Safety assessment of genetically modified crops

Keith T. Atherton

Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield SK10 4TJ, UK

Abstract

The development of genetically modified (GM) crops has prompted widespread debate regarding both human safety

and environmental issues. Food crops produced by modern biotechnology using recombinant techniques usually differ

from their conventional counterparts only in respect of one or a few desirable genes, as opposed to the use of traditional

breeding methods which mix thousands of genes and require considerable efforts to select acceptable and robust hybrid

offspring. The difficulties of applying traditional toxicological testing and risk assessment procedures to whole foods

are discussed along with the evaluation strategies that are used for these new food products to ensure the safety of these

products for the consumer.

# 2002 Published by Elsevier Science Ireland Ltd.

Keywords: Genetically modified crops; Protein safety evaluation; Risk assessment

1. Introduction

Man has cultivated plants for thousands of

years, during which time crop plants have been

continually selected for improved yield, growth,

disease resistance or other useful characteristics.

Plant breeding is an exceptionally successful en-

terprise that has fashioned the raw material of

unimproved germplasm into the modern high-

yielding crop and pasture varieties on which we

now depend. Until recently plant breeders had to

depend on empirical methods to reach their goals.

However, the discovery of plant transformation is

changing the way that breeders approach the

challenge of creating new varieties to fulfil specific

needs. Directed genetic changes provides an im-

portant new tool and allows the use of genetic

information from almost any life form to be

introduced into crop plants and produce desirable

new characteristics.

A long history of producing new varieties of

crop plants by conventional breeding has rarely

resulted in forms that have had to be withdrawn

from the market because of health concerns. Plant

breeders have introduced thousands of new crop

varieties that have had little, if any, effect on food

safety. The concern over genetically modified

(GM) crops stems mainly from the fact that plant

breeders now have access to genetic information

from any living organism or indeed to synthetic

DNA sequences. The developers of new plant

varieties using genetic engineering have the re-

sponsibility of establishing that the newly intro-

duced varieties, and the food products developed

from these are as safe and nutritious as their

traditional counterparts. This article discusses theE-mail address: keith.atherton@syngenta.com (K.T.

Atherton).

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types of new plant varieties being developed usinggenetic modification techniques and the strategies

being used to ensure the safety of these products

for the consumer.

2. Current and future GM crops

Improvements in agronomic traits such as yieldand disease resistance continue to be driving forces

behind today’s seed industry, but, increasingly,

attention is also focussed on speciality traits,

including high oilseed grains, low saturated fat

oilseeds and delayed ripening fruits and vegetables.

Such traits command premium values in the

market place. Whilst plant breeders have used

conventional breeding methods coupled with tech-niques such as tissue culture and mutagenesis to

produce such lines (Swanson et al., 1989), genetic

engineering now provides a real alternative. The

first generation of GM crop varieties, which have

been altered for agronomic traits are generally

encoded by a single gene, such as virus-, insect-, or

herbicide resistance. A comprehensive list of these

can be found on the Biotechnology IndustryOrganisation website (http://www.bio.org).

Over the last few years, it can be seen that the

genetic alterations in new plant varieties under

development are becoming more complex with

more genes being involved in the modification.

Also, it can be seen that a larger variety of

metabolic pathways is being modified for traits

that relate to food quality, nutritional character-istics and other perceived consumer benefits. A

good example of this can be seen in the recent

developments to increase the nutritional value of

rice (Ye et al., 2000).

3. Approaches to the nutritional and food safety

evaluation of GM foods

For many years the practical difficulties of

obtaining meaningful information from conven-

tional toxicology studies on the safety of whole

foods have been well recognised (OECD, 1996).

The limitations of conventional toxicological stu-

dies became particularly apparent when animal

feeding studies were used to assess the safety ofirradiated foods.

Animal studies are a major element in the safety

assessment of many compounds such as pesticides,

pharmaceuticals, industrial chemicals and food

additives. In most cases however, the test sub-

stance is well characterised, of known purity, of no

particular nutritional value and human exposure is

generally low. It is therefore relatively straightfor-ward to feed such compounds to animals at a

range of doses, some several orders of magnitude

greater than the expected human exposure levels,

in order to identify any potential adverse health

effects. In this way it is possible to determine levels

of exposure at which adverse effects are not

observed, and so set safe upper limits by the

application of appropriate safety factors.By contrast, foods are complex mixtures of

compounds characterised by wide variation in

composition and nutritional value. Due to their

bulk they can usually only be fed to animals at low

multiples of the amounts that might be present in

the human diet. In addition, a key factor to

consider in conducting animal studies on foods is

the nutritional value and balance of diets used, totry and avoid the induction of adverse effects,

which are not related to the material itself.

Detecting any potential adverse effects and relat-

ing these conclusively to an individual character-

istic of food can therefore be extremely difficult.

The difficulties of applying traditional toxicolo-

gical testing and risk assessment procedures to

whole foods, meant that an alternative approachwas required for the safety assessment of GM

foods. This led to the concept of substantial

equivalence (OECD, 1993). Substantial equiva-

lence determination is recognised as not being a

safety assessment per se (FAO, 1996), but through

an establishment that the characteristics and

composition of the new GM food is equivalent

to those of a familiar, conventional food which hasa history of safe consumption, it does permit

inference that the new food under consideration

will be no less safe than the conventional food

under conditions of similar exposure, consumption

patterns and processing practices.

In assessing substantial equivalence a wide

range of biochemical, agronomic and phenotypic

K.T. Atherton / Toxicology 181�/182 (2002) 421�/426422

characteristics of the GM crop are compared toexisting unmodified varieties of the crop grown

and harvested under identical climatic and geo-

graphic conditions.

The assessment process for the determination of

substantial equivalence may reach three possible

conclusions (Jonas et al., 1996). A product may be

determined to be substantially equivalent to its

conventional counterpart, whereby the productcould be considered as safe as that counterpart.

Secondly, a product may be determined to be

substantially equivalent except for defined differ-

ences. In such instances, further safety assessment

can be focussed on the identified differences,

narrowing the range of issues to be included in

the assessment. The third possible conclusion is

that the product is not substantially equivalent toits comparator, and therefore the history of safe

use of the comparator cannot be used in establish-

ing the safety of the new product under considera-

tion. In this case, the conclusion does not mean

that the new product is inherently unsafe, rather, it

simply requires that the new product be directly

assessed for safety through the application of

traditional safety assessment approaches.

4. GM crop safety evaluation strategies

In many cases, the modified crop is substantially

equivalent to the unmodified parent except in

respect of one or a limited number of identifiable

traits (such as the presence of proteins conferringinsect resistance or herbicide tolerance) that are

the result of the genetic modification. In these

circumstances, it is sufficient to demonstrate the

safety of the novel trait in order to conclude that

the modified crop or the food/feed products

derived from it are safe. In most cases to date,

the novel trait has been the presence of a particular

protein and safety considerations have been ad-dressed by determining the safety of this protein.

4.1. Protein safety evaluation

The safety assessment of proteins will be based

on their structure, function, bioavailability, speci-

ficity and potential allergenicity. In general, it is

not considered that proteins in the diet willrepresent a significant hazard to human health,

since nearly all proteins that are ingested are

destroyed in the digestive tract by proteases.

However, there are certain adverse effects asso-

ciated with proteins which must be considered and

for which specific safety evaluation strategies must

be considered. For example, some of the most

potent toxins know to man are proteins (Rappuoliand Montecucco, 1997). In addition to acute

toxicity, the other main adverse effects associated

with proteins are anti-nutrient effects (e.g. soybean

trypsin inhibitors), effects on the immune system

(e.g. lectins) and allergenicity (Taylor and Lehrer,

1996).

As an example of the safety evaluation of an

introduced protein, the assessment of the CP4EPSPS enzyme introduced into soybean to pro-

duce a herbicide tolerant crop has been described

(Harrison et al., 1996). CP4 EPSPS protein was

shown to be: (1) readily degraded in simulated

digestive fluids; (2) non-toxic when administered

orally to mice at an acute dose 1000s of times

higher than potential human exposure to CP4

EPSPS in foods; and (3) not structurally orfunctionally related to known protein allergens

or toxins based on amino acid sequence homology

searches.

4.2. Protein allergenicity

With the development of GM crop plants there

has been a growing interest in the approaches

available to confirm or otherwise the lack ofallergenicity of novel gene products (Kimber et

al., 2000). In a comprehensive analysis carried out

by the International Life Sciences Institute (ILSI),

Allergy and Immunology Institute and the Inter-

national Food Biotechnology Council, a scheme

for assessing the allergenic potential of foods

derived from GM crops was proposed in the

form of a hierarchical decision tree (Metcalfe etal., 1996) and was further recommended by the

joint FAO/WHO Expert Consultation on Foods

Derived From Biotechnology (FAO/WHO, 2000).

In this scheme, if the food in question contains a

gene from a source considered to be allergenic then

the immunological identity of the novel protein

K.T. Atherton / Toxicology 181�/182 (2002) 421�/426 423

with allergens deriving from the source material is

determined. The purpose here is to protect those

already sensitised from inadvertent exposure to the

inducing allergens. An example of the successful

utility of this approach derives from investigations

of modified soybean expressing the Brazil nut 2S

storage protein. It was found that sera from eight

of nine subjects with confirmed Brazil nut sensiti-

sation contained IgE antibody reactive with the 2S

protein (Nordlee et al., 1996). If, however, the

protein of interest is the product of a gene derived

from a source which is not normally associated

with allergy, or where there is no widespread

human consumption, then an alternative strategy

is recommended. This is based on considerations

of sequence homology with known allergens and

of protein stability (Gendel, 1998). Linear se-

quence homology of eight or more contiguous

amino acids (based on the minimum peptide length

for immune recognition) between the test protein

and one or more know human allergens is

indicative of sufficient immunological identity to

be of concern. If such linear sequence homology is

identified and/or if there exist other structural

similarities between the test protein and known

human allergens, then it is appropriate to investi-

gate immunological identity as described above. In

the other approach, the stability (digestibility) of

the protein to a simulated gastric fluid (SGF)

containing relevant protelotic enzymes is examined

(Astwood et al., 1996). The assumption is that

proteins that are rapidly digested will not provoke

an immune response, and indeed the data available

indicate that many food allergens are relatively

resistant to digestion in SGF (Astwood et al.,

1996; Metcalfe et al., 1996). Useful as the correla-

tions between sequence homology and stability are

as part of an overall safety assessment, nether

approach provides direct evidence of allergic

potential. For this reason, a number of labora-

tories are developing appropriate animal models

(Atkinson et al., 1996; Dearman et al., 2000;

Knippels et al., 1998; Sampson, 1999). A recent

review has led to a modified strategy for the

prediction of allergenicity being proposed (FAO/

WHO, 2001).

4.3. Requirement for animal studies

If the characterisation of the food indicates that

the available data are insufficient for a thorough

safety assessment, animal testing (despite the

difficulties referred to above) may be deemed

necessary. This would particularly be the case if

the food were expected to make a significant

dietary contribution, if the gene product is stableand if there is no history of its consumption or if

the modification affects several metabolic path-

ways. The studies should be designed in such a way

to specifically address the safety aspects that are

related to the difference between the transgenic

and the parental crops or between their derived

foods. The objective is to ensure that there is no

concern for adverse health effects for humans oranimals after prolonged consumption of GM

crops and their derived foods.

Where toxicology studies are considered neces-

sary to assess the safety of long-term consumption

of food in the diet, it is generally considered that a

sub-chronic study of 90 days duration is the

minimum requirement to demonstrate the safety

of repeated consumption of a food in the diet(FAO/WHO, 2000). This may need to be preceded

by a pilot study of short duration to ensure that

the diet is palatable to the test species and that the

levels of incorporation of the test article are

appropriate. The highest dose level used in any

animal study should be the maximum achievable

without causing nutritional imbalance, while the

lowest level should be comparable to the antici-pated human intake (FAO/WHO, 2000).

The need for additional toxicological tests

should be considered on a case-by-case basis

taking into account the results of the 90-day study

and other studies. For example, proliferative

changes in tissues during the 90-day study may

indicate the need for a longer-term toxicity study

(FAO/WHO, 2000).In addition to animal studies designed specifi-

cally for safety evaluation, nutritional or whole-

someness testing may be performed to determine

whether the food or feed product of the GM crop

poses any nutritional problems in comparison with

the unmodified parent crop (Hammond et al.,

1996). These studies will involve the administra-

K.T. Atherton / Toxicology 181�/182 (2002) 421�/426424

tion of the food or feed product to a suitable test

species in quantities representative of anticipated

use. The best species will normally be that which

would consume the food or feed or may be chosen

because of especially high growth rates which

would result in an increased sensitivity to any

nutritional problems. The studies would typically

be of 28- or 90-day duration and the end points are

generally indices of growth and nutrition such as

food consumption, general condition, weight gain,

milk yield and composition (cattle), laying perfor-

mance (hens), or food conversion efficiency and

body composition (fish). In some studies, observa-

tions would also include some simple pathological

endpoints such as carcass quality and organ

weights and their macroscopic appearance post-

mortem. Whilst these studies should not be con-

fused with toxicology studies since they are not

necessarily optimal for safety evaluation, they do

provide useful data.

5. Conclusions

This paper has attempted to give a representa-

tive overview of the principles of evaluation

followed to assure safe consumption of GM crops

by humans and animals. The safety assessment

strategies have given satisfactory results for GM

crops and novel foods which are substantially

equivalent to their conventional counterparts or

substantially equivalent except for a limited num-

ber of defined differences (often the expression of

one or small number of new proteins). Much less

experience is available with the safety evaluation

of crops, which are not substantially equivalent to

conventional counterparts, and efforts are under-

way to evaluate satisfactory safety testing schemes

for such products. In any event, the scrutiny by

which the safety testing of GM crops is carried out

provides more assurance of safety to human health

than for crops which have been obtained through

traditional breeding which have been subject to

little, if any, toxicological evaluation, yet are

generally accepted as being safe to eat.

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