In assessing the likelihood of adverse impacts due to toxicity or allergenicity of the GM papayas on the health and safety of humans and other organisms, a number of factors were considered including;
the toxicity and allergenicity of non-GM papaya;
the toxicity and allergenicity of the purified form of the introduced proteins; and
the toxicity and allergenicity of the GM papayas (the GMOs) proposed for release;
Reference to these factors are made throughout the assessment.
Section 2.1 Toxicity and allergenicity of non-GM papaya
Numerous studies have demonstrated that leaf, fruit and/or root extracts of non-GM papaya have inherent toxicological or allergenic properties, many of which apparently relate to the complex, largely uncharacterised, chemical composition of papaya latex. Although a range of toxicological studies on papaya have been reported, many of these are associated with impacts on mammalian (including human) fertility and reproduction. In terms of allergenicity, sensitisation to papain among workers in the industry is well known (Baur et al. 1988; Iliev & Elsner 1997). More detailed information on the inherent toxicity and allergenicity of papaya can be found in a review document ‘The Biology and Ecology of Papaya (paw paw), Carica papaya L., in Australia’ that was produced in order to inform this risk assessment process. This document is available at the OGTR website (http://www.ogtr.gov.au)
The range of toxic and allergenic properties associated with unripe non-GM papaya fruit suggest that in the event of commercial production of the GM papayas in the proposed release (which would require a separate application and assessment process), the potential for the genetic modifications to alter papaya’s inherent toxic and allergenic properties must be fully addressed. This consideration is especially important in light of changes in latex levels in the fruit during the fruit ripening process.
At this stage of evaluating the GM papayas proposed for release, these data were unavailable and will not be available until the first fruit are produced by the GM trees. As noted earlier, none of the GM papaya fruit will be available for human consumption. Food Standards Australia New Zealand (FSANZ) is responsible for human food safety assessment.
Section 2.2 Toxicity and allergenicity of the introduced proteins and of GM papaya fruit
The GM papayas in the proposed release are expected to vary from non-GM papaya either in the expression of additional proteins, ETR1-1, GUS and NPTII, or in the reduction in expression of the naturally occurring ACC synthase enzymes, encoded by the capacs 1 and capacs 2 genes. It is also possible that some GM papaya plants would have increased expression of the ACC synthase enzymes, due to the presence of additional copies of the capacs 1 or capacs 2 genes in the sense orientation. Reduction in the activity of the ACC synthase enzymes could also potentially lead to accumulation of the substrate of the ACC synthase enzyme, S-adenosyl-L-methionine, because this substrate is normally utilised by ACC synthase. Reduction in ACC synthase activity may also perturb other aspects of the ethylene biosynthesis pathway.
None of the proteins/enzymes to be expressed, or the naturally occurring proteins to be down-regulated in the GM papaya, have any known intrinsic toxicity or allergenicity. The intrinsic toxic and allergenic properties of papaya are associated with other enzymes and compounds, which are unlikely to be affected by the genetic modifications.
2.2.1 Acute Toxicity Studies
Due to the widespread use of both the NPTII and GUS proteins in GM plants currently approved for human consumption, data is available on the toxicology of these proteins. These data consistently demonstrate that the NPTII and GUS proteins are not toxic for mammals. For example, an acute oral toxicity study in mice, in which purified NPTII protein was fed, at doses up to 5000 mg/kg (2500 mg/kg administered twice, four hours apart), did not show any adverse effects (Berberich et al. 1993). There were no treatment-related differences in mortality, weight gain, food consumption, behaviour, clinical signs or gross pathology. Likewise, acute oral toxicity studies in mice, with purified GUS protein at doses of up to 100 mg/kg, did not show any adverse effects (Naylor 1992; ANZFA 2001). The bla gene, conferring resistance to the antibiotic ampicillan in bacteria, will not be expressed in the GM papaya plants and protein encoded by this gene will not be present.
At this stage of the evaluation process for the GM papayas proposed for release, acute toxicological data is not available for the ETR1-1 protein, the ACC synthase enzymes, or other components of the plant ethylene biosynthesis pathway, the levels of which in the plants could be altered by changed ACC synthase activity. Such data would be required for evaluation by the OGTR before the GM papaya could be considered for a larger scale release.
2.2.2 Stability of the introduced proteins in the human digestive tract
Current scientific knowledge suggests that common food allergens tend to be resistant to degradation by heat, acid, and proteases (Astwood et al. 1996). This is because it is necessary that a protein is sufficiently stable to reach and cross the mucosal membrane for it to stimulate an allergenic response following oral ingestion (Kimber et al. 1999).
The NPTII protein is heat labile and degrades rapidly in simulated human gastric fluid. (Fuchs et al. 1993) reported that no NPTII was detected 10 seconds after addition of simulated gastric fluid as measured by both Western blot and enzymatic activity. Likewise, exposure of the GUS protein to simulated mammalian digestive systems resulted in its rapid degradation (ANZFA 2001). Additionally, the enzymatic activity of NPTII requires the co-factor ATP, which is unstable at the low pH of the digestive system (Flavell et al. 1992).
There are no data currently available on the stability of the ETR1-1 protein, ACC synthase enzymes or other components of the plant ethylene biosynthesis pathway in the human digestive tract. As with the acute toxicity studies, this information would be required before the GM papayas could be considered for larger scale releases.
2.2.3 Homology with known allergens
The ETR1-1 protein and the ACC synthase enzymes are not derived from known allergen or allergenic organisms. ETR1-1 encodes a non-functional protein with similarity to a group of bacterial proteins involved in signal transduction known as ‘two-component receptor proteins’. The ETR1-1 protein is composed of multiple parts, each likely to have different functions. No other two-component receptor proteins, or proteins similar to the various parts of the ETR1-1 protein, are currently listed as known food or non-food allergens (The Biotechnology Information for Food Safety Database, National Centre for Food Safety and Technology, USA and The Structural Database of Allergen Proteins, The University of Texas Medical Branch).
ACC synthase enzymes are homologous to pyridoxal-5-phosphate (PLP)-dependent aminotransferases (Alexander & Grierson 2002). No other ACC synthase enzymes or PLP-dependent aminotransferases are currently listed as known food or non-food allergens (The Biotechnology Information for Food Safety Database, National Centre for Food Safety and Technology, USA and The Structural Database of Allergen Proteins, The University of Texas Medical Branch).
A full assessment of the potential allergenicity of the ETR1-1 protein and ACC synthase enzymes would require analysis of DNA or protein sequence homology to all known allergens in the Genbank, EMBL, Pir and Swiss-Prot databases. Food Standards Australia New Zealand (FSANZ), is responsible for human food safety assessment. Currently the applicant has not applied to FSANZ for evaluation of material from the GM papaya for use in human food. However, FSANZ approval for use of the GM papaya in human food would be required before commercialisation of any GM papayas.
The NPTII protein does not display characteristics common to known food allergen proteins (US FDA 1998; Fuchs et al. 1993). NPTII is not derived from a known allergen and shows no significant DNA or protein sequence homology to known allergens in the Genbank, EMBL, Pir and Swiss-Prot databases.
The GUS protein is also unlikely to be a major allergen and does not display the characteristics common to known allergen proteins (ANZFA 2001). The GUS protein does not have chemical or physical characteristics that are typical of known food allergens and does not share significant amino acid sequence similarity with known allergens.
2.2.4 Other sources of the introduced proteins in food
The ETR1-1 protein and the ACC synthase enzymes are components of ethylene perception and biosynthesis pathways that are conserved among many plant species. Proteins with a high degree of sequence homology (similarity in terms of amino acid sequence) and with similar function to ETR1-1 and the ACC synthase enzymes are present in many commonly eaten plant products, especially fresh fruits such as tomatoes, melon, cucumber and citrus (Alexander & Grierson 2002).
The NPTII and GUS proteins are ubiquitous in the environment and in food chains, in naturally occurring microorganisms found in soil and water and in mammalian digestive systems (Flavell et al. 1992; Gilissen et al. 1998). Both are recognised as commonly present on fresh food.
Humans continually ingest kanamycin-resistant microorganisms, some containing the NPTII gene. The diet, especially raw salad, is the major source: at a conservative estimate, each human ingests 1.2 x 106 kanamycin-resistant microorganisms daily (Flavell et al. 1992). With 1012 kanamycin- or neomycin-resistant bacteria already in the gut of each person, the addition of NPTII through GM papaya food products will be of negligible significance. All human-health analyses need to be viewed against this knowledge.
2.2.5 Assessment by other agencies
GM papaya with delayed fruit ripening has not been assessed by other regulatory agencies at this stage. However, other fruits in which similar modifications have been made have been assessed for health and safety hazards for humans and other organisms by regulatory agencies in other countries. It is likely that GM papaya with modified fruit ripening will be similar, in terms of generic biochemistry and metabolism, to other fruit crops with similar genetic modifications.
In the USA and Canada, tomatoes modified for delayed fruit ripening by down-regulation of ethylene biosynthesis have been approved for human food since the mid 1990s (Chapter 1, Section 2.4). Health Canada assessed these tomatoes as being as safe and nutritious as currently available commercial tomato cultivars (FD/OFB-095-306-A, http://www.hc-sc.gc.ca). Health Canada considered that, other than reduced ACC synthase activity, the disease, pest and other agronomic characteristics of the transgenic line was comparable to the unmodified parent line. They also concluded that analysis of nutrients did not reveal any significant differences in the levels of macro- and micronutrients between the GM line and its unmodified parent line. Additionally, the introduction of the truncated ACC synthase gene was not judged to have any potential for additional human toxicity or allergenicity. The same GM tomato line is approved for unregulated commercial use by the USDA Animal and Plant Health Inspection Service (APHIS) (Payne 1995) and is considered to be a safe human food by the US FDA. The USDA/APHIS also concluded that this tomato line exhibits no plant pathogenic properties and is unlikely to harm beneficial organisms, such as bees.
In other lines of GM tomatoes down-regulation of ethylene biosynthesis has been achieved in a slightly different way to the GM papayas proposed for release, by insertion of genes encoding enzymes that degrade essential components of ethylene biosynthesis in the plant. USDA/APHIS also concludes that these GM tomatoes are as safe to grow as traditionally bred tomato lines. The US FDA also considers these GM tomato lines to be a safe human food.
The use of the NPTII and GUS proteins in food crops has previously been assessed by regulatory agencies both in Australia and in other countries. Use of the NPTII enzyme in tomatoes, canola and cotton has been previously evaluated by the US FDA. The FDA concluded that this enzyme does not have any of the recognised characteristics of food allergens or any attributes that would distinguish it toxicologically from other phosphorylating enzymes in the food supply (FDA 1994), cited in (ANZFA 1999).
In their draft risk analysis report for application A378 ‘Food derived from glyphosate-tolerant sugarbeet line 77 (GTSB77)’ ANZFA concluded that food derived from this plant, which expresses the GUS protein, was safe for human consumption. The US Environmental Protection Agency (US EPA) does not consider GUS to be toxic for mammals and has approved its exemption from the requirement to establish tolerance levels.
Food Standards Australia New Zealand (FSANZ), is responsible for human food safety assessment. Currently the applicant has not applied to FSANZ for evaluation of material from the GM papaya for use in human food. FSANZ approval would need to be obtained before it could be used in human food.
2.2.6 Toxicity to invertebrates and microorganisms
The potential exists for the GM papayas in the proposed release to have altered responses to pathogenic or non-pathogenic microorganisms (see Appendix 1). This impact is unlikely to occur as a direct result of toxic effects of the introduced proteins or the GM papaya to microorganisms but rather, as a result of increased or decreased resistance in the GM papaya plants to certain microorganisms.
The applicant is required to report any observed alterations in the responses of the GM papaya plants to pathogenic or non-pathogenic microorganisms as a condition of the licence, if it is issued.
ETR1-1 and the ACC synthase enzymes are the only novel proteins expressed in the GM papayas, other than GUS and NPTII, both of which has been extensively studied in the context of other GMOs containing these proteins. The potential for persistence and/or accumulation in the environment of ETR1-1, the ACC synthase enzymes or other components of ethylene biosynthesis in the GM papayas is unknown. However, the ETR1-1 protein, the ACC synthase enzymes and other components of ethylene biosynthesis are naturally occurring plant proteins and are not inherently toxic, the release is on a limited scale and all fruit from the release will be harvested. Thus, any potential persistence and/or accumulation of the novel proteins is unlikely to have an adverse impact on the environment, should this occur.