing maize ears were bagged before silk emergence to avoid pollen contamination and then self‐pollinated by hand and rebagged. The trials were maintained according to agricultural practices normally employed in the particular regions, including the use of pesticides needed to maintain plant health. Fifteen self‐pollinated ears per plot were harvested at maturity and dried to not more than 18% moisture, the grain was removed from the ears, and a well‐mixed sample from each plot of approximately 500 g was retained. The samples were stored at −20 °C ± 10 °C until they were analysed.

###### 

Composition field trial years and locations

  Event or stack                                Years                                         Locations                                            Total no. of environments[a](#pbi12551-note-0009){ref-type="fn"}
  --------------------------------------------- --------------------------------------------- ---------------------------------------------------- ------------------------------------------------------------------
  Bt11 × MIR162 × MIR604 × GA21                 2006                                          Illinois (2), Indiana, Iowa, Minnesota, Nebraska     6
  Bt11 × MIR162 × GA21                          2006                                          Illinois (2), Indiana, Iowa, Minnesota, Nebraska     6
  Bt11 × MIR604 × GA21                          2006                                          Illinois (2), Indiana, Iowa, Minnesota, Nebraska     12
                                                2008                                          Spain (3), Romania (3)                               
  Bt11 × MIR604                                 2005                                          Illinois (2), Iowa, Minnesota, Nebraska, Wisconsin   6
  Bt11 × GA21                                   2005                                          Illinois (3), Iowa, Nebraska, Wisconsin              12
                                                2008                                          Spain (3), Romania (3)                               
  MIR604 × GA21                                 2005                                          Illinois (2), Iowa, Nebraska, Wisconsin              5
  MIR162                                        2005                                          Illinois (2), Iowa, Nebraska, Wisconsin              9
                                                2009                                          Illinois, Iowa, Nebraska (2)                         
  MIR604                                        2003[b](#pbi12551-note-0010){ref-type="fn"}   Illinois (2), Iowa (3), Minnesota (2)                18
                                                2003[b](#pbi12551-note-0010){ref-type="fn"}   Illinois (2), Iowa (3)                               
                                                2008                                          Spain (3), Romania (3)                               
  GA21                                          2005                                          Illinois (3), Iowa, Minnesota, Nebraska              18
                                                2006                                          Spain (3), Romania (3)                               
                                                2007                                          Spain (3), Romania (3)                               
  Bt11[c](#pbi12551-note-0011){ref-type="fn"}   1995[b](#pbi12551-note-0010){ref-type="fn"}   Iowa (2), Ohio (2), Wisconsin (2)                    15
                                                1995[b](#pbi12551-note-0010){ref-type="fn"}   Iowa (2), Ohio (2), Wisconsin (2)                    
                                                1998[d](#pbi12551-note-0012){ref-type="fn"}   France (3)                                           

Environment = combination of location, year, and dgenetic background.

The event was in two genetic backgrounds but grown at all or most of the same locations in separate field trials.

Only one plot at each location.

The event was in two different genetic backgrounds at one site and in a third genetic background at another site.

John Wiley & Sons, Ltd

Levels of key nutritional components recommended by the Organization for Economic Cooperation and Development (OECD [2002](#pbi12551-bib-0033){ref-type="ref"}) were measured in maize grain. The components measured were crude protein, fat, carbohydrates (by subtraction), acid detergent fibre, neutral detergent fibre, starch, minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, selenium and zinc), fatty acids (five most abundant in maize), vitamins (β‐carotene, thiamine, riboflavin, niacin, pyridoxine, folic acid and α‐tocopherol), important nutritional secondary metabolites (ferulic acid, inositol and ρ‐coumaric acid) and antinutrients (phytic acid, trypsin inhibitor and furfural).

All samples were analysed by Covance Laboratories, Inc. (Madison, WI) using published industry‐standard analytical methods (e.g. AOAC International [2005](#pbi12551-bib-0002){ref-type="ref"}) or methods developed and validated by Covance Laboratories. The component levels were converted to equivalent units of dry weight (DW) based on the moisture content of each sample. Means, standard deviations (SD) and ranges were calculated across trials for each event or stack. Based on the compiled data for each single event, prediction intervals were calculated for the single events as a means of assessing the consistency of the data from stacks containing that single event. These prediction intervals predict with 95% confidence the range within which an additional observation would fall.

Protein expression {#pbi12551-sec-0013}
------------------

Protein expression data were compiled from 26 studies including eight different field trial locations in four different countries over an eight‐year period (2002--2010) (Table [3](#pbi12551-tbl-0003){ref-type="table-wrap"}). Comparative protein expression studies included both the stacked GM event and component single events in the same genetic background, grown in five replicate plots arranged in a randomized complete block design. In addition to the comparative protein expression studies, the single events were also grown in trials at different locations in different seasons and in different germplasm.

###### 

Protein expression field trial years and locations

  Event or Stack                  Locations (year)
  ------------------------------- --------------------------------------------------------------------------------------------------------------------
  Bt11 × MIR162 × MIR604 × GA21   Illinois (2006)
  Bt11                            
  MIR162                          
  MIR604                          
  GA21                            
  Bt11 × MIR162 × GA21            Illinois (2006)
  Bt11                            
  MIR162                          
  GA21                            
  Bt11 × MIR604 × GA21            Illinois (2006), Spain (2008), Romania (2008)
  Bt11                            
  MIR604                          
  GA21                            
  Bt11 × MIR604                   Illinois (2005)
  Bt11                            
  MIR604                          
  Bt11 × GA21                     Illinois (2005), Spain (2008), Romania (2008), Republic of South Africa (2009)
  Bt11                            
  GA21                            
  MIR604 × GA21                   Illinois (2005)
  MIR604                          
  GA21                            
  MIR162                          Illinois (2005), Nebraska (2005), Hawaii (2008), Iowa (2009), Nebraska (2010)
  MIR604                          Illinois (2002), Illinois (2007), Iowa (2009)
  GA21                            Illinois (2004), Spain (2007 & 2008), Illinois (2007), Hawaii (2008), Florida (2006), Iowa (2009), Nebraska (2010)
  Bt11                            Illinois (2002), Illinois (2007), Hawaii (2008), Florida (2006), Iowa (2009), Nebraska (2010)

Each location represents a separate field trial that included all the event(s) and the stack listed in the corresponding table row.

John Wiley & Sons, Ltd

For all protein expression studies, each grain sample consisted of all the kernels from a single ear collected at reproductive growth stage six (Abendroth *et al*., [2011](#pbi12551-bib-0001){ref-type="ref"}). Each sample was placed immediately on dry ice upon removal from the cob and stored frozen. Grain samples were ground to a powder in the presence of dry ice, using a commercial food processor.

Proteins were extracted by homogenization in an appropriate buffer and each extract was analysed by an enzyme‐linked immunosorbent assay (ELISA) specific to the transgenic protein of interest. Concentrations of the target protein were interpolated from a standard curve then converted to μg of protein per gram of sample. Data for each transgenic protein in each single event or stack were combined across germplasm, season and location, and means, medians and ranges, displayed in box plots.

Molecular characterization {#pbi12551-sec-0014}
--------------------------

Southern analyses were performed using standard molecular biology techniques (Chomczynski, [1992](#pbi12551-bib-0003){ref-type="ref"}). Genomic DNA samples from each of the individual events, the stacks and nontransgenic controls were digested with restriction enzymes that resulted in a characteristic DNA hybridization pattern for each probe. For analysis of Bt11, MIR162 and MIR604 inserts, plasmid DNA representing one copy per maize genome, based on plasmid size, was included to serve as a positive control for each of the genes of interest. For the GA21 maize insert analysis, a portion of a restriction enzyme‐generated fragment containing the *mepsps* gene, representing one copy per maize genome, was used as a positive control for the *mepsps*‐specific probe. A nontransgenic control was included on each Southern blot to identify any endogenous maize sequences that could cross‐hybridize with the element‐specific probes.

The authors wish to thank the numerous contributors of the data presented in this manuscript and Dwight Fisher (statistician, Syngenta Product Safety) for assistance with the statistical approach. Publication of the manuscript does not represent a conflict of interest for any of the authors, although they are all employees of Syngenta.

<http://www.syngenta-us.com/Agrisure/agrisure-viptera.aspx>
