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Possibilities and Limitations of Safety Evaluations in Biological Systems

E. G. Jarchow and P. Ahl Goy, CibaGeigy Ltd., Seeds Division, 4002Basle, Switzerland

Insect damages to crops provoke important yield losses worldwide. The development of insect tolerant crops occupies therefore a central position among the practical application of genetic engineering to plant breeding.

Safety aspects play an important role particularly when new technologies are used. The research relating to risk assessment has never been as extensive as today. The same is also true for discussions about the extend and validity of this risk research. This article is looking at the possibilities and limitations of safety evaluations in biological systems and at the approach Ciba Seeds is taking with the development and safety evaluation of insect tolerant maize, with focus of the products' interaction with organisms in the ecosystem.

Safety Evaluation in Biological Systems Possibilities and Limitations

In contrast to the risk evaluation of mechanical machines such as a motor, risk assessment in the field of new technologies like genetic engineering is much more complex. First of all the products are living organisms. Secondly, in the case of plants they are grown in the open field, a part of our highly complex ecosystem. As a consequence we are facing limitations and consequently dilemmas when we evaluate the safety of seed products. One inherent limitation of the system comes forward from the fact that we are dealing with living organisms that might change. Whereas the engine once constructed will still be the same in 50 years, plants undergo mutations. These mutations then underlie the laws of evolution. This actually is the 'Dilemma of life' which does not allow us to guarantee the absolute 100% safety of a product such as a plant. It is not a new dilemma brought about by the use of genetic engineering but a dilemma we are dealing with also when agricultural plants are developed with classical breeding tools. The second limitation relates to the fact that plants are grown in nature, a highly complex system. As we can not look into all aspects of the system, risk research is based on representative examples that are studied, to then judge the safety of a given seed product. In addition to carrying out specific experiments, data already available in literature and experience are taken into account. However, this results in a second dilemma as the relevance of the chosen representative examples are subject to different rankings.

The view points people take in the discussions regarding safety evaluation vary extensively. Overall they can be assigned to three categories/concepts of thinking. The 'Conservative' wants to allow new developments only if the absence of any new risk is guaranteed. The 'Progressive' trust that any new development has come forward to solve an existing problem and thus is advantageous, the 'Balanced' see that new developments should be allowed depending on the benefit risk evaluation.

At Ciba we take what we refered to as the balanced view. If a product brings advantages to our customers and society while limiting or reducing risk Ciba will pursue its development.

Ciba Seeds' Approach Benefit and Safety Evaluation [1]

Ciba Seeds has developed maize tolerant to the European Corn Borer (ECB, Ostrinia nubilalis). The tolerance is due to the insertion of a synthetic, truncated version of a gene encoding the ?endotoxin CrylA(b) from the bacterium Bacillus thuringiensis subsp. kurstaki, strain HD1. Endotoxins from B. thwingiensis are already used in many biological products to control insect pests. When ingested by insects, these endotoxins undergo cleavage, leading to activated proteins which bind to specific receptors present in the insect midgut. This brings about cell lysis and subsequent death of the insect by cessation of feeding. In addition, the ECB tolerant maize from Ciba Seeds contains a selectable marker, a phosphinothricin acetyltransferase from the bacterium Streptomyces hygroscopicus, conferring tolerance to phosphinothricin, the active moiety of glufosinate ammonium herbicide [2].

Benefit Evaluation Field studies with ECB tolerant maize

The production of the CrylA(b) protein in ECB tolerant maize is tissue specific, with preferential expression in green tissues and in pollen. The level of the CrylA(b) protein varies during the plant life cycle, with the highest amount detected at anthesis, about 1.5 gg CrylA(b)/gfw in leaves and 1.8 ?g CrylA(b)/gfw in pollen (mean values of numerous determinations).

The survival of ECB larvae on ECB tolerant maize is greatly reduced. Extensive field evaluations with artificial infestations showed rapid mortality of the larvae, accompanied by a significant decrease in damage to the plants [2, 3]. Under strong insect infestation, yield losses are highly reduced on ECB tolerant maize compared to control maize (maize plants lacking the protection mechanism), whereas yields are similar in the absence of infestation [4]. In comparison with other control methods (chemical and biological) the integrated protection mechanism of ECB tolerant maize proved to be superior.

Safety Evaluation Example: Organisms in the Ecosystem

Specificity of the insecticidal protein expressed by ECB tolerant maize

One question raised by the development of ECB tolerant maize concerns the specificity of the insecticidal protein. Although the specificity of the native bacterial CrylA(b) protein is well documented [4], the specificity of the truncated form of the protein encoded by the plant was also verified. Three different types of studies were conducted, which are briefly outlined below.

1) In vitro comparison of the activity of native bacterial Cry1Mb) and of Cry1Mb) protein expressed by ECB tolerant maize

The susceptibility of neonate larvae of five lepidopterous insects to the native bacterial CrylA(b) protein and to the protein expressed by ECB tolerant maize were very similar and the ranking of the insects according to their susceptibility was identical (table 1). The somewhat higher activity observed with the plant protein was anticipated, as the protein expressed in maize is a truncated version of the native protein and therefore contains more active endotoxin per unit weight of protein.

Table 1:In vitro susceptibility of lepidopterous insects to the CrylA(b) protein

¹⁾ the proteins were added to standard diets for each species and the LC50 values determined (30 replicates).

These results indicate that the protein expressed by ECB tolerant maize possesses a similar activity as the native bacterial protein.

2) Field monitoring of the entompfauna present in ECB tolerant maize and control maize.

A field study conducted in Bloomington in 1993 (Illinois, USA) showed no difference in the kind and number of insects associated with ECB tolerant maize, compared to the entornofauna associated with control maize. The monitoring encompassed phytophagous and entomophagous insects, including beneficial predators and parasites, and was conducted weekly over a 10week period. In contrast, treatment with a conventional insecticide, permethrin, showed a diminution of the coleopteran population following the treatments on all plots (ECB tolerant maize and control maize). Table 2 shows as an example the monitoring results obtained in early August.

Table 2: Entomofauna associated with ECB tolerant maize and control maize

¹⁾Chr = chrysomelidae (coleoptera); Coc = coccinellidae (coleoptera); Oth = other coleoptera; Dip = diptera; Thy = thysanoptera; Hom = homoptera; Hem = hemiptera; Hym = hymenoptera. The insects were collected using Scentry Multigard yellow sticky traps; 2 traps per plot and 6 plots per type of maize, from which 3 were treated with permethrin (Pounce) at 225 g/ha, on July 29 and August 23.

²⁾"control maize 1" = negative segregants from ECB tolerant maize; "control maize 2" = wild type maize.

³⁾values statistically different from the corresponding control values (P<0.05).

These results confirm the high specificity of ECB tolerant maize towards target pests, compared to what can be achieved using a conventional insecticide.

3) Toxicity studies on selected organisms with ECB tolerant maize

Several toxicity studies were conducted on selected organisms, from which two are presented below.

A inhive test showed no effect of pollen from ECB tolerant maize on the development of honeybees (Apis mellifera). The survival of larvae and the emergence to adults were similar for honeybees receiving pollen from ECB tolerant maize or not treated, whereas honeybees treated with Carbaryl insecticide as a positive control showed high mortality (table 3). The cause for the slightly reduced emergence of honeybees treated with pollen from control maize was unclear but is likely to be due to differences in hive vigor or genetic variability.

Table 3:Development of honeybee larvae treated with pollen from ECB tolerant maize

¹⁾brood frames with young larvae were removed from the beehives, treated in the laboratory with pollen from ECB tolerant maize or from control maize, at the concentration of 1 mg in 1 drop of water per cell, and returned to the beehives after allowing time for the pollen to be consumed. Carbaryl, 200 ppm, was used as positive control (25 honeybees per hive, 4 hives per treatment).

Earthworms (Eisenia foetida) were selected as a second organism for a toxicity study. Extracts from leaves of ECB tolerant maize or from control maize showed no effects on the survival and development of earthworms during a 14day toxicity study conducted in an artificial soil. All earthworms survived the end of the study and weight gain during the test period were similar. The concentration of CrylA(b) protein used in the study (0.35 mg CrylA(b)/kg soil) represents a much higher concentration than that to which earthworms are likely to be exposed under field conditions. Calculations based on CrylA(b) concentration in ECB tolerant maize showed that the concentration used in the test is approximately 785 times higher than the expected concentration in the soil, when maize plants will be incorporated into the soil after harvest.

Additional toxicity studies carded out on various organisms supported the lack of toxicity of the CrylA(b) protein expressed by ECB tolerant maize to nontarget organisms. A single study, conducted with a soil invertebrate, showed some potential activity of ECB tolerant maize. This does not represent a safety issue as the effect seen appeared only in a concentration range well above that present in ECB tolerant maize cultivated soils.

Conclusion

ECB tolerant maize developed by Ciba Seeds will represent a significant new method for control of ECB damage in maize, and has proven to be highly effective. Three kinds of tests were conducted to assess the specificity of the insecticidal protein expressed in ECB tolerant maize: in vitro dietary tests with selected lepidopterans, field monitoring of insect populations and toxicity studies on selected organisms. As stated earlier there are limitations to the safety evaluation of a seed product in the complex ecosystem, also out of lack of reference data available. But all data available today support the safe use of the Ciba Seeds' ECB tolerant maize within the ecosystem.

References

Patricia Ahl Goy, Gregory Warren, James White, Laura Privalle, Patricia Fearing and D. Viachos 1995. Interaction of an insect tolerant maize with organisms in the ecosystem. Proceedings of Workshop on 'Key Safety Aspects of Genetically Modified Organisms, Braunschweig April 10 11, 1995.

Koziel M.G., Beland G.L., Bowman C., Carozzi N.B., Crenshaw R., Crossland L., Dawson J., Desai N., Hill W Kadwell S., Launis K, Lewis K, Maddox D., McPherson K, Meghji M.R., Merlin E., Rhodes R., Warren G.W., Wright M. & Evola S.V. 1993. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/Technology 11: 194 200.

Labatte J.M., Meusnier S., Migeon A. & Got B. 1994. Field evaluation of and modeling the impact of three control methods on the larval dynamics of Ostrinia nubilalis (Lepidoptera: Pyralidae): a chemical insecticide, the biopesticide Beauveria bassiana Vuil. (Deutoromycotina: hyphomycete) and a transgenic corn hybrid. J. Econ. Entomol., in press).

Christensen D., Beland G. & Meghji M. 1993. Yield loss due to European corn borer in normal and transgenic hybrids. Proceedings of the 48th annual corn & sorghum research conference, pp. 43 52.

Lereclus D., De16cluse A. & Lecadet M. 1993. Diversity of Bacillus thuringiensis toxins and genes. In: Bacillus thuringiensis, an environmental biopesticide: theory and practice, edited by Entwistle P.F., Cory J.S., Bailey M.J & Higgs S., John Wiley and Sons PubL, NY, pp. 37 69.