1. Risk assessment practice in the context of legislation and guidelines Armin Spök Inter-University Research Centre for Technology, Work and Culture (IFF/IFZ) Graz Presentation held at the Workshop “Scrutinizing GMO Risk Assessment” Vienna, 10 December 2003

2. spoek@ifz.tugraz.at Questions that will be dealt with  What was the regulatory context of the investigated dossiers?  What’s about the major changes in the regulatory context?  Is everything settled now? - Putting the project results into a changing context  Overall Conclusions

3. spoek@ifz.tugraz.at  What was the regulatory context of the dossiers?

4. spoek@ifz.tugraz.at EU Regulatory Context of Directive 90/220 Dossiers investigated SCP Guidance Novel Food Regulation

5. spoek@ifz.tugraz.at EU Regulatory Context of Novel Food Dossiers investigated Novel Food Regulation SCF Guidance SSC Opinion

6. spoek@ifz.tugraz.at  What’s about the major changes in the regulatory context?

7. spoek@ifz.tugraz.at EU Regulatory Context: Summary  For the dossiers investigated  Directive 90/220/ECC (Directives 94/15/EC, 97/35/EC, Decision 92/146/EEC, Guidance Notes), SCP Guidance (1998)  Novel Food Regulation, SCF Guidance (97/618/EC)  Present/upcoming context  Directive 2001/18/EC (Decision 2002/811/EC), SSC Guidance (2003)  Novel Food Regulation (Regulation 1829/2003), SSC Guidance (2003) SSC Guidance was not completed before March 2003

8. spoek@ifz.tugraz.at Was: Regulatory Context 1  Directive 90/220/EEC: environmental risk assessment mandatory  No details for toxicity/allergenicity safety requirements specified: “toxic or allergenic effects of the non-viable GMOs and/or their metabolic products“ (Decision 92/146/EC Annex, 40 (a))  „covers effects on all classes of organisms“/“all vertebrate animals“ (Document XI/619/91-Rev.3 )  SCP guidance (1998) provides some details  Insect resistant and herbicide resistant GMP: „Data should be provided on the toxicity [...] of compounds with plant protective properties as expressed in GM plants“  „In the case of new metabolites appropriate toxicity studies should be carried out with respect to the assessment of animal and human safety as laid down in the Directive 91/414“

9. spoek@ifz.tugraz.at Is: Regulatory Context 2  Directive 2001/18/EC  Includes “direct or indirect, immediate, delayed or unforeseen effects“  Potential adverse affects should not be discount if they are unlikely to occur  Decision 2002/811/EC of 24 July 2002 establishing guidance notes supplementing Annex II to Directive 2001/18/EC of the European Parliament and of the Council on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC  For more details the SSC Guidance (2003) applies  EU Common catalogue of varieties of agricultural plant species  No toxicity/allergenicity safety requirements specified  Sectoral EU legislation: feed-stuff, seeds,  No toxicity/allergenicity safety requirements specified

10. spoek@ifz.tugraz.at Was: Regulatory Context 3  Novel Food Regulation  does not specify requirements for toxicity and allergenicity assessment  Recommendation 97/618/EC (29 July 1997)  toxicity assessment guided by substantial equivalence  full substantial equivalence: not toxicity testing required?  partial substantial equivalence: focus on deviating traits  no substantial equivalence: case-by-case evaluation –chemical structure, physico-chemical properties of the NF” “source, composition, potential intake” –“toxicity studies in vitro and in vivo including mutagenicity studies, reproduction and teratogenicity studies as well as long term feeding studies” –studies on potential allergenicity.

11. spoek@ifz.tugraz.at Is: Regulatory Context 4  Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed  Already come into force  Shall apply from 18 April 2004  No toxicity/allergenicity safety requirements specified  For more details the SSC Guidance (2003) applies  Updated /new guidance in preparation?

12. spoek@ifz.tugraz.at  Given these changes in legislation and on top of that the new Guidance Document – Is everything settled now ?  Not at all!

13. spoek@ifz.tugraz.at Comparing the Project Proposals to the SSC Guidance  Some suggestions are already included in the SSC Guidance  E.g. stand-alone dossiers  Some are partly considered  E.g. GLP demanded for toxicity testing only  Some points are simply reiterating prior suggestions  E.g. Homology studies are still encouraged – without any qualification or specification  Others are still ambiguous  E.g. toxicity testing requirements  Others are contrasting prior recommendations  E.g. in-vitro digestibility studies

14. spoek@ifz.tugraz.at Comparing the Project Recommendations to those of the SSC Guidance  Toxicity testing requirements (new proteins)  In-vitro digestibility studies (new proteins) SSC says... - 28 d sub-acute, repeated dose - Disregards acute toxicity tests - still ambiguous if toxicity tests will be required in any case We say... - subchronic, repeated dose tests - in any case SSC says... - in-vitro studies required We urge for... - further research & clarification

15. spoek@ifz.tugraz.at Acute vs. Subchronic Testing  No subchronic toxicity testing because  Proteins are only known to act as acute toxicants – if toxic at all  “Absence of evidence” does not preclude the “evidence of absence”  No acute toxicity  From of absence of acute toxicity one cannot infer the absence of subchronic/chronic effects  Proteins are easily degraded in the digestive tract  In-vitro tests are not fully representative of digestibility in vivo  Protein might survive in vivo (e.g. Chowdhury et al. 2003)  No homology to known toxins  Significance of homology studies are questionable

16. spoek@ifz.tugraz.at Toxicity testing requirements: Overview SourceRequirements (new proteins) SCF (1997)Not specified SCP (1998)Not specified In practice (90/220/EEC)14 day acute toxicity, single dose In practice (Novel Food)14 day acute toxicity, single dose SSC (2003)28 day repeated dose (not standard) EuropaBio (2003)Acute toxicity NL Biosafety Council (2003)28 day acute toxicity, repeated dose Royal Society (Canada)90 day, sub-chronic, repeated dose

17. spoek@ifz.tugraz.at Toxicity testing requirements 2 SourceRequirements (substance) Food Additives Food Enzymes90d on rats Feed Enzymes90d on rats Biotech products in CosmeticSub-acute toxicity Biotech PesticidesAcute toxicity Biotech Pharmaceuticals90 d repeated dose (ICH 1997) Herbal MedicinesSub-chronic, chronic

18. spoek@ifz.tugraz.at SCF on Food Additives “Any new additive should normally be tested in subchronic oral toxicity studies in two laboratory species, usually rodent and non-rodent, for a period of at least 90 days. Preceding feeding studies conducted for 14 or 28 days can provide an indication of target organs and help predict appropriate doses for 90-day studies, but, in general, studies of shorter duration are not sufficient, by themselves, for evaluation of potential subchronic toxicity.” (SCF 2000: 23)

19. spoek@ifz.tugraz.at SSC on GMO Food and Feed “An acute, single dose test with a 14-day observation period, is inadequate to detect possible toxicity arising from repeated dosing. Furthermore, this test does not provide information on the dose-response relationship and is designed to examine only a few endpoints (mortality, morbidity, clinical observation and gross necropsy) and not the broad range of endpoints required to be investigated in repeated dose studies, such as haematology, clinical chemistry, urine analysis, organ weights and histopathological examination of organs and tissues.” (SSC 2003: 19)

20. spoek@ifz.tugraz.at In-Vivo vs. In-Vitro Digestibility Studies: Summary  In-vivo digestibility studies required: yes, yes but..., probably, rather not, not at all…  “ The use of in vitro simulation of gastric and intestinal digestion of the gene product should be considered supplementary to in-vivo experiments… “ (SCP 1998)  Disregarded digestibility for toxicity studies in general (SSC 2000)  In-vitro studies criticised (OECD 2000)  In-vitro studies suggested (FAO/WHO 2001, SSC 2003)

21. spoek@ifz.tugraz.at In-vivo vs. in-vitro digestibility studies 2 “Evidence of degradation of the introduced gene products should be based on data obtained in vivo by feeding the GM plant material or its derived products to the intended target animal. [...] The use of in vitro simulation of gastric and intestinal digestion of the gene product should be considered supplementary to in vivo experiments designed to measure the survival of the gene products when fed to animals as an integral part of the GM plant. Isolated proteins are known which are fully degraded in the simulated gastric system but survive gut passage intact when fed as part of a normal diet.” (SCP 1998)

22. spoek@ifz.tugraz.at In-vivo vs. in-vitro digestibility studies 3 “There are arguments that the currently used tests of gastric and intestinal protein resistance to gastric and intestinal hydrolysis represent a ‚best case‘ situation and do not reflect the digestive capacity of the very young and those with pancreatic and severe gastric disorders. The fact that a protein is digested does not preclude for some eventual pharmacological/toxicological properties of derived peptides. Such properties have been observed for conventional foods and thus should be anticipated in all foods, whether they are derived from GM protein or from non-GM protein.” (SSC 2000)

23. spoek@ifz.tugraz.at In-vivo vs. in-vitro digestibility studies 4  “…the simulated gastric fluid (SGF) test, an artificial system for testing proteins’ digestibility, does not mimic exactly the physiological conditions in the digestive tract. Such testing may not always provide clear evidence of the possible toxic or allergenic potential of peptides formed as breakdown products in the test system.” (p.14)  “Tests normally used to assess toxicity include in vitro digestibility of the protein which is used to compare the properties of the novel gene product to the characteristics of known proteins. This test is not intended to detect any potential toxicity in the very young, the elderly, and that segment of the population which is unable to produce stomach acid. Test methods should be designed to evaluate potential risks for those subjects when the gene product is similar to a chemical that is implicated in having unique toxicity when not digested.” (p.26-27) (OECD 2000)

24. spoek@ifz.tugraz.at In-vivo vs. in-vitro digestibility studies 4  “Data concerning the resistance of the novel protein to proteolytic enzymes (e.g. pepsin) should be obtained, e.g. by in vitro investigations using appropriate and validated tests.” (p.19) (SSC 2003)  “An in vitro digestibility assay in simulated gastric and/or intestinal fluids is required. It is important to note the at resistance to in vitro digestion is not a toxicity endpoint by itself, but simply an indication that the protein warrants closer examination and perhaps different types of testing. On a case-by-case basis, also an ex-vivo gastric fluid test (e.g. pig, cattle, dog) or in vivo models may be required.” (p.11) (Dutch Biosafety Council 2003)

25. spoek@ifz.tugraz.at In-vivo vs. in vitro digestibility studies 5  In vitro studies do not consider  That proteins can be ‘protected’ by food  The potential effect of anti-nutrients (proteinase-inhibitor, phytic acid etc.) included in the food  Potential effects of peptides as breakdown products  Studies that show that proteins might pass the stomach in vivo without breakdown (e.g. Chowdhury et al. 2003)  The wide use of proton-pump inhibitors to reduce gastric acidity  That the stomach acidity of elderly people and infants is much lower  That proteins used in these studies are usually produced from micro-organisms and might therefore have an altered stability

26. spoek@ifz.tugraz.at  Overall Conclusions

27. spoek@ifz.tugraz.at Overall Conclusion 1  The study clearly shows differences in risk assessment between dossiers  Particular differences occur because of lack of detailed guidance  They might affect the level of safety provided  Points to a need to  Standardize/harmonize risk assessment requirements where there is a scientific consensus  Clarify or at least pursue research where there is no scientific consensus and agree on interim requirements

28. spoek@ifz.tugraz.at Overall Conclusions 2  The study also shows possible shortcomings in risk assessment practice  Practice seems to be sometimes not in line  with state of the art knowledge  with standards of risk assessment practice in other regulatory contexts  Points to a need of a debate on possible improvements  The recommendations presented can be seen as an input to both to standardize and to improve risk assessment

29. spoek@ifz.tugraz.at Overall Conclusions 3  The recommendations point to a level of detail that also applies despite the changed regulatory contexts  Some recommendations are even strengthened by the more explicit precautionary approach laid down in directives and regulations  The recommendations are only partly precluded by the SSC Guidance  Many details and more fundamental questions remain to be dealt with

30. spoek@ifz.tugraz.at Thank you for your attention!