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Possibilities and Limitations of Safety Evaluations in Biological
E. G. Jarchow and P. Ahl Goy, CibaGeigy Ltd., Seeds Division, 4002Basle,
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
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
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
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 .
Benefit Evaluation Field studies with ECB tolerant
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
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 . 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
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 , 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
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
||native bacterial CrylIA(b):
(LC5o in ng/cm2 of diet) 1)
|CrylA(b) from ECB
(LC5o in ng/cm2 of diet)1)
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
Chr Coc 1 Oth Dip
|insects per trap')
Thy 1 Horn Hem 1 Hym
|ECB tolerant maize"
|control maize 12)
|control maize 22)
|ECB tolerant maize
|control maize 12)
|control maize 22)
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
3) Toxicity studies on selected organisms with ECB tolerant
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
||Survival (%, days after
|pollen from ECB tolerant maize
|pollen from control 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
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.
Patricia Ahl Goy, Gregory Warren, James White, Laura Privalle, Patricia
Fearing and D. Viachos 1995. Interaction of an insect tolerant maize with
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thuringiensis toxins and genes. In: Bacillus thuringiensis, an
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