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3-Food: Austrian study on toxicological and allergological safetyevaluation of GMO



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TITLE:  Toxicological and allergological safety evaluation of GMO
SOURCE: Federal Environment Agency, Austria, Monograph 109
        English Summary
        http://www.ubavie.gv.at
DATE:   2003

------------------ archive: http://www.gene.ch/genet.html ------------------


German Title:
Toxikologie und Allergologie von GVO-Produkten. Empfehlungen zur
Standardisierung der Sicherheitsbewertung von gentechnisch veränderten
Pflanzen auf Basis der Richtlinie 90/220/EWG (2001/18/EG)


Toxicological and allergological safety evaluation of GMO - Summary
Spoek A., Hofer H., Valenta R., Kienzl-Plochberger K., Lehner P., Gaugitsch H.

A high safety standard for genetically modified organisms (GMO) and
products derived therefrom constitutes an important factor in order to
materialize the innovational and economic potential of modern biotechnology.

Toxicological and allergological safety evaluation of GMO is presently
being conducted under Directive 90/220/EEC or - from October 2002 on -
under Directive 2001/18/EC. However, toxicological and allergological
safety requirements are not determined in detail in these directives and
related documents. Consequently, the margins for putting safety
evaluation into practice are rather wide.

In the course of the project described here the requirements for
toxicological and allergological safety evaluation for genetically
modified plants (GMP) and products derived therefrom as they appear in
the regulatory framework of the EU were investigated. Subsequently, the
practice of toxicological and allergological safety evaluation as
demonstrated in eleven application dossiers for placing on the market of
GMP and products derived therefrom was thoroughly reviewed in order to
investigate the practice of safety evaluation in general and the
differences between different dossiers in particular. As in most dossiers
the concept of substantial equivalence was referred to, the practice of
substantial equivalence was also investigated. Based on the results of
the review recommendations were derived in order to put into concrete
terms and standardize toxicological and allergological safety evaluation
and - to some extent - also the practice of substantial equivalence.

Regulatory Context

The protection of human and animal health and the environment against
potential risks of GMO and products containing GMO is mainly regulated in
Directive 90/220/EEC (Directive 2001/18/EC). These directives cover the
placing on the market of GMO products, which mainly means import,
cultivation, production of seeds or feed stuff, handling, further
processing, and storage. Before placing on the market the GMO has to be
authorised as prescribed by these directives. In order to get an approval
for a GMO an environmental risk assessment has to be carried out.
Toxicological and allergological requirements for safety evaluation are
mentioned in the directives but are not specified in detail. Directive
90/220/EEC merely demands information on potentially toxic effects to
human and the environment caused by the recipient or parental plants, as
well as toxic effects specifically attributed to the GMP. Interpretative
documents and documents specifying safety evaluation in greater detail
only exist for Directive 90/220/EEC, so far. These documents further
specified that allergic properties referring to Vertebrata including
humans and toxic properties referring to all organisms have to be
evaluated. Furthermore, notifiers have to provide data on toxicity and
allergenicity of the resulting GMO as well as on metabolites. As a
consequence, the requirements for toxicological and allergological safety
evaluation in the context of EU regulation still remain to be detailed.

The Scientific Committee on Plant issued guidelines which specify some
more details of toxicological safety evaluation: the "object" of safety
evaluation (target protein, GMP or particular parts of the GMP),
preparation of samples (herbicide resistant plants cultivated with
herbicide applied, proof of equivalence if protein is purified from
another source, e.g. prokaryotic micro-organisms), considering exposition
(recommendation of organisms to be looked at, consideration of probably
different expression levels in different parts of plant). In case of feed
stuff, if deviations in the content of particular compounds attributable
to the genetic modification are identified, further toxicological tests
focussing on macro nutrients and known toxins is required.

Directive 2001/18/EC puts more emphasise on toxicological and
allergological requirements than Directive 90/220/EEC. In case of feed
stuff potentially toxic and allergic effects on animals are especially
considered. An important difference in comparison to Directive 90/220/EEC
is that immediate and delayed, direct and indirect (including cumulative)
effects have to be considered. Furthermore, potential adverse effects
have to be considered even if such effects seem to be unlikely. This
considerable extension in safety evaluation for toxicological and
allergological testing might presumably result e.g. in long-term rather
than acute toxicity testing. If probable but unlikely indirect effects
have to be considered one might have to take into account also effects
via food chain, following gene transfer to non-target organism, or caused
by genetic instability. However, toxicological and allergological
requirements for safety evaluation remains to be specified also in
Directive 2001/18/EC.

According to a proposal issued by the European Commission food and feed
stuff might be covered by the same piece of EC regulation in the near
future. This proposal, however, does not touch upon toxicological or
allergological requirements. Seeds from GMP will also be covered by a
separate regulation, though a proposal still has to be issued. Until such
sectoral regulation will come into force, feed stuff and seeds from GMO
are regulated by Directive 90/220/EEC (Directive 2001/18/EC).

Some aspects of the interplay of horizontal and sectoral regulations are
regulated in interpretative documents: in case of import, cultivation,
seed, processing, and feed stuff potentially toxic effects to humans,
animals and the environment have to be evaluated under Directive 90/220/
EEC, if these products are not intended to be used in food as well. If
this is the case it is up to the notifier whether the safety evaluation
will be carried out under Directive 90/220/EEC or under the Novel Food
Regulation. GMO products solely intended for use in or as food have to be
evaluated solely under the Novel Food Regulation. However, particular
toxicological safety requirements or the problem of distinction between
the extent of safety requirements regarding to human health under
Directive 90/220/EEC and those under the Novel Food Regulation are not
touched upon.

Besides the GMO specific regulations mentioned above, also non-GMO
specific regulations aiming at the protection of human and animal health
and the environment are covering products from plants or GMP, especially
in case of feed stuff and seeds. However, toxicological and
allergological safety requirements for feed stuff are presently (until a
separate regulation for feed stuff from GMO will come into force) almost
exclusively regulated under Directive 90/220/EEC (Directive 2001/18/EC).
In case of seeds from GMP no toxicological requirements are demanded in
the context of seed testing with the possible exception that the level of
known toxins in plants is considered.

Results of Analysis of the Directive 90/220/EEC-Dossiers

Out of 28 applications for placing on the market of GMP which are
presently under review or are already approved, eleven applications were
selected: applications for intended use for cultivation and as feed stuff
(RR-fodder beet A5/15, potato EH92-527-1, Bt-cotton 531, RR-cotton 1445),
"twin applications" (first application for import, second application for
cultivation; maize Bt11, RR-maize GA 21), one application intended for
cultivation as well as use as food and feed stuff (rape Topas 19/2),
applications for use as ornamental plants (carnation 66, carnation 959A
etc.). Besides the actual application dossiers also correspondence,
additional information from the notifiers, opinions of the national
competent authorities as well as the Scientific Committee on Plants, and
- if available - decisions of the European Commission were considered.

Toxicology: In general toxicological information is rather a minor part
of the dossiers. Differences in the intended use of the GMP do not affect
the extent of the toxicological evaluations. Most toxicity tests are
displayed as summaries or are just references to the literature and can
therefore not be verified and reviewed. Internal references are often
used improperly. Statements which are closely related to each other are
sometimes scattered over the dossier.

Apparently, toxicological tests were carried out rather sporadically,
most likely in cases of Bt-plants, as Bt-toxins had already been approved
before as an insecticide in some countries. Data on the toxicity of the
whole GMP are not provided in any dossier. Toxicological acceptance is
often justified by three arguments: low toxicity of the gene product,
substantial equivalence of the GMP to their conventional counterparts,
and low exposure. Potentially toxic effects resulting as a secondary
effect from the gene insertion are not considered in any case.

Most of the toxicological testing were not carried out in compliance with
quality assurance programs such as Good Laboratory Practise (GLP).

GMP are very often declared as being safe just by assumption based
reasoning. Furthermore these assumptions are sometimes not easily or not
at all verifiable. Risk assessment procedures which are carried out in a
systematic way consisting of a hazard assessment of the GMP on one hand
and of an analysis of exposure on the other hand, are lacking in the dossiers.

Allergology: No direct testing of potentially allergic properties of GMP
and products derived therefrom was carried out. The absence of allergenic
properties was justified solely in an either argumentative way and/or by
giving rather indirect evidence (e.g., digestion studies, sequence
homology comparisons). Some quotations of literature intended to confirm
the safety of the GMP in the dossiers are cited wrongly or are outdated
or are even suspected to be selectively quoted. The usual way of arguing
is as follows: (i) no homology could be detected between the newly
introduced protein and known allergens, (ii) the expression level of
target protein in the GMP is rather low, (iii) the protein will rapidly
be digested in the intestine, (iv) the newly introduce protein originates
from a non-allergenic source, (v) the protein is not glycosylated and
will therefore less likely exhibit allergic properties, (vi) the protein
will less likely exhibit allergenic properties because it is not new.

Each of these arguments and their underlying assumptions have to be
questioned in the light of recent scientific data. Furthermore,
unintended secondary effects possibly caused by the gene insertion, such
as the possible upregulated expression of other allergens through
insertion and expression of the foreign gene in the GMP, are not
considered at all. A safety evaluation which is based exclusively on the
above described approaches is insufficient.

Substantial Equivalence: Analysis and comparisons of plant compounds are
part of each dossier with the exception of carnation. However, no
connection can be established between the nature and extent of these
analysis and the intended use of the GMP or GMP products. Compositional
analysis are largely restricted to macro-nutrients and known plant
specific anti-nutrients as well as known toxins. A detailed
characterisation of macrocompounds is however, rarely done.

Substantial equivalence is referred to in each dossier in order to argue
for the safety of the particular GMP. The parameters chosen in
composition analysis are however, not comprehensive enough to justify
substantial equivalence and/or to detect probable unintended secondary
effects. In each dossier some significant differences between the GMP and
conventional counterparts were either reported or could be found by
reviewing the displayed data. However, these differences did not lead to
a repetition of the analysis including an extension of parameters
investigated. In contrast, these differences were argumentatively
attributed to naturally occurring ranges, effects from back-crossing,
climate conditions etc.

Detailed descriptions of cultivation conditions, single examination
sheets and statistical data interpretation, information on storage and
preparation of samples as well as detailed data on the results of
compound analysis are lacking in most cases. Detailed explanations on
summaries of compound analysis are often fragmentary or even missing. On
the ground of information given and data shown, substantial equivalence
often cannot be verified. In case of herbicide resistant GMP it is often
not quite clear if the herbicide was applied during cultivation.

As a matter of comparing average values of different cultivation sites
the variance of analysed compounds is sometimes quite high, and might be
covering any unintended secondary effects e.g. resulting in changes in
plant metabolism.

Nutritional considerations in general and especially with respect to
substantial equivalence (e.g. vitamin profiles, characterisation of
fibre, analyses of different types of proteins) apparently do not play a
role in the dossiers and are just occasionally considered in comparative
composition analysis.

Composition of food products derived from animals fed on GMP was not
considered in any dossier.

Recommendations for Standardisation

On the basis of the results of the review of the GMP dossiers as
described above, a number of more general recommendations are drawn
aiming at a specification and standardisation of toxicological and
allergological safety evaluation. These recommendations are partly
inspired by authorisation practices of products other than GMO. In detail
these recommendations aim at simplifying the evaluation of the dossiers,
by checking in a more systematic way comprehensiveness and acceptability
of tests displayed in the dossiers (e.g. formal and thematic structure of
dossiers, use of references, display of results). In general all dossiers
should be stand-alone. In case of Bt- or herbicide resistant GMP these
dossiers should include the approval of the plant protection product. All
safety testing should be performed according to GLP.

Toxicology: From a toxicological perspective the toxicological
examination of the newly introduced gene products should be substantially
extended in order to elucidate not just acute but also sub-chronic,
mutagenic, reproductive, and eco-toxic effects. Furthermore, the toxicity
of the whole plant should be comprehensively studied in order to detect
any possible non-intended secondary effects. However, suitable and
standardised toxicological methods for detecting and investigating such
effects are still lacking. Therefore, such methods should be elaborated
and further developed.

For each GMP a set of basic toxicity data should be elaborated.
Additional testing may be required depending on the intended use of the
plant and the particular exposure.

Allergology: The approach to allergological safety evaluation used so far
is mainly of argumentative nature including at maximum indirect evidences
from sequence homology and digestion studies. This approach is
insufficient and should be complemented or perhaps better replaced by
other methods: Sera from allergic patients should be used to study the
gene product as well as the whole GMP and the corresponding parental line
regarding IgE antibody reactivity. Immunization studies carried out with
extracts prepared from the GMP and the parental line would allow to
compare the allergenic activity (i.e. induction of allergic sensitization
in a naive host) of both organisms.

The exclusive investigation of the gene product is insufficient because
complex changes caused via secondary effects cannot be detected. This
test allows to estimate the number of allergic patients who exhibit IgE
reactivity to the GMP versus the parental line. Furthermore it will be
possible to determine by IgE inhibition studies if the GMP contains more
IgE reactive determinants than the parental line. In order to compare the
allergic potential of the GMP and the parental line, immunisation studies
and further estimation of IgE level could be carried out in mice using
extracts from the GMP and the parental line. On the basis of these
results the risk for a de-novo sensitisation in a not jet sensitised
individual can be estimated.

Substantial Equivalence: Recommendations for field trails and composition
analysis carried out to investigate substantial equivalence are given as
follows:

In order to thoroughly and comparatively investigate different cultivars,
all factors that might affect this investigation have to be considered.
The conditions used for field trials should be very close or even
identical to conditions in normal agricultural practice of that
particular crop. The field trials should be carried out on at least three
different sites and during at least two growing seasons. Time of
cultivation and harvesting, on-site cultivation conditions, experimental
plots and sampling should be thoroughly described. Furthermore, climate
conditions should be reported.

As it is rather difficult to distinguish between natural variation and
variations probably induced by the inserted genes, only isogenic lines
should be used as conventional counterparts. Harmonised lists of key
components (nutrients and anti-nutrients) that need to be investigated by
the applicants should be established for each particular crop. Likewise,
content and ranges of these key components should be agreed for each
particular crop and should in any case include not only macrocomponents
but also microcomponents.

In order to improve the basis of comparative analysis, all relevant data
on the nutritional aspects of each particular compound and the natural
variability in content (e.g. depending on the nature of the site,
climate, agricultural management and cultivars used) should be
systematically collected in international databases. Furthermore, data on
human consumption should be gathered in order to estimate more accurately
the exposure of humans. Such database could be established and ran by
FAO/WHO or OECD as well as by the newly established European Food Safety
Authority.

In order to overcome the current methodological limits of composition
analysis, new methods that are not focussing on particular compounds but
on whole profiles of compounds (e.g. DNA-array, mRNA-fingerprinting,
proteomics, chemical-fingerprinting) should be established, further
developed and used in routine testing.

Finally, the comparative analysis of cultivars and the evaluation of
results should be carried out by an independent institution or panel of
experts.

In order to elucidate possible effects - especially indirect and
unintended ones - of GMP on the environment under conditions of normal
agricultural practice, a research-oriented monitoring programme is
recommended that compares approved GMP, conventional counterparts and
organic farming.

The results and recommendations of this study are intended to contribute
to national and international discussion on the improvement of safety
evaluation of GMP and products derived therefrom. Consequently, these
results and recommendations should be introduced into the relevant
working groups of e.g. the European Commission, OECD, Codex, Cartagena
Protocol.



Order Information:
Federal Environment Agency - Austria
Spittelauer Laende 5, A-1090 Wien, Austria
Tel.: +43 1 31304-0
Fax: +43 1 31304-540
URL: -------------------------------- GENET-news
--------------------------------

TITLE:  Toxicological and allergological safety evaluation of GMO
SOURCE: Federal Environment Agency, Austria, Monograph 109
        English Summary
DATE:   2003

------------------ archive: http://www.gene.ch/genet.html ------------------


German Title:
Toxikologie und Allergologie von GVO-Produkten. Empfehlungen zur
Standardisierung der Sicherheitsbewertung von gentechnisch veränderten
Pflanzen auf Basis der Richtlinie 90/220/EWG (2001/18/EG)


Toxicological and allergological safety evaluation of GMO - Summary
Spoek A., Hofer H., Valenta R., Kienzl-Plochberger K., Lehner P., Gaugitsch H.

A high safety standard for genetically modified organisms (GMO) and
products derived therefrom constitutes an important factor in order to
materialize the innovational and economic potential of modern biotechnology.

Toxicological and allergological safety evaluation of GMO is presently
being conducted under Directive 90/220/EEC or - from October 2002 on -
under Directive 2001/18/EC. However, toxicological and allergological
safety requirements are not determined in detail in these directives and
related documents. Consequently, the margins for putting safety
evaluation into practice are rather wide.

In the course of the project described here the requirements for
toxicological and allergological safety evaluation for genetically
modified plants (GMP) and products derived therefrom as they appear in
the regulatory framework of the EU were investigated. Subsequently, the
practice of toxicological and allergological safety evaluation as
demonstrated in eleven application dossiers for placing on the market of
GMP and products derived therefrom was thoroughly reviewed in order to
investigate the practice of safety evaluation in general and the
differences between different dossiers in particular. As in most dossiers
the concept of substantial equivalence was referred to, the practice of
substantial equivalence was also investigated. Based on the results of
the review recommendations were derived in order to put into concrete
terms and standardize toxicological and allergological safety evaluation
and - to some extent - also the practice of substantial equivalence.

Regulatory Context

The protection of human and animal health and the environment against
potential risks of GMO and products containing GMO is mainly regulated in
Directive 90/220/EEC (Directive 2001/18/EC). These directives cover the
placing on the market of GMO products, which mainly means import,
cultivation, production of seeds or feed stuff, handling, further
processing, and storage. Before placing on the market the GMO has to be
authorised as prescribed by these directives. In order to get an approval
for a GMO an environmental risk assessment has to be carried out.
Toxicological and allergological requirements for safety evaluation are
mentioned in the directives but are not specified in detail. Directive
90/220/EEC merely demands information on potentially toxic effects to
human and the environment caused by the recipient or parental plants, as
well as toxic effects specifically attributed to the GMP. Interpretative
documents and documents specifying safety evaluation in greater detail
only exist for Directive 90/220/EEC, so far. These documents further
specified that allergic properties referring to Vertebrata including
humans and toxic properties referring to all organisms have to be
evaluated. Furthermore, notifiers have to provide data on toxicity and
allergenicity of the resulting GMO as well as on metabolites. As a
consequence, the requirements for toxicological and allergological safety
evaluation in the context of EU regulation still remain to be detailed.

The Scientific Committee on Plant issued guidelines which specify some
more details of toxicological safety evaluation: the "object" of safety
evaluation (target protein, GMP or particular parts of the GMP),
preparation of samples (herbicide resistant plants cultivated with
herbicide applied, proof of equivalence if protein is purified from
another source, e.g. prokaryotic micro-organisms), considering exposition
(recommendation of organisms to be looked at, consideration of probably
different expression levels in different parts of plant). In case of feed
stuff, if deviations in the content of particular compounds attributable
to the genetic modification are identified, further toxicological tests
focussing on macro nutrients and known toxins is required.

Directive 2001/18/EC puts more emphasise on toxicological and
allergological requirements than Directive 90/220/EEC. In case of feed
stuff potentially toxic and allergic effects on animals are especially
considered. An important difference in comparison to Directive 90/220/EEC
is that immediate and delayed, direct and indirect (including cumulative)
effects have to be considered. Furthermore, potential adverse effects
have to be considered even if such effects seem to be unlikely. This
considerable extension in safety evaluation for toxicological and
allergological testing might presumably result e.g. in long-term rather
than acute toxicity testing. If probable but unlikely indirect effects
have to be considered one might have to take into account also effects
via food chain, following gene transfer to non-target organism, or caused
by genetic instability. However, toxicological and allergological
requirements for safety evaluation remains to be specified also in
Directive 2001/18/EC.

According to a proposal issued by the European Commission food and feed
stuff might be covered by the same piece of EC regulation in the near
future. This proposal, however, does not touch upon toxicological or
allergological requirements. Seeds from GMP will also be covered by a
separate regulation, though a proposal still has to be issued. Until such
sectoral regulation will come into force, feed stuff and seeds from GMO
are regulated by Directive 90/220/EEC (Directive 2001/18/EC).

Some aspects of the interplay of horizontal and sectoral regulations are
regulated in interpretative documents: in case of import, cultivation,
seed, processing, and feed stuff potentially toxic effects to humans,
animals and the environment have to be evaluated under Directive 90/220/
EEC, if these products are not intended to be used in food as well. If
this is the case it is up to the notifier whether the safety evaluation
will be carried out under Directive 90/220/EEC or under the Novel Food
Regulation. GMO products solely intended for use in or as food have to be
evaluated solely under the Novel Food Regulation. However, particular
toxicological safety requirements or the problem of distinction between
the extent of safety requirements regarding to human health under
Directive 90/220/EEC and those under the Novel Food Regulation are not
touched upon.

Besides the GMO specific regulations mentioned above, also non-GMO
specific regulations aiming at the protection of human and animal health
and the environment are covering products from plants or GMP, especially
in case of feed stuff and seeds. However, toxicological and
allergological safety requirements for feed stuff are presently (until a
separate regulation for feed stuff from GMO will come into force) almost
exclusively regulated under Directive 90/220/EEC (Directive 2001/18/EC).
In case of seeds from GMP no toxicological requirements are demanded in
the context of seed testing with the possible exception that the level of
known toxins in plants is considered.

Results of Analysis of the Directive 90/220/EEC-Dossiers

Out of 28 applications for placing on the market of GMP which are
presently under review or are already approved, eleven applications were
selected: applications for intended use for cultivation and as feed stuff
(RR-fodder beet A5/15, potato EH92-527-1, Bt-cotton 531, RR-cotton 1445),
"twin applications" (first application for import, second application for
cultivation; maize Bt11, RR-maize GA 21), one application intended for
cultivation as well as use as food and feed stuff (rape Topas 19/2),
applications for use as ornamental plants (carnation 66, carnation 959A
etc.). Besides the actual application dossiers also correspondence,
additional information from the notifiers, opinions of the national
competent authorities as well as the Scientific Committee on Plants, and
- if available - decisions of the European Commission were considered.

Toxicology: In general toxicological information is rather a minor part
of the dossiers. Differences in the intended use of the GMP do not affect
the extent of the toxicological evaluations. Most toxicity tests are
displayed as summaries or are just references to the literature and can
therefore not be verified and reviewed. Internal references are often
used improperly. Statements which are closely related to each other are
sometimes scattered over the dossier.

Apparently, toxicological tests were carried out rather sporadically,
most likely in cases of Bt-plants, as Bt-toxins had already been approved
before as an insecticide in some countries. Data on the toxicity of the
whole GMP are not provided in any dossier. Toxicological acceptance is
often justified by three arguments: low toxicity of the gene product,
substantial equivalence of the GMP to their conventional counterparts,
and low exposure. Potentially toxic effects resulting as a secondary
effect from the gene insertion are not considered in any case.

Most of the toxicological testing were not carried out in compliance with
quality assurance programs such as Good Laboratory Practise (GLP).

GMP are very often declared as being safe just by assumption based
reasoning. Furthermore these assumptions are sometimes not easily or not
at all verifiable. Risk assessment procedures which are carried out in a
systematic way consisting of a hazard assessment of the GMP on one hand
and of an analysis of exposure on the other hand, are lacking in the dossiers.

Allergology: No direct testing of potentially allergic properties of GMP
and products derived therefrom was carried out. The absence of allergenic
properties was justified solely in an either argumentative way and/or by
giving rather indirect evidence (e.g., digestion studies, sequence
homology comparisons). Some quotations of literature intended to confirm
the safety of the GMP in the dossiers are cited wrongly or are outdated
or are even suspected to be selectively quoted. The usual way of arguing
is as follows: (i) no homology could be detected between the newly
introduced protein and known allergens, (ii) the expression level of
target protein in the GMP is rather low, (iii) the protein will rapidly
be digested in the intestine, (iv) the newly introduce protein originates
from a non-allergenic source, (v) the protein is not glycosylated and
will therefore less likely exhibit allergic properties, (vi) the protein
will less likely exhibit allergenic properties because it is not new.

Each of these arguments and their underlying assumptions have to be
questioned in the light of recent scientific data. Furthermore,
unintended secondary effects possibly caused by the gene insertion, such
as the possible upregulated expression of other allergens through
insertion and expression of the foreign gene in the GMP, are not
considered at all. A safety evaluation which is based exclusively on the
above described approaches is insufficient.

Substantial Equivalence: Analysis and comparisons of plant compounds are
part of each dossier with the exception of carnation. However, no
connection can be established between the nature and extent of these
analysis and the intended use of the GMP or GMP products. Compositional
analysis are largely restricted to macro-nutrients and known plant
specific anti-nutrients as well as known toxins. A detailed
characterisation of macrocompounds is however, rarely done.

Substantial equivalence is referred to in each dossier in order to argue
for the safety of the particular GMP. The parameters chosen in
composition analysis are however, not comprehensive enough to justify
substantial equivalence and/or to detect probable unintended secondary
effects. In each dossier some significant differences between the GMP and
conventional counterparts were either reported or could be found by
reviewing the displayed data. However, these differences did not lead to
a repetition of the analysis including an extension of parameters
investigated. In contrast, these differences were argumentatively
attributed to naturally occurring ranges, effects from back-crossing,
climate conditions etc.

Detailed descriptions of cultivation conditions, single examination
sheets and statistical data interpretation, information on storage and
preparation of samples as well as detailed data on the results of
compound analysis are lacking in most cases. Detailed explanations on
summaries of compound analysis are often fragmentary or even missing. On
the ground of information given and data shown, substantial equivalence
often cannot be verified. In case of herbicide resistant GMP it is often
not quite clear if the herbicide was applied during cultivation.

As a matter of comparing average values of different cultivation sites
the variance of analysed compounds is sometimes quite high, and might be
covering any unintended secondary effects e.g. resulting in changes in
plant metabolism.

Nutritional considerations in general and especially with respect to
substantial equivalence (e.g. vitamin profiles, characterisation of
fibre, analyses of different types of proteins) apparently do not play a
role in the dossiers and are just occasionally considered in comparative
composition analysis.

Composition of food products derived from animals fed on GMP was not
considered in any dossier.

Recommendations for Standardisation

On the basis of the results of the review of the GMP dossiers as
described above, a number of more general recommendations are drawn
aiming at a specification and standardisation of toxicological and
allergological safety evaluation. These recommendations are partly
inspired by authorisation practices of products other than GMO. In detail
these recommendations aim at simplifying the evaluation of the dossiers,
by checking in a more systematic way comprehensiveness and acceptability
of tests displayed in the dossiers (e.g. formal and thematic structure of
dossiers, use of references, display of results). In general all dossiers
should be stand-alone. In case of Bt- or herbicide resistant GMP these
dossiers should include the approval of the plant protection product. All
safety testing should be performed according to GLP.

Toxicology: From a toxicological perspective the toxicological
examination of the newly introduced gene products should be substantially
extended in order to elucidate not just acute but also sub-chronic,
mutagenic, reproductive, and eco-toxic effects. Furthermore, the toxicity
of the whole plant should be comprehensively studied in order to detect
any possible non-intended secondary effects. However, suitable and
standardised toxicological methods for detecting and investigating such
effects are still lacking. Therefore, such methods should be elaborated
and further developed.

For each GMP a set of basic toxicity data should be elaborated.
Additional testing may be required depending on the intended use of the
plant and the particular exposure.

Allergology: The approach to allergological safety evaluation used so far
is mainly of argumentative nature including at maximum indirect evidences
from sequence homology and digestion studies. This approach is
insufficient and should be complemented or perhaps better replaced by
other methods: Sera from allergic patients should be used to study the
gene product as well as the whole GMP and the corresponding parental line
regarding IgE antibody reactivity. Immunization studies carried out with
extracts prepared from the GMP and the parental line would allow to
compare the allergenic activity (i.e. induction of allergic sensitization
in a naive host) of both organisms.

The exclusive investigation of the gene product is insufficient because
complex changes caused via secondary effects cannot be detected. This
test allows to estimate the number of allergic patients who exhibit IgE
reactivity to the GMP versus the parental line. Furthermore it will be
possible to determine by IgE inhibition studies if the GMP contains more
IgE reactive determinants than the parental line. In order to compare the
allergic potential of the GMP and the parental line, immunisation studies
and further estimation of IgE level could be carried out in mice using
extracts from the GMP and the parental line. On the basis of these
results the risk for a de-novo sensitisation in a not jet sensitised
individual can be estimated.

Substantial Equivalence: Recommendations for field trails and composition
analysis carried out to investigate substantial equivalence are given as
follows:

In order to thoroughly and comparatively investigate different cultivars,
all factors that might affect this investigation have to be considered.
The conditions used for field trials should be very close or even
identical to conditions in normal agricultural practice of that
particular crop. The field trials should be carried out on at least three
different sites and during at least two growing seasons. Time of
cultivation and harvesting, on-site cultivation conditions, experimental
plots and sampling should be thoroughly described. Furthermore, climate
conditions should be reported.

As it is rather difficult to distinguish between natural variation and
variations probably induced by the inserted genes, only isogenic lines
should be used as conventional counterparts. Harmonised lists of key
components (nutrients and anti-nutrients) that need to be investigated by
the applicants should be established for each particular crop. Likewise,
content and ranges of these key components should be agreed for each
particular crop and should in any case include not only macrocomponents
but also microcomponents.

In order to improve the basis of comparative analysis, all relevant data
on the nutritional aspects of each particular compound and the natural
variability in content (e.g. depending on the nature of the site,
climate, agricultural management and cultivars used) should be
systematically collected in international databases. Furthermore, data on
human consumption should be gathered in order to estimate more accurately
the exposure of humans. Such database could be established and ran by
FAO/WHO or OECD as well as by the newly established European Food Safety
Authority.

In order to overcome the current methodological limits of composition
analysis, new methods that are not focussing on particular compounds but
on whole profiles of compounds (e.g. DNA-array, mRNA-fingerprinting,
proteomics, chemical-fingerprinting) should be established, further
developed and used in routine testing.

Finally, the comparative analysis of cultivars and the evaluation of
results should be carried out by an independent institution or panel of
experts.

In order to elucidate possible effects - especially indirect and
unintended ones - of GMP on the environment under conditions of normal
agricultural practice, a research-oriented monitoring programme is
recommended that compares approved GMP, conventional counterparts and
organic farming.

The results and recommendations of this study are intended to contribute
to national and international discussion on the improvement of safety
evaluation of GMP and products derived therefrom. Consequently, these
results and recommendations should be introduced into the relevant
working groups of e.g. the European Commission, OECD, Codex, Cartagena
Protocol.



Order Information:
Federal Environment Agency - Austria
Spittelauer Laende 5, A-1090 Wien, Austria
Tel.: +43 1 31304-0
Fax: +43 1 31304-540
URL: http://www.ubavie.gv.a
Contact: Helmut Gaugitsch


-------------------------------- GENET-news --------------------------------

TITLE:  Toxicological and allergological safety evaluation of GMO
SOURCE: Federal Environment Agency, Austria, Monograph 109
        English Summary
DATE:   2003

------------------ archive: http://www.gene.ch/genet.html ------------------


German Title:
Toxikologie und Allergologie von GVO-Produkten. Empfehlungen zur
Standardisierung der Sicherheitsbewertung von gentechnisch veränderten
Pflanzen auf Basis der Richtlinie 90/220/EWG (2001/18/EG)


Toxicological and allergological safety evaluation of GMO - Summary
Spoek A., Hofer H., Valenta R., Kienzl-Plochberger K., Lehner P., Gaugitsch H.

A high safety standard for genetically modified organisms (GMO) and
products derived therefrom constitutes an important factor in order to
materialize the innovational and economic potential of modern biotechnology.

Toxicological and allergological safety evaluation of GMO is presently
being conducted under Directive 90/220/EEC or - from October 2002 on -
under Directive 2001/18/EC. However, toxicological and allergological
safety requirements are not determined in detail in these directives and
related documents. Consequently, the margins for putting safety
evaluation into practice are rather wide.

In the course of the project described here the requirements for
toxicological and allergological safety evaluation for genetically
modified plants (GMP) and products derived therefrom as they appear in
the regulatory framework of the EU were investigated. Subsequently, the
practice of toxicological and allergological safety evaluation as
demonstrated in eleven application dossiers for placing on the market of
GMP and products derived therefrom was thoroughly reviewed in order to
investigate the practice of safety evaluation in general and the
differences between different dossiers in particular. As in most dossiers
the concept of substantial equivalence was referred to, the practice of
substantial equivalence was also investigated. Based on the results of
the review recommendations were derived in order to put into concrete
terms and standardize toxicological and allergological safety evaluation
and - to some extent - also the practice of substantial equivalence.

Regulatory Context

The protection of human and animal health and the environment against
potential risks of GMO and products containing GMO is mainly regulated in
Directive 90/220/EEC (Directive 2001/18/EC). These directives cover the
placing on the market of GMO products, which mainly means import,
cultivation, production of seeds or feed stuff, handling, further
processing, and storage. Before placing on the market the GMO has to be
authorised as prescribed by these directives. In order to get an approval
for a GMO an environmental risk assessment has to be carried out.
Toxicological and allergological requirements for safety evaluation are
mentioned in the directives but are not specified in detail. Directive
90/220/EEC merely demands information on potentially toxic effects to
human and the environment caused by the recipient or parental plants, as
well as toxic effects specifically attributed to the GMP. Interpretative
documents and documents specifying safety evaluation in greater detail
only exist for Directive 90/220/EEC, so far. These documents further
specified that allergic properties referring to Vertebrata including
humans and toxic properties referring to all organisms have to be
evaluated. Furthermore, notifiers have to provide data on toxicity and
allergenicity of the resulting GMO as well as on metabolites. As a
consequence, the requirements for toxicological and allergological safety
evaluation in the context of EU regulation still remain to be detailed.

The Scientific Committee on Plant issued guidelines which specify some
more details of toxicological safety evaluation: the "object" of safety
evaluation (target protein, GMP or particular parts of the GMP),
preparation of samples (herbicide resistant plants cultivated with
herbicide applied, proof of equivalence if protein is purified from
another source, e.g. prokaryotic micro-organisms), considering exposition
(recommendation of organisms to be looked at, consideration of probably
different expression levels in different parts of plant). In case of feed
stuff, if deviations in the content of particular compounds attributable
to the genetic modification are identified, further toxicological tests
focussing on macro nutrients and known toxins is required.

Directive 2001/18/EC puts more emphasise on toxicological and
allergological requirements than Directive 90/220/EEC. In case of feed
stuff potentially toxic and allergic effects on animals are especially
considered. An important difference in comparison to Directive 90/220/EEC
is that immediate and delayed, direct and indirect (including cumulative)
effects have to be considered. Furthermore, potential adverse effects
have to be considered even if such effects seem to be unlikely. This
considerable extension in safety evaluation for toxicological and
allergological testing might presumably result e.g. in long-term rather
than acute toxicity testing. If probable but unlikely indirect effects
have to be considered one might have to take into account also effects
via food chain, following gene transfer to non-target organism, or caused
by genetic instability. However, toxicological and allergological
requirements for safety evaluation remains to be specified also in
Directive 2001/18/EC.

According to a proposal issued by the European Commission food and feed
stuff might be covered by the same piece of EC regulation in the near
future. This proposal, however, does not touch upon toxicological or
allergological requirements. Seeds from GMP will also be covered by a
separate regulation, though a proposal still has to be issued. Until such
sectoral regulation will come into force, feed stuff and seeds from GMO
are regulated by Directive 90/220/EEC (Directive 2001/18/EC).

Some aspects of the interplay of horizontal and sectoral regulations are
regulated in interpretative documents: in case of import, cultivation,
seed, processing, and feed stuff potentially toxic effects to humans,
animals and the environment have to be evaluated under Directive 90/220/
EEC, if these products are not intended to be used in food as well. If
this is the case it is up to the notifier whether the safety evaluation
will be carried out under Directive 90/220/EEC or under the Novel Food
Regulation. GMO products solely intended for use in or as food have to be
evaluated solely under the Novel Food Regulation. However, particular
toxicological safety requirements or the problem of distinction between
the extent of safety requirements regarding to human health under
Directive 90/220/EEC and those under the Novel Food Regulation are not
touched upon.

Besides the GMO specific regulations mentioned above, also non-GMO
specific regulations aiming at the protection of human and animal health
and the environment are covering products from plants or GMP, especially
in case of feed stuff and seeds. However, toxicological and
allergological safety requirements for feed stuff are presently (until a
separate regulation for feed stuff from GMO will come into force) almost
exclusively regulated under Directive 90/220/EEC (Directive 2001/18/EC).
In case of seeds from GMP no toxicological requirements are demanded in
the context of seed testing with the possible exception that the level of
known toxins in plants is considered.

Results of Analysis of the Directive 90/220/EEC-Dossiers

Out of 28 applications for placing on the market of GMP which are
presently under review or are already approved, eleven applications were
selected: applications for intended use for cultivation and as feed stuff
(RR-fodder beet A5/15, potato EH92-527-1, Bt-cotton 531, RR-cotton 1445),
"twin applications" (first application for import, second application for
cultivation; maize Bt11, RR-maize GA 21), one application intended for
cultivation as well as use as food and feed stuff (rape Topas 19/2),
applications for use as ornamental plants (carnation 66, carnation 959A
etc.). Besides the actual application dossiers also correspondence,
additional information from the notifiers, opinions of the national
competent authorities as well as the Scientific Committee on Plants, and
- if available - decisions of the European Commission were considered.

Toxicology: In general toxicological information is rather a minor part
of the dossiers. Differences in the intended use of the GMP do not affect
the extent of the toxicological evaluations. Most toxicity tests are
displayed as summaries or are just references to the literature and can
therefore not be verified and reviewed. Internal references are often
used improperly. Statements which are closely related to each other are
sometimes scattered over the dossier.

Apparently, toxicological tests were carried out rather sporadically,
most likely in cases of Bt-plants, as Bt-toxins had already been approved
before as an insecticide in some countries. Data on the toxicity of the
whole GMP are not provided in any dossier. Toxicological acceptance is
often justified by three arguments: low toxicity of the gene product,
substantial equivalence of the GMP to their conventional counterparts,
and low exposure. Potentially toxic effects resulting as a secondary
effect from the gene insertion are not considered in any case.

Most of the toxicological testing were not carried out in compliance with
quality assurance programs such as Good Laboratory Practise (GLP).

GMP are very often declared as being safe just by assumption based
reasoning. Furthermore these assumptions are sometimes not easily or not
at all verifiable. Risk assessment procedures which are carried out in a
systematic way consisting of a hazard assessment of the GMP on one hand
and of an analysis of exposure on the other hand, are lacking in the dossiers.

Allergology: No direct testing of potentially allergic properties of GMP
and products derived therefrom was carried out. The absence of allergenic
properties was justified solely in an either argumentative way and/or by
giving rather indirect evidence (e.g., digestion studies, sequence
homology comparisons). Some quotations of literature intended to confirm
the safety of the GMP in the dossiers are cited wrongly or are outdated
or are even suspected to be selectively quoted. The usual way of arguing
is as follows: (i) no homology could be detected between the newly
introduced protein and known allergens, (ii) the expression level of
target protein in the GMP is rather low, (iii) the protein will rapidly
be digested in the intestine, (iv) the newly introduce protein originates
from a non-allergenic source, (v) the protein is not glycosylated and
will therefore less likely exhibit allergic properties, (vi) the protein
will less likely exhibit allergenic properties because it is not new.

Each of these arguments and their underlying assumptions have to be
questioned in the light of recent scientific data. Furthermore,
unintended secondary effects possibly caused by the gene insertion, such
as the possible upregulated expression of other allergens through
insertion and expression of the foreign gene in the GMP, are not
considered at all. A safety evaluation which is based exclusively on the
above described approaches is insufficient.

Substantial Equivalence: Analysis and comparisons of plant compounds are
part of each dossier with the exception of carnation. However, no
connection can be established between the nature and extent of these
analysis and the intended use of the GMP or GMP products. Compositional
analysis are largely restricted to macro-nutrients and known plant
specific anti-nutrients as well as known toxins. A detailed
characterisation of macrocompounds is however, rarely done.

Substantial equivalence is referred to in each dossier in order to argue
for the safety of the particular GMP. The parameters chosen in
composition analysis are however, not comprehensive enough to justify
substantial equivalence and/or to detect probable unintended secondary
effects. In each dossier some significant differences between the GMP and
conventional counterparts were either reported or could be found by
reviewing the displayed data. However, these differences did not lead to
a repetition of the analysis including an extension of parameters
investigated. In contrast, these differences were argumentatively
attributed to naturally occurring ranges, effects from back-crossing,
climate conditions etc.

Detailed descriptions of cultivation conditions, single examination
sheets and statistical data interpretation, information on storage and
preparation of samples as well as detailed data on the results of
compound analysis are lacking in most cases. Detailed explanations on
summaries of compound analysis are often fragmentary or even missing. On
the ground of information given and data shown, substantial equivalence
often cannot be verified. In case of herbicide resistant GMP it is often
not quite clear if the herbicide was applied during cultivation.

As a matter of comparing average values of different cultivation sites
the variance of analysed compounds is sometimes quite high, and might be
covering any unintended secondary effects e.g. resulting in changes in
plant metabolism.

Nutritional considerations in general and especially with respect to
substantial equivalence (e.g. vitamin profiles, characterisation of
fibre, analyses of different types of proteins) apparently do not play a
role in the dossiers and are just occasionally considered in comparative
composition analysis.

Composition of food products derived from animals fed on GMP was not
considered in any dossier.

Recommendations for Standardisation

On the basis of the results of the review of the GMP dossiers as
described above, a number of more general recommendations are drawn
aiming at a specification and standardisation of toxicological and
allergological safety evaluation. These recommendations are partly
inspired by authorisation practices of products other than GMO. In detail
these recommendations aim at simplifying the evaluation of the dossiers,
by checking in a more systematic way comprehensiveness and acceptability
of tests displayed in the dossiers (e.g. formal and thematic structure of
dossiers, use of references, display of results). In general all dossiers
should be stand-alone. In case of Bt- or herbicide resistant GMP these
dossiers should include the approval of the plant protection product. All
safety testing should be performed according to GLP.

Toxicology: From a toxicological perspective the toxicological
examination of the newly introduced gene products should be substantially
extended in order to elucidate not just acute but also sub-chronic,
mutagenic, reproductive, and eco-toxic effects. Furthermore, the toxicity
of the whole plant should be comprehensively studied in order to detect
any possible non-intended secondary effects. However, suitable and
standardised toxicological methods for detecting and investigating such
effects are still lacking. Therefore, such methods should be elaborated
and further developed.

For each GMP a set of basic toxicity data should be elaborated.
Additional testing may be required depending on the intended use of the
plant and the particular exposure.

Allergology: The approach to allergological safety evaluation used so far
is mainly of argumentative nature including at maximum indirect evidences
from sequence homology and digestion studies. This approach is
insufficient and should be complemented or perhaps better replaced by
other methods: Sera from allergic patients should be used to study the
gene product as well as the whole GMP and the corresponding parental line
regarding IgE antibody reactivity. Immunization studies carried out with
extracts prepared from the GMP and the parental line would allow to
compare the allergenic activity (i.e. induction of allergic sensitization
in a naive host) of both organisms.

The exclusive investigation of the gene product is insufficient because
complex changes caused via secondary effects cannot be detected. This
test allows to estimate the number of allergic patients who exhibit IgE
reactivity to the GMP versus the parental line. Furthermore it will be
possible to determine by IgE inhibition studies if the GMP contains more
IgE reactive determinants than the parental line. In order to compare the
allergic potential of the GMP and the parental line, immunisation studies
and further estimation of IgE level could be carried out in mice using
extracts from the GMP and the parental line. On the basis of these
results the risk for a de-novo sensitisation in a not jet sensitised
individual can be estimated.

Substantial Equivalence: Recommendations for field trails and composition
analysis carried out to investigate substantial equivalence are given as
follows:

In order to thoroughly and comparatively investigate different cultivars,
all factors that might affect this investigation have to be considered.
The conditions used for field trials should be very close or even
identical to conditions in normal agricultural practice of that
particular crop. The field trials should be carried out on at least three
different sites and during at least two growing seasons. Time of
cultivation and harvesting, on-site cultivation conditions, experimental
plots and sampling should be thoroughly described. Furthermore, climate
conditions should be reported.

As it is rather difficult to distinguish between natural variation and
variations probably induced by the inserted genes, only isogenic lines
should be used as conventional counterparts. Harmonised lists of key
components (nutrients and anti-nutrients) that need to be investigated by
the applicants should be established for each particular crop. Likewise,
content and ranges of these key components should be agreed for each
particular crop and should in any case include not only macrocomponents
but also microcomponents.

In order to improve the basis of comparative analysis, all relevant data
on the nutritional aspects of each particular compound and the natural
variability in content (e.g. depending on the nature of the site,
climate, agricultural management and cultivars used) should be
systematically collected in international databases. Furthermore, data on
human consumption should be gathered in order to estimate more accurately
the exposure of humans. Such database could be established and ran by
FAO/WHO or OECD as well as by the newly established European Food Safety
Authority.

In order to overcome the current methodological limits of composition
analysis, new methods that are not focussing on particular compounds but
on whole profiles of compounds (e.g. DNA-array, mRNA-fingerprinting,
proteomics, chemical-fingerprinting) should be established, further
developed and used in routine testing.

Finally, the comparative analysis of cultivars and the evaluation of
results should be carried out by an independent institution or panel of
experts.

In order to elucidate possible effects - especially indirect and
unintended ones - of GMP on the environment under conditions of normal
agricultural practice, a research-oriented monitoring programme is
recommended that compares approved GMP, conventional counterparts and
organic farming.

The results and recommendations of this study are intended to contribute
to national and international discussion on the improvement of safety
evaluation of GMP and products derived therefrom. Consequently, these
results and recommendations should be introduced into the relevant
working groups of e.g. the European Commission, OECD, Codex, Cartagena
Protocol.



Order Information:
Federal Environment Agency - Austria
Spittelauer Laende 5, A-1090 Wien, Austria
Tel.: +43 1 31304-0
Fax: +43 1 31304-540
URL: http://www.ubavie.gv.at
Contact: Helmut Gaugitsch



Contact: Helmut Gaugitsch