The goxv247 gene is derived from O. anthropi strain LBAA (formerly Achromobacter sp.), a bacterium commonly found in the soil. The goxv247 gene encodes the GOXv247 protein that differs from the original O. anthropi enzyme by three amino acids.
O. anthropi is an opportunistic human pathogen (Alnor et al. 1994; Mahmood et al. 2000). However, the gox gene represents a very small proportion of the pathogen genome and is not, in itself, infectious or pathogenic. The bacterial GOX protein is highly specific for its substrate, glyphosate (OECD 1999a), hence it is unlikely to be involved in human pathogenesis.
The cp4 epsps gene is derived from another common soil bacteria, Agrobacterium sp. strain CP4 (Padgette et al. 1995), which is widespread in the environment and can be found on plant produce (especially raw vegetables). The CP4 EPSPS protein is functionally and structurally similar to EPSPS proteins naturally present in canola and in human food and animal feed derived from other plant and microbial sources (Nair et al. 2002).
CP4 EPSPS has been used extensively in GM plants as a selectable marker or a source of field resistance to glyphosate herbicides. Consequently, the Regulator has approved several GM plants expressing cp4 epsps for limited and controlled release (for example, see DIR 074/2007, pima cotton; DIR 082, perennial ryegrass and tall fescue; and DIR 101, cotton). The Regulator has also approved GM cotton lines expressing cp4 epsps for commercial release under licences DIR 012/2002, DIR 023/2003, DIR 059/2005 and DIR 066/2006. Both the GOXv247 and CP4 EPSPS proteins are present in GM canola approved for limited and controlled release under DIR 011/2001 and DIR 104, and for commercial release under DIR 020/2002. Therefore, the toxicity and allergenicity of GM plants expressing the GOXv247 and CP4 EPSPS proteins has been previously assessed by the Regulator and the assessments concluded that they are unlikely to be toxic or allergenic.
The amino acid sequences of both CP4 EPSPS (Mitsky 1993; Harrison et al. 1996) and GOX (Astwood 1995) were compared to the amino acid sequences of known protein toxins and allergens and no significant homology was found. Further bioinformatic studies using updated databases have confirmed that the GOXv247 and CP4 EPSPS proteins do not share any similarity with any known toxins or allergens (EFSA 2009d). The GOXv247 and CP4 EPSPS proteins are readily inactivated by heat and rapidly degraded by simulated mammalian digestive conditions (Harrison et al. 1996; OECD 1999a; Chang et al. 2003).
Acute oral toxicity studies using CP4 EPSPS and GOXv247 proteins produced by bacterial expression systems are described in the RARMP for DIR 020/2002. In summary, high doses of the CP4 EPSPS and GOXv247 proteins fed to mice had no adverse effects on food consumption, survival, body weight or gross pathology (Naylor 1994a; Harrison et al. 1996).
Feeding studies in animals using seed from GM canola lines containing the CP4 EPSPS and GOXv247 proteins are discussed in Section 128.
Food from Roundup Ready® canola has been approved for human consumption by FSANZ (ANZFA 2000). Food derived from GM soybean, cotton, sugarbeet, maize and lucerne lines that express the cp4 epsps gene have also been considered safe for human consumption by FSANZ (FSANZ 2005a; FSANZ 2005b; FSANZ 2006; FSANZ 2007).
A number of international regulatory bodies have also assessed Roundup Ready® canola GT73 with regard to toxicity and allergenicity. These include the United States Environmental Protection Agency (EPA 1996; EPA 1997a), the United Stated Food and Drug Administration (FDA 1995), Health Canada (Health Canada 1999a), the Canadian Food Inspection Agency (Canadian Food Inspection Agency 1995b) and the European Food Safety Authority (EFSA 2009d). These agencies have concluded that the presence of EPSPS and GOX proteins in food does not pose a significant toxicity or allergenicity risk. The EPA considers these proteins as inert ingredients (EPA 1996; EPA 1997a).
Toxicity of herbicide metabolites
The potential toxicity of herbicide metabolites is considered by the the Australian Pesticides and Veterinary Medicines Authority (APVMA) in its assessment of a new use pattern for particular herbicides, in this case glyphosate and glufosinate ammonium on InVigor® x Roundup Ready® canola. The issue is summarised below.
Glyphosate metabolites
There is no difference in the metabolic fate of glyphosate in non-GM canola and in GM canola expressing goxv247 and cp4 epsps. In the case of CP4 EPSPS, no new metabolic products are formed as the only difference from the native enzyme is the reduced affinity for glyphosate (OECD 1999a).
In glyphosate-sensitive plants very little of the glyphosate that is applied would be broken-down. The presence of the GOXv247 protein confers glyphosate tolerance by increasing the rate of breakdown of glyphosate to glyoxylate and aminomethylphosphonic acid (AMPA). Glyoxylate is a common metabolite in plants and forms part of the biochemical pathway that allows synthesis of carbohydrates from fat (the glyoxylate cycle).
AMPA is the most frequently detected metabolite of glyphosate in soil, water and plants (Reddy et al. 2008). Despite the faster breakdown of glyphosate, AMPA does not accumulate to higher levels in GM canola expressing GOX than in soybean that does not contain an introduced gox gene (Nandula et al. 2007; Duke 2010). AMPA is either non-selectively bound to natural plant constituents, conjugated with naturally occurring organic acids to give trace level secondary metabolites, or further degraded to one-carbon fragments that are incorporated into a variety of natural products and plant constituents (FAO & WHO 1998b).
Glyphosate and AMPA have similar toxicological profiles and both exhibit low toxicity (EPA 1997a; Williams et al. 2000; WHO 2005), although AMPA was shown to be genotoxic (able to change DNA) in a recent study using a very sensitive test (Manas et al. 2009). The APVMA sets maximum residue limits (MRLs) for agricultural and veterinary chemicals in agricultural produce, particularly produce entering the food chain. MRLs are set to reflect the legal use of a chemical and to ensure a safe food supply, and are set well below the level that would be harmful. The residue definition for glyphosate includes the metabolite AMPA (APVMA 2011).
