The cp4 epsps gene introduced into Roundup Ready® canola GT73 was isolated from the soil bacterium Agrobacterium sp. strain CP4 and encodes an EPSPS protein with naturally reduced affinity for glyphosate relative to endogenous plant EPSPS enzymes. The presence of CP4 EPSPS in Roundup Ready® canola allows the plants to complete the shikimate pathway even in the presence of glyphosate.
The cp4 epsps gene encodes a protein of 47.6 kD consisting of a single polypeptide of 455 amino acids. The nucleotide sequence of the bacterial cp4 epsps gene was modified for plant-preferred codon usage, but these nucleotide substitutions did not alter the amino acid sequence of the encoded protein.
The barnase and barstar genes that comprise the hybrid breeding system were derived from the common soil bacterium, B. amyloliquefaciens. This bacterium is used commercially as a source of industrial enzyme production, particularly α-amylase, and is also used in the food industry for brewing and bread-making. Although some Bacillus species have been implicated as the causal agents of human diseases, B. amyloliquefaciens is not known to be allergenic or pathogenic towards humans.
GM InVigor® canola lines incorporating the MS and RF hybrid breeding system have been approved for limited and controlled release under DIRs 010/2001, 032/2002, 057/2004, 069/2006 and 104, and for commercial release under DIR 021/2002. Therefore, the toxicity and allergenicity of the BARNASE and BARSTAR proteins have been previously assessed by the Regulator and the assessments concluded that they are unlikely to be toxic or allergenic.
No sequence homology was found between BARNASE or BARSTAR and known toxins or allergens (see DIR 069/2006; Van den Bulcke 1997). Further bioinformatic studies using updated databases have confirmed these results (EFSA 2009b). BARNASE and BARSTAR do not have characteristics typical of known protein allergens (Van den Bulcke 1997) and no matches with known IgE epitopes were found (Kleter & Peijnenburg 2002). Both proteins are rapidly degraded in simulated gastric juices (0.32% pepsin and acidic pH) with complete protein degradation within five minutes (Van den Bulcke 1997), showing that these proteins would not survive in the digestive tract.
Feeding studies in animals using seed from GM canola lines expressing barnase and barstar are discussed in Section 128.
Food derived from GM InVigor® canola lines MS1, MS8, RF1, RF2 and RF3, which expresses BARNASE and BARSTAR proteins, has been considered safe for human consumption by FSANZ (ANZFA 2001b), and InVigor® canola has been grown commercially in North America since 1995 without reports of toxicity or allergenicity associated with the introduced genes.
BARNASE degrades ribonucleic acid into its component ribonucleotides. Ribonucleotides are ubiquitous in nature and are not considered toxic or allergenic. BARSTAR does not possess enzymatic activity but, instead, exerts its action by binding to the BARNASE enzyme to form an inactive complex. Therefore, the products of the enzymatic reactions catalysed by the novel proteins are also unlikely to be toxic or allergenic.
PAT protein
The bar and pat genes were obtained from the common soil bacteria S. hygroscopicus and S. viridochromogenes, respectively. These species of Streptomyces are saprophytic, soil-borne microbes that are not considered pathogens of plants, humans or other animals (OECD 1999b).
The bar and pat genes have both been used extensively in the production of GM plants as selectable markers in the laboratory or to provide herbicide tolerance in the field. Consequently, PAT proteins have been used in several GM plants approved by the Regulator for limited and controlled release (for example, see DIR 71/2006, DIR 86/2008 and DIR 100). In addition, GM canola (DIR 021/2002), GM Liberty Link® cotton (DIR 062/2005) and GM WideStrike® cotton (DIR 091) expressing the bar or pat genes have been approved for commercial release. Therefore, the toxicity and allergenicity of PAT proteins have been previously assessed by the Regulator and the assessments concluded that they are unlikely to be toxic or allergenic.
A review of published literature and experimental studies was used to evaluate the safety of the PAT proteins encoded by the pat and bar genes (Herouet et al. 2005). The authors concluded that there is a reasonable certainty of no harm resulting from including the PAT proteins in human food or animal feed.
No sequence homology has been found between PAT and any known toxic or allergenic proteins (Van den Bulcke 1997; Herouet et al. 2005; EFSA 2009b). The PAT proteins do not possess any of the characteristics associated with food allergens and they are not stable in simulated gastric or intestinal fluid conditions (Wehrmann et al. 1996; OECD 1999b; ANZFA 2001b; Herouet et al. 2005) hence the potential for the PAT protein to be a food allergen is minimal (EPA 1997b). In addition, PAT proteins are inactivated by heat, low pH and during processing of canola (Wehrmann et al. 1996; EPA 1997b; European Scientific Committee on Plants 1998a; OECD 1999b).
There is no evidence that the PAT proteins encoded by the bar and pat genes are toxic to either humans or other animals. The potential for PAT to be toxic has been addressed via acute toxicity studies, as detailed in the RARMP for DIR 021/2002. In summary, 14-day acute oral toxicity studies in mice and rats found no treatment-related significant effects (Merriman 1996; Bremmer & Leist 1996). A more recent study found no toxic effect in mice after acute intravenous administration of the PAT proteins at up to 10 mg/kg body weight (Herouet et al. 2005).
Feeding studies in animals using seed from GM canola lines containing PAT proteins are discussed in Section 128.
FSANZ has approved the use of food derived from GM plants containing either the bar or pat gene, including GM canola, cotton, maize, rice and soybean, concluding that the PAT proteins are not toxic (ANZFA 2001a; ANZFA 2001b; FSANZ 2004; FSANZ 2005c; FSANZ 2008).
A number of international regulatory bodies have also assessed the PAT proteins expressed in GM plants as safe. These include the United Stated Food and Drug Administration (FDA 1996; FDA 1997; FDA 1998), Health Canada (Health Canada 1997; Health Canada 1999b), the Canadian Food Inspection Agency (Canadian Food Inspection Agency 1995c; Canadian Food Inspection Agency 1996), the European Commission (European Scientific Committee on Plants 1998a) and the European Food Safety Authority (EFSA 2008; EFSA 2009b). The United States Environmental Protection Agency has determined that PAT, and the genetic material necessary for its production, is exempt from the requirement to establish a maximum permissible level for residues in plants (EPA 1997b).
