Subchronic feeding study of DAS-59122-7 maize grain in Sprague-Dawley rats

Abstract

59122 is a transgenic maize line containing event DAS-59122-7 that expresses the corn rootworm (CRW) specific pesticidal Cry34Ab1 and Cry35Ab1 proteins from Bacillus thuringiensis (Bt) Berliner strain PS149B1 and the phosphinothricin-N-acetyltransferase (PAT) protein from Streptomyces viridochromogenes for tolerance to the herbicidal ingredient glufosinate-ammonium. For the current study, 59122 maize grain, non-transgenic near-isogenic maize grain (091), and a commercially available non-transgenic reference maize grain (33R77) were grown under conditions simulating commercial farming practices. Adult Sprague-Dawley rats (12/sex/group) were fed diets formulated with 35% maize grain from either 59122, 091, or 33R77, or one of two separate lots of commercially available rodent chow prepared with commercially available corn (35%) in accordance with the standards of Purina Mills Labdiet^® 5002 for approximately 90 days. All diets possessed similar nutritional and contaminant profiles. The transgenic proteins were detected only in diets prepared with 59122 maize grain and were stable over the course of the study. Compared to control groups, no adverse diet-related differences were observed in rats fed diets formulated with 59122 maize grain with respect to body weight/gain, food consumption/efficiency, clinical signs of toxicity, mortality, ophthalmology, neurobehavioral (FOB and motor activity) assessments, clinical pathology (hematology, clinical chemistry, coagulation, and urinalysis), and pathology (organ weights and gross and microscopic pathology). Results from this study indicate that 59122 maize grain is nutritionally equivalent to and as safe as conventional maize grain.

Introduction

It has been estimated that Western and Northern corn rootworms (Diabrotica virgifera virgifera LeConte and Diabrotica barberi Smith and Lawrence, collectively; CRW) are responsible for $1 billion in maize crop damage and control annually in the US (Metcalfe, 1986). In-field pesticide treatments with organophosphates and pyrethroids have been used to control the growth and impact of these organisms. However, both classes of pesticides risk affecting non-target species and have been proposed to promote the development of pesticide-resistant populations (Wright et al., 2000, Zhou et al., 2003). The potential benefits of CRW resistant maize produced through biotechnology have been discussed (Oehme and Pickrell, 2003).

The transgenic maize line 59122, containing event DAS-59122-7, was produced by genomic integration of the cry34Ab1 and cry35Ab1 genes from Bacillus thuringiensis (Bt) Berliner strain PS149B1 and the pat gene (phosphinothricin-N-acetyltransferase) from Streptomyces viridochromogenes. The Cry34Ab1 and Cry35Ab1 proteins (14 and 44 kDa, respectively) are normally found in the parasporal inclusion bodies of Bt PS149B1 and when present together cause a selective toxicity toward CRW that appears to be attributable to pathological changes in the CRW midgut epithelium (Ellis et al., 2002). The efficacy of maize containing the cry34Ab1 and cry35Ab1 transgenes expressed in planta toward controlling the growth of CRW has been demonstrated (Moellenbeck et al., 2001). The pat gene in the 59122 maize line is expressed as a protein (PAT) with a molecular mass of 21 kDa and confers tolerance to the herbicidal active ingredient glufosinate-ammonium.

Guidelines for the mammalian safety assessment of crops modified through biotechnology include an evaluation of the possible toxicity and allergenicity of the transgenic proteins as well as an evaluation of the transgenic crop for nutritional equivalence (WHO, 1991, WHO, 1995, FAO, 1996, OECD, 1993, OECD, 1996, OECD, 1997, ILSI, 1997). Amino acid sequence similarity between transgenic proteins and known allergens and toxins is an indicator of potential adverse effects that may be attributable to the transgenic proteins; guidelines have been published with regard to sequence similarity comparisons to known allergens (FAO/WHO, 2001).

The safety assessment process also includes in vitro and in vivo studies conducted with recombinant forms of the transgenic proteins (Harrison et al., 1996). These studies include an evaluation of the stability of the protein in simulated gastric fluid as a part of a weight of evidence approach for allergenicity assessment (Astwood et al., 1996). Acute oral toxicity studies are also conducted because toxic proteins act through acute mechanisms (Sjoblad et al., 1992). In the case of 59122 maize grain, the amino acid sequences of the Cry34Ab1, Cry35Ab1, and PAT proteins are not homologous to known mammalian protein toxins or allergens and the protein products are labile in simulated gastric fluid (Herman et al., 2003, Herouet et al., 2005). Additionally, no adverse effects were observed in mice following acute oral exposure to Cry34Ab1 (2700 mg/kg) or Cry35Ab1 (1850 mg/kg) proteins singly or in combination (482 [Cry34Ab1] and 1520 mg/kg [Cry35Ab1] Brooks and DeWildt, 2000b, Brooks and DeWildt, 2000c) nor with the PAT protein following intravenous exposure to 10 mg/kg (Herouet et al., 2005).

Because xenobiotic substances often contribute little to the overall caloric intake of laboratory animals, toxicology studies with these substances can evaluate multiple doses over a relatively wide dose range. However, commercial rodent diets contain a substantial amount of maize grain (33–38%) so evaluating the dose–response relationship of transgenic maize grain as would be done in a typical toxicology study is not possible. The highest possible concentration of maize grain that could be fed to laboratory animals (i.e., 100%) will result in a diet that is nutritionally imbalanced. Further, this would only represent a factor of approximately three times greater than the concentration of maize grain in normal commercial diets. It is likely that any effect observed under these conditions that could be associated with the introduced molecular construct would be obscured. The concept of substantial equivalence (rather than absolute safety) has been established to evaluate the comparative safety of transgenic crops (OECD, 1993, FAO, 1996). Establishment of substantial equivalence of GM crops includes an analytical comparison of the nutritional, anti-nutritional, and selected secondary metabolite profiles of GM crops to non-GM references. Previous investigations have indicated that 59122 maize grain and conventional maize grain are compositionally equivalent (Herman et al., 2007). Another component of substantial equivalence is a comparison of the performance of GM crops to non-GM reference crops in nutritional equivalence studies that are typically conducted in broiler chickens (Taylor et al., 2003a, Taylor et al., 2003b). Similarly, safety studies have been conducted in rats by comparing the consumption of standard rodent diets produced with GM grains with diets produced with non-transgene containing counterparts in subchronic feeding studies (Hammond et al., 2004). The current study was conducted to evaluate the nutritional performance in Sprague-Dawley rats and search for any possible unintended toxicological properties of 59122 maize grain.