To further develop this proposal on transportability of confined field trial data, this paper reviewed the results of the confined field trials for three GM corn events conducted both in the US and Japan as case studies. The goal of reviewing these data is to illustrate how results from the US confined fields trials are relevant for conducting the ERA of GM crops for an import country like Japan.
Specifically, this paper reviewed ERA data for Lysine maize LY038, lepidopteran insect-protected corn MON 89034, and drought-tolerant corn MON 87460, all of which have previously been deregulated in the US and approved in Japan under the Cartagena Law. The submission documents for the three GM corn varieties are available in the websites of both United States Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) and Japan Biosafety Clearing House (J-BCH) (APHIS 2015; J-BCH 2015).
Lysine maize LY038 was developed through the use of recombinant DNA techniques, to integrate the cordapA coding sequence into the maize genome. The cordapA sequence is under the control of the maize Glb1 promoter to direct expression of the Corynebacterium glutamicum-derived lysine-insensitive dihydrodipicolinate synthase (cDHDPS) enzyme predominantly in the germ, resulting in increased levels of lysine in grain for animal feed applications.
Lepidopteran insect-protected corn MON 89034 expresses Cry1A.105 and Cry2Ab2 insecticidal proteins and is protected from feeding damage caused by European corn borer (Ostrinia nubilialis) and other lepidopteran insect pests. Cry1A.105 is a modified Bacillus thuringiensis (Bt) Cry1A protein with 93.6 % overall amino acid sequence homology to the Cry1Ac protein. Cry2Ab2 is also a Bt (subsp. kurstaki) protein. The combination of the Cry1A.105 and Cry2Ab2 insecticidal proteins in a single plant provides broad spectrum of insect control and offers an enhanced insect-resistance management tool.
Drought-tolerant corn MON 87460 expresses a cold shock protein B (CSPB) produced from the inserted B. subtilis-derived gene. In bacteria, the CSPB protein helps preserve normal cellular functions during certain stresses by binding cellular RNA and unfolding non-translatable secondary structures affecting RNA stability and translation. During product development, MON 87460 exhibited reduced yield loss under water-limited conditions compared to conventional corn. Like conventional corn, MON 87460 is still subject to yield loss under water-limited conditions, particularly during flowering and grainfill periods when corn yield potential is most sensitive to stress as a result of disrupted kernel development (Monsanto Company 2009).
As summarized in the Table 1, confined field data were obtained from multiple locations and multiple years in the US. For example, phenotypic and agronomic data for Lysine maize LY038 were obtained at 10 and seven sites in 2002 and 2003, respectively, in Missouri, Illinois, Indiana, Iowa, and Nebraska (Monsanto Company 2004). These diverse locations provided a range of environmental and agronomic conditions representing major US corn-growing regions where commercial production of the GM crops would be expected. Notably, drought-tolerant corn MON 87460 was tested in more diverse field conditions such as (1) well-watered, (2) both well-watered and water-limited treatments established in the same field, or (3) water managed according to typical agronomic practices, which included typical amounts of supplemental irrigation at relevant sites. Because MON 87460 reduces yield loss under water-limited conditions, field studies were designed to evaluate the environmental consequences of MON 87460 performance across a broad range of soil moisture and environmental conditions (Sammons et al. 2014).
In Japan, data from a confined field trial obtained at a single location and a single year is accepted for both cultivation and import approval (Table 2). Furthermore, the tassels of GM corn are usually cut off or covered by paper bags because it is difficult to ensure sufficient isolation distance to limit cross-pollination with conventional corn varieties which grow in neighborhoods in Japan; while isolation distances can be established and managed in the US field trials. This measure to avoid cross-pollination in Japan makes it difficult to obtain reliable data from field trials for the ERA of GM crops.
Regarding the data requirements for the ERA of GM corn and cotton, some differences exist between the US and Japan (Table 3). For example, “tolerance to low or high temperature of immature plants” and “the overwintering or over summering ability of the mature plant” are not requested for any GM crops in the US. However, it is usually the case that GM crops tested in the US are exposed to a wide range of field temperatures by testing the crop at multiple locations covering the major US corn-growing regions as described above, thereby effectively addressing these Japanese requirements. Additionally, ERAs that are science-based should be hypothesis driven, and therefore abiotic stress tolerance studies, including cold stress, are conducted in the US based on the characteristics of the inserted gene(s). For example, drought, cold, heat, and salt stress studies were conducted under controlled environmental conditions, such as greenhouses and growth chambers, for drought-tolerant corn MON 87460 in the US, because cold shock proteins are known to mitigate multiple abiotic stressors in both bacteria and plants (Castiglioni et al. 2008). Results support the conclusion that the abiotic stress tolerance of MON 87460 during young plant growth stages is not meaningfully different compared to conventional corn (Monsanto Company 2009). Consistent with a hypothesis driven approach for the ERA of GM crops, these comprehensive studies to confirm abiotic stress tolerance were not conducted for non-stress-tolerant events such as LY038 and MON 89034.
Evaluation of “potential production of harmful substance” and its effects on other plants and soil microorganisms is also requested in Japan regardless of the characteristics of the inserted gene(s) (Table 3). Although these data are not obtained in the US, ecological interaction data are assessed qualitatively for every GM crop during the growing season. This study assesses plant interactions with insect pests and disease, as well as plant responses to abiotic stressors. The results of the ecological interaction study are relevant for assessing the release of harmful substances from GM crops and, if meaningful differences were detected between a GM crop and its conventional control further analysis may be needed to inform the ERA. Furthermore, more detailed and targeted NTO studies were conducted for lepidopteran insect-protected corn MON 89034 because insecticidal proteins such as Cry1A.105 and Cry2Ab2 expressed in MON 89034 could negatively affect the diversity and abundance of non-target arthropod communities including predators, parasitoids, and other ecologically important non-target arthropods. The assessment took into consideration several components, including the familiarity with the mode of action of Cry proteins, the activity spectra of the Cry1A.105 and Cry2Ab2 proteins, the expression levels of the two proteins in MON 89034, the environmental fate of the proteins, any potential interaction between the two proteins, and feeding tests of the two proteins or MON 89034 corn materials to representative NTOs. As the result of the comprehensive assessment of the potential impact of MON 89034 and the introduced proteins on NTOs and endangered species, it was concluded that environmental risk to these organisms from the use of MON 89034 was negligible (Monsanto Company 2006). These comprehensive studies to confirm the impact on NTOs and endangered species were not conducted for non-insect-protected events such as LY038 and MON 87460, because they do not have insecticidal activity.
As described above, there are some differences in the data requirements for GM corn and cotton between the US and Japan. However, additional data such as abiotic stress tolerance and release of harmful substance are obtained in the US depending on the characteristics of the inserted gene(s) and/or results obtained from the confined field trials.
Both the US and Japan evaluate plant characteristics that may be related to weediness potential regardless of the characteristics of inserted gene(s). For example, seed dormancy, plant lodging, and seed pod shattering are recognized as important characteristics related to weediness potential of soybean in the US (Horak et al. 2015). Seed dormancy would be required for a seed to over-winter or establish self-sustaining populations over several seasons. In addition, plant lodging and seed pod shattering could potentially be associated with aspects of seed dispersal. The mature seeds would need to be dispersed to favorable niches for the plant to function as a weed outside of cultivation or in an agronomic setting and not be harvested at the end of the growing season. In the US, these plant characteristics, including dropped ears, stalk lodged plants, yield and germination of harvested seed, are evaluated for each GM crop product regardless of the characteristics of the inserted gene(s) as a part of the agronomic/phenotypic evaluation. Similarly the characteristics related to seed productivity (e.g. number of grain rows), seed shattering, and germination of harvested seed are always evaluated in Japan (Table 3). As the seed shattering in Japan is compared between GM corn and non-GM control by visual analysis, no statistical comparison is conducted for this endpoint.
This paper evaluates the following selected plant characteristics for the assessment of weediness potential for LY038, MON 89034, and MON 87460 from the US: dropped ears (#/plot), yield (bu/a), stalk lodged plants (#/plot) and germination of harvested seed (%) (Table 4), and from Japan: number of grain rows, 100 grain weight (g), number of grains per ear and germination of harvested seed (%) (Table 5). All of these endpoints are recognized as important characteristics related to weediness potential of corn.
In the US, statistical differences were observed in MON 89034 (2004) and MON 87460 grown in Chile (Water-limited treatment) in the comparison of stalk lodged plants (#/plot) and yield (bu/a), respectively. The mean value of stalk lodged plants for MON 89034 (0.8) was, however, within the range of the value of the reference varieties (0.0–6.0) planted at the same locations. The mean value of yield for MON 87460 grown in Chile (114.5 bu/a) was also within the range of values of the reference varieties (56.4–167.6 bu/a) planted at the same locations (Table 4). The increase in yield for MON 87460 under stress conditions in Chile was expected and proved the efficacy of MON 87460.
In Japan, statistical differences between the GM crop and conventional control were observed in the comparison of number of grain rows, number of grains per ear, 100 grain weight (g) and germination of harvested seeds (%). However, when the mean values of GM events (number of grain rows: 14.7 and 14.3 for LY038-A and LY038-B, respectively, number of grains per ear; 584.1 and 663.6 for LY038-B and MON 89034, respectively, 100 grain weight: 30.7 for LY038-B, germination of harvested seeds: 97.8 for MON 89034) were compared with the range of the minimum and maximum mean values of the non-GM controls used in previous confined field trials (number of grain rows: 12.3–16.9, number of grains per ear; 549.2–728.6, respectively, 100 grain weight: 22.3–43.9, germination of harvested seeds: 86.7–100.0), all GM values were found to be within the reference ranges (Table 5).
As described above, both the US and Japan use the concept of familiarity to interpret the statistical differences identified between the GM crop and non-GM control. However, it would appear that the US undergoes a more rigorous process than Japan to interpret statistical differences by conducting confined field trials at multiple locations and by obtaining the range of values of the reference varieties which were planted at the same locations. In the US, data from GM crops and non-GM controls are compared at a single location (pollen study) or across locations (germination study and growth and development studies) (Steps 1 and 2 in Fig. 1) as the initial steps (Horak et al. 2007, 2015). If a statistically significant difference between the GM crops and non-GM controls is detected, the mean value of GM crop is compared with the range of means obtained for the reference varieties grown in that study (Step 3 in Fig. 1). If the means of the GM crop is outside of the range of the means of the reference varieties, the GM crops’ mean characteristic value is considered in the context of published literature values for the characteristics for commercial varieties of the crop. If the GM crop mean value for a particular characteristic is outside the published characteristic value for commercial varieties, (Step 4 in Fig. 1), the characteristic would be assessed for the magnitude of the change and for whether or not it is adverse in terms of weediness potential or other ecological impact (Step 5 in Fig. 1) (Horak et al. 2007, 2015). In the case of confined field trials in Japan, there are often an insufficient number of non-GM control values to allow the development of a reference range. In this case, the GM crop mean value is directly assessed for the magnitude of the change and for whether or not it was adverse in terms of weediness potential or other ecological impact.
As described above, confined field trials in the US are conducted in diverse geographies representing a broad range of environmental conditions and agricultural ecosystems for which the crops is grown (Horak et al. 2015). Given the similarity of the assessment endpoints, such as the reduction in abundance of a valued species and the process by which this assessment is made, results from confined field trials in the US can be considered relevant to identify any potential ecological hazards of GM crops for FFP use in Japan. To facilitate data transportability more efficiently across different geographies, this paper advocates harmonization of protocols for confined field trials.