Characteristics and safety assessment of intractable proteins in genetically modified crops.

Genetically modified (GM) crops may contain newly expressed proteins that are described as "intractable". Safety assessment of these proteins may require some adaptations to the current assessment procedures. Intractable proteins are defined here as those proteins with properties that make it extremely difficult or impossible with current methods to express in heterologous systems; isolate, purify, or concentrate; quantify (due to low levels); demonstrate biological activity; or prove equivalency with plant proteins. Five classes of intractable proteins are discussed here: (1) membrane proteins, (2) signaling proteins, (3) transcription factors, (4) N-glycosylated proteins, and (5) resistance proteins (R-proteins, plant pathogen recognition proteins that activate innate immune responses). While the basic tiered weight-of-evidence approach for assessing the safety of GM crops proposed by the International Life Sciences Institute (ILSI) in 2008 is applicable to intractable proteins, new or modified methods may be required. For example, the first two steps in Tier I (hazard identification) analysis, gathering of applicable history of safe use (HOSU) information and bioinformatics analysis, do not require protein isolation. The extremely low level of expression of most intractable proteins should be taken into account while assessing safety of the intractable protein in GM crops. If Tier II (hazard characterization) analyses requiring animal feeding are judged to be necessary, alternatives to feeding high doses of pure protein may be needed. These alternatives are discussed here.

E. coli Lipopolysaccharide: acute oral toxicity study in mice.

A single dose of endotoxin (lipopolysaccharide) from a common laboratory cloning and expression strain (Escherichia coli BL21[DE3]) was administered to groups of male and female CD-1 mice (n=5/group) at doses up to 1,000,000 endotoxin units (EU) per mouse by oral gavage. The mice were observed for mortality, body weight effects, and clinical signs for 14 days after which they were sacrificed for gross organ necropsy. All mice survived until the scheduled sacrifice, no clinical signs of toxicity were observed, no test substance-related body weight losses occurred and no gross lesions were present at necropsy. Under the conditions of this study, oral administration of E. coli BL21(DE3) endotoxin to mice at a dose of up to 1,000,000 EU/mouse produced no evidence of toxicity.

Toxicology studies with N-acetylglycine.

N-acetylglycine (NAGly) has been identified as a minor constituent of numerous foods. The current paper reports the outcome of in vitro and in vivo genotoxicity, acute oral and repeated dose dietary toxicology studies conducted with NAGly. No evidence of genotoxicity was observed with NAGly in vitro bacterial tester strains or in vivo bone marrow micronucleus studies conducted in mice. No mortalities or evidence of adverse effects were observed in Sprague-Dawley rats following acute oral gavage with NAGly at a dose of 2000 mg/kg of body weight or following repeated dose dietary exposure to NAGly at targeted doses of 100, 500, or 1000 mg/kg of body weight/day for 28 days. No biologically significant or test substance related differences were observed in body weights, feed consumption, or clinical pathology response variables in any of the treatment groups. Based on these results it was concluded that NAGly is not genotoxic or acutely toxic. Further, the no-observed adverse-effect-level (NOAEL) for systemic toxicity from repeated dose dietary exposure to NAGly was 898.9 mg/kg of body weight/day for male rats and 989.9 mg/kg of body weight/day for female rats.

N-acetyl-glutamic acid: evaluation of acute and 28-day repeated dose oral toxicity and genotoxicity.

N-acetyl-glutamic acid (NAG) is an endogenously produced mammalian substance and minor constituent of commonly consumed foods. This paper reports the outcome of genotoxicity and acute and repeated dose (28-day) oral toxicology studies conducted with NAG. No evidence of genotoxicity was observed with NAG in in vitro or in vivo studies. No mortalities or evidence of adverse effects was observed in Sprague-Dawley rats following acute oral gavage with NAG at a dose of 2000 mg/kg of body weight. No adverse effects were observed in rats following repeated dose dietary exposure to NAG at target concentrations corresponding to doses of 100, 500, or 1000 mg/kg of body weight/day for 28 days. All rats survived until scheduled sacrifice and no biologically significant or test substance related differences were observed in body weights, feed consumption, clinical signs, functional observational battery (FOB), ophthalmology, hematology, coagulation, clinical chemistry, organ weights or histopathology of any of the treatment groups. Based on the observed results it is concluded that NAG is not genotoxic or acutely toxic. The no-observed-adverse-effect-level (NOAEL) for systemic toxicity from repeated dose (28-day) dietary exposure to NAG was 914 mg/kg of body weight/day for male rats and 1007 mg/kg of body weight/day for female rats.