Insect-protected event DAS-81419-2 soybean (Glycine max L.) grown in the United States and Brazil is compositionally equivalent to nontransgenic soybean.

The transgenic soybean event DAS-81419-2 contains genes that encode the Cry1F, Cry1Ac, and PAT proteins. Cry1F and Cry1Ac provide protection against key lepidopteran insect pests, while PAT confers tolerance to the herbicide glufosinate. To satisfy regulatory requirements for the safety evaluation of transgenic crops, studies were conducted in the United States and Brazil to evaluate the nutrient and antinutrient composition of event DAS-81419-2 soybean. On the basis of the results of these studies, event DAS-81419-2 soybean is compositionally equivalent to nontransgenic soybean. This conclusion concurs with numerous other published studies in soybean and other crops where compositional equivalence between the transgenic crop and its nontransgenic comparator has been demonstrated.

Introduction

Soybean event DAS-81419-2 (Conkesta seed) [Conkesta is a registered trademark of The Dow Chemical Company (“DOW”) or an affiliated company of Dow] was created via Agrobacterium-mediated transformation and expresses the Cry1Ac, Cry1F, and PAT proteins, which are derived from the Gram-positive bacteria Bacillus thuringiensis subspecies kurstaki, Bacillus thuringiensis subspecies aizawai, and Streptomyces viridochromogenes, respectively. The Cry1Ac and Cry1F proteins provide plant protection against certain lepidopteran insect pests,[1−3] while PAT (used as a selectable marker during the development of event DAS-81419-2 soybean) confers tolerance to the herbicide glufosinate.[4] The protection against key lepidopteran soybean insect pests such as soybean looper (Chrysodeixis includens), velvetbean caterpillar (Anticarsia gemmatalis), fall armyworm (Spodoptera frugiperda), and tobacco budworm (Heliothis virescens) that is provided by the Cry1Ac and Cry1F proteins in event DAS-81419-2 will offer soybean producers an invaluable insect management tool that does not require the use of traditional insecticides to control these pests.

Conventional plant breeding involves a multitude of techniques such as direct plant selections, tissue culture, and mutagenesis, and is often associated with mutations, deletions, insertions, and rearrangements with little or no knowledge of the underlying genetic changes that occurred to achieve the desired trait(s).[5] Transgenesis, unlike conventional breeding, consists of the insertion of a known sequence of DNA at a specific insertion point; hence, desirable traits are achieved through a clearly understood mechanism (often the production of a protein that confers insect protection or herbicide tolerance).[6] The development of transgenic crop plants includes extensive research that is conducted to elucidate the genetic changes that are caused by the transgenesis and to investigate any disruption that might occur in native genes or regulatory elements.

The nondisruptive nature of transgenesis has been demonstrated repeatedly. In 86 published studies that were conducted to assess the composition of transgenic crops, compositional equivalence between the transgenic crop and an appropriate nontransgenic comparator was demonstrated.[7−92] Regardless of this amassed body of scientific evidence, government regulations continue to require the conduct of composition studies as part of the safety evaluation of transgenic crops.[93,94]

Previous composition studies have been conducted with soybean expressing Cry1Ac and PAT, cotton expressing Cry1F, Cry1Ac, and PAT, and corn expressing Cry1F; in each of these studies, compositional equivalence between the transgenic crop and the nontransgenic crop was concluded.[11,89,91,95] The objective of this research was to determine whether the composition of forage and seed from event DAS-81419-2 soybean expressing Cry1Ac, Cry1F, and PAT is equivalent to that of nontransgenic soybean.

Materials and Methods

Soybean Samples for Compositional Analyses

Field studies were conducted in the United States (U.S.) and Brazil to produce soybean forage and seed samples of event DAS-81419-2, a near-isogenic nontransgenic comparator of the same variety that was never transformed (isoline), and commercially available nontransgenic varieties for compositional analyses. The U.S. study was conducted in 2011 at 10 field sites near Richland, Iowa, Atlantic, Iowa, Carlyle, Illinois, Wyoming, Illinois, Frankfort, Indiana, Fisk, Missouri, La Plata, Missouri, York, Nebraska, Brunswick, Nebraska, and Germansville, Pennsylvania. The Brazil study was conducted in 2012–2013 at two field sites near Indianópolis, Minas Gerais, and Montividiu, Goiás. The experimental design was a randomized complete block with four replicate plots of each entry at each field site in the U.S. study and three replicate plots of each entry at each field site in the Brazil study. In the U.S. study, plots were four rows wide by 7.6 m long with 6 cm seed spacing and 76 cm row spacing. Each four row plot was separated from adjacent plots by two border rows of a commercially available nontransgenic soybean variety of similar maturity. In the Brazil study, plots were 10 rows wide by 10 m long with 10 cm seed spacing and 50 cm row spacing. In addition to DAS-81419-2 and the isoline, a total of five nontransgenic commercial varieties were used (DSR 75213-72, HiSOY 38C60, IL3503, Porter 75148, and Williams 82) in the U.S. study; three out of the five varieties were chosen at random and planted at each field site. In the Brazil study, the nontransgenic commercial variety CD 215 was planted at both field sites. Standard commercial agronomic practices (e.g., insect and weed control) were implemented at each field site (uniformly across the entire trial) to produce a commercially acceptable crop. Forage and seed samples were collected at the R3 (beginning pod) and R8 (full maturity) growth stages, respectively, and shipped to Covance Laboratories Inc. (Madison, Wisconsin) for compositional analysis. Samples were shipped frozen and were placed in freezer storage at the analytical laboratory where they remained until removal for preparation or analysis.

Compositional Analyses

At Covance Laboratories composition samples were cryogenically ground to a homogeneous state using a blender and liquid nitrogen prior to assay. Analytes in forage samples (9 total) included proximates (moisture, carbohydrates, ash, crude fat, and protein), fiber [acid detergent fiber (ADF) and neutral detergent fiber (NDF)], and minerals (calcium and phosphorus); seed analytes (80 total) included proximates (moisture, carbohydrates, ash, crude fat, and protein), fiber (ADF, NDF, crude fiber, and total dietary fiber), minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, selenium, sodium, and zinc), amino acids (alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), fatty acids (8:0 caprylic, 10:0 capric, 12:0 lauric, 14:0 myristic, 14:1 myristoleic, 15:0 pentadecanoic, 15:1 pentadecenoic, 16:0 palmitic, 16:1 palmitoleic, 17:0 heptadecanoic, 17:1 heptadecenoic, 18:0 stearic, 18:1 oleic, 18:2 linoleic, 18:3 linolenic, 18:3 γ-linolenic, 20:0 arachidic, 20:1 eicosenoic, 20:2 eicosadienoic, 20:3 eicosatrienoic, 20:4 arachidonic, and 22:0 behenic), vitamins [β-carotene, vitamin B1 (thiamine hydrochloride), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine hydrochloride), vitamin B9 (folic acid), vitamin C (ascorbic acid), tocopherols (α, β, γ, δ, total)], and bioactives (lectin, phytic acid, raffinose, stachyose, trypsin inhibitor, total daidzein equivalent, total genistein equivalent, and total glycitein equivalent). With the exception of crude fiber and selenium, samples were assayed using previously published methods.[23] Crude fiber was determined gravimetrically as the loss on ignition of dried residue remaining after digestion of the samples with 1.25% sulfuric acid and 1.25% sodium hydroxide solutions under specific conditions. Selenium samples were closed-vessel microwave digested with nitric acid (HNO3) and water. After digestion, the solution was brought to a final volume with water. To normalize the organic contribution between samples and standards, a dilution was prepared for analysis that contained methanol. The selenium concentration was determined with Se[78] using an inductively coupled plasma-mass spectrometer (ICP-MS) with a dynamic reaction cell (DRC) by comparing the counts generated by standard solutions.

Statistical Analysis

At Dow AgroSciences, analysis of variance (ANOVA) was conducted across field sites within the U.S. study and within the Brazil study using a mixed model where entry was considered a fixed effect and location, block within location, and location-by-entry were designated as random effects.[96] Paired contrasts were performed between DAS-81419-2 and the isoline using t tests, and a false-discovery rate adjustment was applied to the P-values to account for multiplicity.[97−99] Differences were considered significant at the 95% confidence level. Analytes were excluded from the statistical analysis if more than 50% of the results were less than the limit of quantitation (fatty acids 8:0 caprylic, 10:0 capric, 12:0 lauric, 14:1 myristoleic, 15:0 pentadecanoic, 15:1 pentadecenoic, 17:1 heptadecenoic, 18:3 γ-linolenic, 20:2 eicosadienoic, 20:3 eicosatrienoic, and 20:4 arachidonic. Additionally, β-carotene and the fatty acids 14:0 myristic, 16:1 palmitoleic, and 17:0 heptadecanoic were excluded from analysis of the U.S. data, and the mineral selenium was excluded from analysis of the Brazil data. The range of values observed in the nontransgenic commercial soybean varieties was included to put any statistically significant differences into context, and ranges for nontransgenic soybean in the literature were also compiled for further reference.[11,20,28,30,35,39,59,85,100−106]

Results and Discussion

The composition of DAS-81419-2 soybean forage and seed was compared with that of the isoline at 10 field sites in the U.S. and two field sites in Brazil. In addition to DAS-81419-2 and the isoline, the U.S. study included five commercially available nontransgenic soybean varieties, and the Brazil study included one commercially available nontransgenic soybean variety. Composition analytes evaluated in forage (9) included proximates, fiber, and minerals, while analytes evaluated in seed (80) included proximates, fiber, minerals, amino acids, fatty acids, vitamins, antinutrients, and bioactives. Of the 80 seed analytes, 18 were excluded from the statistical analysis of the U.S. and/or Brazil data sets because more than 50% of the results were

No statistically significant differences were detected between DAS-81419-2 and the isoline in both the U.S. and Brazil studies in all but one (16:0 palmitic acid) of the analytes included in the statistical analysis (Tables 1–7). Moreover, mean results for all analytes fell within the ranges observed in nontransgenic commercial reference varieties and/or the ranges of values for nontransgenic soybean in the literature. In the U.S. study only, a statistically significant difference was detected between the isoline and DAS-81419-2 soybean for 16:0 palmitic acid. Although statistically significant, the difference had no meaningful effect on nutritional value; the mean 16:0 palmitic acid content of DAS-81419-2 was only 4.5% higher than that of the isoline, and the entire range of values observed in DAS-81419-2 was encompassed by the range of values reported in the literature for nontransgenic soybean (Table 5). Furthermore, a difference for 16:0 palmitic acid was not observed in the Brazil study, where the DAS-81419-2 mean was actually slightly lower than the isoline.

Table 1

Proximate, Fiber, and Mineral Composition of DAS-81419-2 Soybean Forage

	U.S. study				Brazil study
analytical componenta	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	literature range min–maxf
ash	0.926	8.96 ± 0.31 7.01–16.9	8.99 ± 0.32 7.51–10.9	6.80–15.2	0.955	9.08 ± 0.72 7.63–10.6	8.88 ± 0.72 7.87–11.1	6.37–7.58	4.68–10.782
carbohydrates	0.693	68.2 ± 1.1 57.2–76.4	67.7 ± 1.1 60.3–75.7	50.1–75.6	0.700	64.6 ± 0.6 62.5–66.5	65.9 ± 0.6 64.7–66.8	69.7–71.5	59.8–80.18
crude protein	0.841	20.4 ± 0.8 13.9–29.8	20.6 ± 0.8 15.8–25.8	14.0–35.5	0.700	23.9 ± 0.6 21.7–25.5	22.7 ± 0.6 21.1–24.6	20.0–21.6	11.2–24.71
moisture	0.719	79.7 ± 0.8 75.8–84.3	79.4 ± 0.8 75.5–83.6	75.3–85.5	0.784	81.0 ± 0.8 80.4–81.6	81.8 ± 0.7 79.7–85.0	72.6–76.9	32.05–84.60
crude fat	0.607	2.49 ± 0.19 0.898–3.89	2.70 ± 0.20 0.857–4.32	0.685–5.32	0.955	2.6 ± 0.2 2.30–3.07	2.57 ± 0.25 1.40–3.71	1.78–2.84	1.01–9.87
ADF	0.611	34.7 ± 2.1 22.4–56.7	33.3 ± 2.1 22.2–45.4	19.4–63.3	0.960	25.1 ± 1.6 21.6-31.4	25.6 ± 1.6 19.7–31.3	24.9–29.8	22.72–59.03
NDF	0.609	41.6 ± 2.6 27.8–70.9	39.9 ± 2.6 27.2–59.3	25.2–82.0	0.955	31.3 ± 1.2 27.7–36.4	31.0 ± 1.2 27.5–34.0	31.2–35.8	19.61–73.05
calcium	0.616	1377 ± 64 940–1840	1401 ± 64 908–1740	874–2000	0.700	1134 ± 61 995–1380	1040 ± 61 950–1090	697–988	NRg
phosphorus	0.841	266 ± 7 206–327	268 ± 7 201–342	187–381	0.912	254 ± 16 211–296	262 ± 16 223–337	173–238	NRg

Moisture = percent fresh weight; calcium and phosphorus = mg/100g dry weight; all others = percent dry weight.

P value (false discovery rate adjusted) for t test comparing DAS-81419-2 with isoline.

U.S. study: mean across 10 field sites with four replicates per site; Brazil study: mean across two field sites with three replicates per site.

Minimum (min) and maximum (max) represent values observed in single replicate plots.

U.S. study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots across five commercially available nontransgenic soybean varieties with three varieties planted at each field site. Brazil study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots of one commercially available nontransgenic soybean variety planted at both field sites.

See Materials and Methods section for reference citations.

NR = not reported.

Table 7

Antinutrient and Bioactive Composition of DAS-81419-2 Soybean Seed

	U.S. study				Brazil study
analytical componenta	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	literature range min–maxf
lectin	0.638	30.8 ± 2.1 13.9–50.1	32.2 ± 2.1 12.4–52.6	7.89–45.2	0.760	9.11 ± 0.86 8.06–10.2	8.50 ± 0.86 6.19–9.98	2.63–6.23	37–323
phytic acid	0.662	1.22 ± 0.06 0.857–2.02	1.24 ± 0.06 0.911–1.52	0.752–1.71	0.972	1.40 ± 0.11 1.22–1.64	1.41 ± 0.11 1.11–1.61	0.998–1.27	0.41–2.68
raffinose	0.674	0.750 ± 0.038 0.505–1.02	0.766 ± 0.038 0.475–0.977	0.570–1.16	0.700	0.840 ± 0.044 0.816–0.903	1.07 ± 0.04 0.930–1.14	0.616–0.773	0.212–1.85
stachyose	0.747	3.68 ± 0.08 3.19–4.14	3.69 ± 0.08 3.15–4.29	3.01–5.28	0.955	3.50 ± 0.04 3.36–3.61	3.49 ± 0.04 3.37–3.54	3.44–3.78	1.21–6.65
total daidzein equivalent	0.609	950 ± 48 462–1200	932 ± 48 504–1190	585–1460	0.760	260 ± 18 207–310	272 ± 18 240–295	504–577	25–2453.5
total genistein equivalent	0.616	1296 ± 63 808–1680	1276 ± 63 922–1620	888–1950	0.760	450 ± 25 358–530	428 ± 25 370–477	614–705	28–2837.2
total glycitein equivalent	0.074	197 ± 6 156–266	180 ± 6 140–237	40.3–259	0.700	160 ± 4 154–171	206 ± 4 196–216	146–168	15.3–349.19
trypsin inhibitor	0.566	29.1 ± 1.2 22.2–46.5	30.2 ± 1.2 21.3–49.9	19.5–41.5	0.700	34.1 ± 4.1 29.3–40.0	38.5 ± 4.1 28.3–46.8	19.0–24.0	18.14–118.68

Lectin = HU/mg protein dry weight; phytic acid, raffinose, and stachyose = % dry weight; isoflavones = μg/g dry weight; trypsin inhibitor = TIU/mg dry weight.

P value (false discovery rate adjusted) for t test comparing DAS-81419-2 with isoline.

U.S. study: mean across 10 field sites with four replicates per site. Brazil study: mean across two field sites with three replicates per site.

Minimum (min) and maximum (max) represent values observed in single replicate plots.

U.S. study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots across five commercially available nontransgenic soybean varieties with three random varieties planted at each field site. Brazil study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots of one commercially available nontransgenic soybean variety planted at both field sites.

See Materials and Methods section for reference citations.

Table 5

Fatty Acid Composition of DAS-81419-2 Soybean Seed

	U.S. study				Brazil study
analytical componenta	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	literature range min–maxf
8:0 caprylic	NAg	NAg <LOQ	NAg <LOQ	<LOQ	NAg	NAg <LOQ	NAg <LOQ	<LOQ	<LOQ–0.148
10:0 capric	NAg	NAg <LOQ	NAg <LOQ	<LOQ	NAg	NAg <LOQ	NAg <LOQ	<LOQ	<LOQ–0.27
12:0 lauric	NAg	NAg <LOQ	NAg <LOQ	<LOQ	NAg	NAg <LOQ	NAg <LOQ	<LOQ	<LOQ–0.132
14:0 myristic	NAg	NAg <LOQg	NAg <LOQg	<LOQ	0.700	0.0988 ± 0.0018 0.0930–0.103	0.103 ± 0.002 0.094–0.108	0.0724–0.0895	<LOQ–0.238
14:1 myristoleic	NAg	NAg <LOQ	NAg <LOQ	<LOQ	NAg	NAg <LOQ	NAg <LOQ	<LOQ	<LOQ–0.125
15:0 pentadecanoic	NAg	NAg <LOQ	NAg <LOQ	<LOQ	NAg	NAg <LOQ	NAg <LOQ	<LOQ	<LOQ
15:1 pentadecenoic	NAg	NAg <LOQ	NAg <LOQ	<LOQ	NAg	NAg <LOQ	NAg <LOQ	<LOQ	<LOQ
16:0 palmitic	<0.001h	11.1 ± 0.1 10.0–11.6	11.6 ± 0.1 10.9–12.3	9.12–11.5	0.749	13.0 ± 0.1 12.8–13.2	12.9 ± 0.1 12.6–13.1	11.3–12.2	1.40–15.77
16:1 palmitoleic	NAg	NAg <LOQ	NAg <LOQ–0.236	<LOQg	0.700	0.108 ± 0.002 0.105–0.111	0.0915 ± 0.0017 0.0871–0.0961	0.111–0.131	<LOQg–0.194
17:0 heptadecanoic	NAg	NAg <LOQ–0.127	NAg <LOQ–0.132	<LOQg–0.133	0.896	0.103 ± 0.003 0.0955–0.111	0.102 ± 0.003 0.0972–0.106	0.0753–0.0877	<LOQg–0.146
17:1 heptadecenoic	NAg	NAg <LOQ	NAg <LOQ	<LOQ	NAg	NAg <LOQ	NAg <LOQ	<LOQ	<LOQ–0.087
18:0 stearic	0.516	4.40 ± 0.12 3.67–5.27	4.46 ± 0.12 3.62–5.22	3.19–5.08	0.760	5.18 ± 0.11 4.85–5.46	5.07 ± 0.11 4.94–5.18	3.65–3.84	0.50–5.88
18:1 oleic	0.439	21.6 ± 0.4 19.6–25.2	21.2 ± 0.4 19.8–25.5	18.8–24.6	0.700	27.7 ± 1.1 26.0–30.4	22.1 ± 1.1 21.5–22.6	25.0–30.0	2.60–45.68
18:2 linoleic	0.476	54.1 ± 0.4 51.2–55.8	53.8 ± 0.4 50.5–55.7	53.6–57.5	0.700	47.5 ± 1.0 45.0–49.2	51.9 ± 1.0 51.5–52.4	47.3–50.7	7.58–58.8
18:3 linolenic	0.314	7.97 ± 0.15 6.91–8.76	8.17 ± 0.15 7.32–8.94	6.58–9.88	0.700	5.14 ± 0.26 4.54–5.64	6.56 ± 0.26 6.06–7.05	6.21–6.95	1.27–12.52
18:3 γ-linolenic	NAg	NAg <LOQ	NAg <LOQ	<LOQ	NAg	NAg <LOQ	NAg <LOQ	<LOQ	<LOQ
20:0 arachidic	0.413	0.319 ± 0.007 0.276–0.370	0.325 ± 0.007 0.282–0.372	0.254–0.383	0.700	0.453 ± 0.012 0.418–0.491	0.471 ± 0.012 0.450–0.484	0.396–0.409	0.038–0.57
20:1 eicosenoic	0.616	0.119 ± 0.019 <LOQ–0.189	0.105 ± 0.019 <LOQ–0.171	<LOQg–0.191	0.700	0.223 ± 0.007 0.210–0.245	0.200 ± 0.006 0.184–0.214	0.205–0.220	0.024–0.35
20:2e icosadienoic	NAg	NAg <LOQ	NAg <LOQ	<LOQ	NAg	NAg <LOQ	NAg <LOQ	<LOQ	<LOQ–0.245
20:3 eicosatrienoic	NAg	NAg <LOQ	NAg <LOQ	<LOQ	NAg	NAg <LOQ	NAg <LOQ	<LOQ	<LOQ
20:4 arachidonic	NAg	NAg <LOQ	NAg <LOQ	<LOQ	NAg	NAg <LOQ	NAg <LOQ	<LOQ	<LOQ
22:0 behenic	0.476	0.317 ± 0.003 0.283–0.347	0.321 ± 0.003 0.263–0.345	0.281–0.390	0.986	0.466 ± 0.018 0.429–0.499	0.465 ± 0.018 0.438–0.490	0.517–0.534	0.043–0.65

Percent of total fatty acids.

P value (false discovery rate adjusted) for t test comparing DAS-81419-2 with isoline.

U.S. study: mean across 10 field sites with four replicates per site. Brazil study: mean across two field sites with three replicates per site.

Minimum (min) and maximum (max) represent values observed in single replicate plots.

U.S. study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots across five commercially available nontransgenic soybean varieties with three varieties planted at each field site. Brazil study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots of one commercially available nontransgenic soybean variety planted at both field sites.

See Materials and Methods section for reference citations.

NA (not available) = analysis not performed: majority of data was

Statistically significant difference when compared with isoline (P < 0.05).

While the composition of isoline and DAS-81419-2 soybean was similar both across and within field sites, results within each entry were highly variable across field sites; ADF and NDF in forage provide examples of this site to site variation (Figure 1). The lack of differences between the isoline and DAS-81419-2 coupled with the wide range of results across field sites in these studies indicates that environment has a substantial effect on crop composition and the effect of transgenesis is undetectable, as others have reported.[7,28,85,89,100,107−109]

Figure 1

Site means for ADF and NDF in soybean forage. The shaded area represents the literature range and locations are represented by the following symbols: ○ = Richland, IA; × = Atlantic, IA; + = Carlyle, IL; Δ = Wyoming, IL; □ = Frankfort, IN;◇ = Fisk, MO; ● = La Plata, MO; ▲ = York, NE; ■ = Brunswick, NE; ◆ = Germansville, PA; # = Montividiu, Goiás; ∼ = Indianópolis, Minas Geráis. “Isoline” = near-isogenic nontransgenic line. Note that each nontransgenic commercial variety was included, on average, at only half of the field sites in the U.S. study, so the range of data for these entries is expected to be narrower than that of the isoline and DAS-81419-2 entries, which were present at all 10 sites.

Conclusions

On the basis of the absence of meaningful differences between the isoline and DAS-81419-2 soybean in studies that were conducted at multiple field sites in two countries, it is concluded that the composition of DAS-81419-2 soybean forage and seed is equivalent to that of nontransgenic soybean. The conclusion of this study is in concordance with 86 other published studies and over 100 regulatory submissions where compositional equivalence between the transgenic crop and nontransgenic crop was concluded.[7−92,110] Collectively, these results indicate that composition studies to detect unintended effects due to transgenesis with traits that are not expected to alter composition are of dubious value in assessing the safety of transgenic crops.

Table 2

Proximate and Fiber Composition of DAS-81419-2 Soybean Seed

	U.S. study				Brazil study
analytical componenta	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	literature range min–maxf
ash	0.233	5.06 ± 0.07 4.62–5.68	5.18 ± 0.07 4.57–6.05	3.79–6.79	0.786	5.35 ± 0.09 5.19–5.45	5.43 ± 0.09 5.14–5.60	4.84–5.15	3.885–6.994
carbohydrates	0.616	38.8 ± 0.7 33.6–41.2	39.0 ± 0.7 33.3–43.3	29.9–40.7	0.700	38.3 ± 2.9 33.7–44.2	32.9 ± 2.9 31.6–34.4	33.8–34.9	29.3–50.2
crude protein	0.662	37.9 ± 0.6 34.3–41.9	38.1 ± 0.6 32.9–42.8	36.5–46.0	0.972	39.9 ± 0.5 39.5–40.6	40.0 ± 0.5 38.9–41.5	38.5–40.4	32–48.4
moisture	0.196	12.3 ± 0.9 8.29–19.2	11.7 ± 0.9 7.56–17.9	7.94–22.7	0.700	36.0 ± 2.9 31.2–42.0	28.7 ± 2.9 25.7–30.6	14.9–18.6	4.7–34.4
crude fat	0.108	18.2 ± 0.5 15.4–21.5	17.7 ± 0.5 14.2–21.0	14.1–21.4	0.700	16.5 ± 2.6 11.1–21.1	21.7 ± 2.6 21.0–22.5	20.6–21.9	8.104–24.7
ADF	0.845	15.3 ± 0.7 10.6–22.8	15.2 ± 0.7 10.5–22.5	10.2–21.0	0.986	11.9 ± 0.9 9.03–15.7	11.9 ± 0.9 10.1–14.1	14.0–16.6	7.81–26.26
NDF	0.719	17.5 ± 0.8 11.7–24.2	17.7 ± 0.8 11.6–25.5	10.6–21.9	0.700	15.0 ± 0.8 12.8–17.6	14.4 ± 0.8 13.0–16.3	14.6–17.2	8.53–23.90
crude fiber		not measured			0.700	6.67 ± 0.99 5.28–8.21	7.72 ± 0.99 6.47–8.82	10.6–12.1	4.12–13.87
total dietary fiber	0.765	23.8 ± 0.9 17.6–29.1	24.0 ± 0.9 17.4–31.3	16.1–29.2	0.700	17.0 ± 0.4 16.4–17.4	17.6 ± 0.4 16.9–18.7	17.5–20.2	NRg

Moisture = percent fresh weight; all others = percent dry weight.

P value (false discovery rate adjusted) for t test comparing DAS-81419-2 with isoline.

U.S. study: mean across 10 field sites with four replicates per site. Brazil study: mean across two field sites with three replicates per site.

Minimum (min) and maximum (max) represent values observed in single replicate plots.

U.S. study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots across five commercially available nontransgenic soybean varieties with three varieties planted at each field site. Brazil study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots of one commercially available nontransgenic soybean variety planted at both field sites.

See Materials and Methods section for reference citations.

NR = not reported.

Table 3

Mineral Composition of DAS-81419-2 Soybean Seed

	U.S. study				Brazil study
analytical componenta	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	literature range min–maxf
calcium	0.476	270 ± 8 233–335	267 ± 8 205–328	181–308	0.700	332 ± 12 318–359	322 ± 12 304–341	264–350	116.55–510
copper	0.747	1.32 ± 0.06 0.922–1.64	1.33 ± 0.06 0.894–1.72	0.693–1.86	0.714	0.947 ± 0.140 0.765–1.13	0.911 ± 0.140 0.694–1.07	0.626–1.06	0.632–1.092
iron	0.693	9.56 ± 1.40 6.61–27.3	10.3 ± 1.4 6.21–42.9	6.33–151	0.700	20.1 ± 3.3 14.0–38.8	13.8 ± 3.3 11.4–14.7	6.34–13.5	3.734–10.954
magnesium	0.747	233 ± 4 204–256	232 ± 4 197–257	205–278	0.700	294 ± 8 281–314	267 ± 8 257–276	280–296	219.40–312.84
manganese	0.719	2.64 ± 0.09 2.13–3.09	2.67 ± 0.09 2.01–3.96	2.22–7.18	0.700	2.30 ± 0.27 1.84–2.71	2.48 ± 0.27 2.13–2.79	1.77–2.88	2.52–3.876
phosphorus	0.476	607 ± 14 536–704	619 ± 14 494–708	471–759	0.700	588 ± 29 539–655	620 ± 29 536–670	509–586	506.74–935.24
potassium	0.478	1799 ± 21 1660–1940	1819 ± 21 1490–1980	1650–2050	0.700	1858 ± 21 1790–1930	1977 ± 21 1910–2050	1760–1840	1868.01–2510
selenium	0.616	460 ± 187 <LOQ–2370	498 ± 187 <LOQ–2560	<LOQ–3060	NAg	NAg <LOQ	NAg <LOQ	<LOQ	NRh
sodium	NA	NAg <LOQ	NAg <LOQ	<LOQ–18.5	NAg	NAg <LOQ	NAg <LOQ	<LOQ	4.05–30
zinc	0.413	4.53 ± 0.15 3.66–5.73	4.63 ± 0.15 3.66–5.83	3.15–6.33	0.700	4.11 ± 0.14 3.72–4.48	4.40 ± 0.14 3.93–5.09	3.66–4.28	4.98–7.578

Selenium = ppb dry weight; all others = mg/100 g dry weight.

P value (false discovery rate adjusted) for t test comparing DAS-81419-2 with isoline.

U.S. study: mean across ten field sites with four replicates per site. Brazil study: mean across two field sites with three replicates per site.

Minimum (min) and maximum (max) represent values observed in single replicate plots.

U.S. study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots across five commercially available nontransgenic soybean varieties with three varieties planted at each field site. Brazil study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots of one commercially available nontransgenic soybean variety planted at both field sites.

See Materials and Methods section for reference citations.

NA (not available) = analysis not performed; majority of data was

NR = not reported.

Table 4

Amino Acid Composition of DAS-81419-2 Soybean Seed

	U.S. study				Brazil study
analytical componenta	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	literature range min–maxf
alanine	0.566	4.57 ± 0.03 4.31–4.76	4.59 ± 0.03 4.30–4.82	4.28–4.75	0.760	4.46 ± 0.03 4.39–4.57	4.49 ± 0.03 4.38–4.61	4.35–4.53	4.16–4.74
arginine	0.476	7.49 ± 0.05 7.04–7.79	7.44 ± 0.05 7.05–7.74	7.22–8.20	0.830	7.59 ± 0.05 7.47–7.68	7.57 ± 0.05 7.49–7.66	7.68–8.03	6.41–8.41
aspartic acid	0.476	11.5 ± 0.01 11.3–11.6	11.5 ± 0.01 11.3–11.8	9.99–11.74	0.700	11.5 ± 0.03 11.4–11.6	11.5 ± 0.03 11.4–11.5	11.4–11.6	11.37–12.68
cystine	0.609	1.61 ± 0.03 1.43–1.95	1.63 ± 0.03 1.45–1.86	1.24–1.79	0.700	1.46 ± 0.10 1.33–1.61	1.52 ± 0.10 1.42–1.62	1.23–1.48	1.02–1.87
glutamic acid	0.476	17.3 ± 0.11 16.3–18.0	17.2 ± 0.1 16.3–18.0	17.0–18.6	0.973	18.0 ± 0.1 17.7–18.3	18.0 ± 0.1 17.9–18.1	17.7–18.4	17.71–20.48
glycine	0.845	4.50 ± 0.20 4.34–4.67	4.50 ± 0.02 4.30–4.66	4.14–4.54	0.700	4.34 ± 0.01 4.31–4.40	4.31 ± 0.01 4.27–4.38	4.28–4.36	4.19–4.62
histidine	0.719	2.71 ± 0.02 2.58–2.82	2.70 ± 0.02 2.17–2.84	2.43–2.78	0.896	2.59 ± 0.01 2.55–2.64	2.60 ± 0.01 2.58–2.64	2.45–2.66	2.49–2.89
isoleucine	0.926	4.80 ± 0.02 4.65–4.96	4.80 ± 0.02 4.52–4.99	4.61–4.98	0.700	4.90 ± 0.02 4.82–5.00	4.83 ± 0.02 4.77–4.89	4.72–4.86	4.13–5.11
leucine	0.732	7.65 ± 0.01 7.51–7.83	7.64 ± 0.01 7.49–7.75	7.49–7.98	0.972	7.66 ± 0.02 7.60–7.70	7.67 ± 0.02 7.63–7.73	7.57–7.79	7.46–8.29
lysine	0.476	6.32 ± 0.08 5.90–7.51	6.44 ± 0.08 5.92–7.41	5.61–7.29	0.700	6.46 ± 0.03 6.40–6.63	6.40 ± 0.03 6.34–6.45	5.87–6.61	6.23–7.38
methionine	0.551	1.42 ± 0.01 1.31–1.61	1.44 ± 0.01 1.33–1.61	1.22–1.62	0.700	1.37 ± 0.02 1.33–1.41	1.35 ± 0.02 1.32–1.39	1.22–1.37	1.18–1.71
phenylalanine	0.108	5.15 ± 0.01 4.99–5.25	5.11 ± 0.01 4.92–5.22	4.88–5.37	0.760	5.23 ± 0.04 5.15–5.31	5.21 ± 0.04 5.15–5.25	5.24–5.46	4.91–5.44
proline	0.566	5.14 ± 0.04 4.86–5.97	5.20 ± 0.04 4.91–5.73	4.80–6.02	0.700	5.17 ± 0.04 5.12–5.27	5.24 ± 0.04 5.20–5.30	5.14–5.32	4.75–5.62
serine	0.670	5.13 ± 0.02 4.74–5.37	5.16 ± 0.02 4.85–5.38	4.81–5.53	0.700	5.02 ± 0.04 4.80–5.18	5.16 ± 0.04 5.10–5.22	4.73–5.14	3.25–6.04
threonine	0.616	4.19 ± 0.03 3.99–4.38	4.20 ± 0.03 3.96–4.44	3.86–4.25	0.760	4.03 ± 0.03 3.96–4.08	4.04 ± 0.03 4.00–4.09	3.94–4.05	3.15–4.24
tryptophan	0.747	1.52 ± 0.02 1.42–1.64	1.52 ± 0.02 1.40–1.65	1.27–1.69	0.714	1.38 ± 0.01 1.33–1.43	1.41 ± 0.01 1.37–1.44	1.33–1.38	0.95–1.49
tyrosine	0.638	3.97 ± 0.01 3.88–4.10	3.96 ± 0.01 3.83–4.06	3.82–4.16	0.786	3.94 ± 0.02 3.86–4.00	3.93 ± 0.02 3.89–4.00	3.99–4.07	2.62–3.72
valine	0.693	4.98 ± 0.02 4.74–5.22	4.96 ± 0.02 4.70–5.32	4.63–5.18	0.700	4.85 ± 0.02 4.79–4.91	4.78 ± 0.02 4.70–4.83	4.66–4.88	4.28–5.57

Unit of measure = percent of total amino acids.

P value (false discovery rate adjusted) for t test comparing DAS-81419-2 with isoline.

U.S. study: mean across 10 field sites with four replicates per site. Brazil study: mean across two field sites with three replicates per site.

Minimum (min) and maximum (max) represent values observed in single replicate plots.

U.S. study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots across five commercially available nontransgenic soybean varieties with three varieties planted at each field site. Brazil study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots of one commercially available nontransgenic soybean variety planted at both field sites.

See Materials and Methods section for reference citations.

Table 6

Vitamin Composition of DAS-81419-2 Soybean Seed

	U.S. study				Brazil study
analytical componenta	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	P valueb	isoline mean ± SEc min–maxd	DAS-81419-2 mean ± SEc min–maxd	reference variety range min–maxe	literature range min–maxf
α-tocopherol (vitamin E)	0.607	14.3 ± 1.3 8.56–29.3	13.6 ± 1.3 9.62–26.6	6.51–25.0	0.700	9.45 ± 0.91 6.98–11.5	12.5 ± 0.9 9.31–15.9	25.2–39.2	1.934–84.9
β-tocopherol	NAg	NAc <LOQ	NAc <LOQ	<LOQ	NAc	NAc <LOQ	NAc <LOQ	<LOQ	NRg
γ-tocopherol	0.616	168 ± 8 97–220	172 ± 8 118–219	77.5–204	0.700	39.4 ± 5.2 28.7–49.1	57.0 ± 5.2 36.7–77.9	176–215	NRg
δ-tocopherol	0.074	69.4 ± 2.7 41.3–83.1	74.7 ± 2.8 45.4–90.8	49.7–104	0.700	26.7 ± 2.1 22.7–33.5	31.6 ± 2.1 23.8–38.8	62.2–76.6	NRg
total tocopherols	0.476	252 ± 8 168–305	261 ± 8 205–301	160–304		not measured			NRg
β-carotene	NA	NAc <LOQ	NAc <LOQ	<LOQ–0.244		not measured			NRg
vitamin B1 (thiamine hydrochloride)	0.662	3.51 ± 0.24 2.35–5.44	3.43 ± 0.24 2.20–5.16	1.82–4.60	0.700	5.69 ± 0.36 5.31–6.07	4.67 ± 0.36 4.08–5.78	3.95–5.40	1.01–2.54
vitamin B2 (riboflavin)	0.476	3.40 ± 0.08 2.63–4.65	3.51 ± 0.08 2.58–4.64	2.42–5.00	0.700	4.98 ± 0.13 4.51–5.24	5.43 ± 0.13 5.05–5.89	4.53–5.70	1.90–3.21
vitamin B3 (niacin)	0.413	25.0 ± 0.7 20.2–30.5	25.6 ± 0.7 20.3–32.1	20.7–29.0	0.700	34.3 ± 1.2 31.7–36.4	36.0 ± 1.2 34.3–37.9	18.4–23.0	NRg
vitamin B5 (pantothenic acid)	0.074	14.8 ± 0.5 12.3–19.5	14.0 ± 0.5 11.8–16.8	8.97–18.0	0.700	14.8 ± 0.7 13.7–15.5	16.2 ± 0.72 14.7–18.3	14.4–16.7	NRg
vitamin B6 (pyridoxine hydrochloride)	0.787	5.23 ± 0.11 4.42–6.43	5.18 ± 0.11 4.53–6.00	3.01–6.36	0.700	4.88 ± 0.19 4.71–5.04	4.31 ± 0.19 3.79–4.60	4.49–5.18	NRg
vitamin B9 (folic acid)	0.732	4.21 ± 0.20 3.05–5.62	4.15 ± 0.20 2.75–5.51	2.94–5.59	0.700	5.08 ± 0.23 4.64–5.72	4.50 ± 0.23 4.16–5.06	4.02–4.42	2.386–4.709
vitamin C (ascorbic acid)	0.476	141 ± 13 75.2–231	133 ± 13 74.0–230	49.2–210	0.700	62.2 ± 9.0 48.0–98.8	46.9 ± 9.0 37.0–72.5	53.7–83.8	NRg

mg/kg dry weight.

P value (false discovery rate adjusted) for t test comparing DAS-81419-2 with isoline.

U.S. study: mean across 10 field sites with four replicates per site. Brazil study: mean across two field sites with three replicates per site.

Minimum (min) and maximum (max) represent values observed in single replicate plots.

U.S. study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots across five commercially available nontransgenic soybean varieties with three random varieties planted at each field site. Brazil study: reference variety range represents minimum (min) and maximum (max) values observed in single replicate plots of one commercially available nontransgenic soybean variety planted at both field sites.

See Materials and Methods section for reference citations.

NR = not reported.