EXECUTIVE SUMMARY. Technical Aspects of Enzymes (Chapter 3)

Executive Summary 5 EXECUTIVE SUMMARY Technical Aspects of Enzymes (Chapter 3) Application of enzymes (Section 3.2) Enzymes are applied in various areas of application, the most important ones are technical use, manufacturing of food and feedstuff, cosmetics, medicinal products and as tools for research and development. Enzymatic processes - usually carried out under mild conditions - are often replacing steps in traditional chemical processes which were carried out under harsh industrial environments (temperature, pressures, ph, chemicals). Technical enzymes are applied in detergents, for pulp and paper applications, in textile manufacturing, leather industry, for fuel production and for the production of pharmaceuticals and chiral substances in the chemical industry. Typically technical enzymes are manufactured and used as bulk enzymes in high volumes compared to other areas of enzyme application. Food enzymes are mainly used in baking industry, for manufacturing fruit juices, in wine making and brewing as well as in cheese manufacturing. An important field of applications in terms of volumes is starch conversion to yield ingredients for foodstuff. The use of enzymes in animal nutrition is an important and growing area of enzyme application, especially for pig and poultry nutrition. Feed enzymes offer the benefit of degrading specific feed components otherwise harmful or of no value to the livestock. Presently, a number of enzymes are or are intended to be applied in cosmetic products. A well documented use of application is skin peeling, future applications may be skin protection and enzyme systems preserving the cosmetic product. Notable medications of enzymes are as digestive aids, for wound cleaning, lysis of vein thromboses, acute therapy of myocardial infarction and as support in the therapy of certain types of leukaemia. Enzymes can be used in chemical analysis and as a research tool in the life sciences. However, the volumes used in this area of applications are regarded as negligible and this type of enzymes is not considered in the course of this study. Manufacturing of enzymes (Section 3.3) The majority of enzymes currently available is manufactured from microorganism. Manufacturing process comprise large-scale fermentation to yield high volumes of microbes. Enzymes are either accumulating inside the cells or are secreted into the media of the fermentation tanks. In subsequent steps the disrupted cells (or the media including the enzymes) are subjected to further purification processes using variety of chemical, mechanical and thermal techniques (concentration, precipitation, extraction, centrifugation, filtration, chromatography). The resulting enzyme concentrate is then formulated to the final ready-to-sell product by adding stabilisers, standardizing agents, preservatives and salts. The final enzyme preparations are usually commercially marketed in granular or liquid forms. The nature of enzyme products (Section 3.4) Within the scope of this study, the terminology for enzymes used by AMFEP was applied. Commercially available enzymes are produced as enzyme concentrates which result from fermentation and subsequent purification steps. The enzyme concentrate contains the active enzyme(s) and various by-products from the fermentation process. The composition and amount of by-products in the enzyme concentrate is extremely variable and depending on the organisms, the media and the conditions applied during fermentation and subsequent

6 Executive Summary downstream processing (30-98% by-products in the enzyme concentrate). Thus, in addition to identification and characterisation of the enzyme as the active substance, parameters applicable for characterisation of enzyme concentrate have to be implemented. Additives are added in a subsequent step depending on the particular application and on customers demands (final ready-for-use enzyme preparation). Enzyme preparations should be regarded as preparations in the sense of Directive 1999/45/EC. Recent developments in enzyme manufacturing (Section 3.5) The application of genetic engineering techniques in enzyme manufacturing is dramatically sparking the exploitation of new enzymes and the development of new enzyme properties. Due to new technologies, new enzymes not accessible before can be cloned into and produced from a well-known host organism. Thereby, enzymes from almost any source in nature become accessible, including enzymes exhibiting unusual properties, such as extreme thermostability. Applying new technologies, the enzyme properties may be efficiently altered which will lead to an increase in the variability of enzymes available and might lead to enzymes not present in nature so far. This mainly concerns enzyme stability, catalytic mechanism, substrate specificity and range, surface activity, folding mechanisms, cofactor dependency, ph- and temperature optima, kinetic parameters. Furthermore, enzymes could be chemically modified. With applying new technologies, the variability in enzyme structure is dramatically increased and enzyme properties are significantly enhanced. Thus, these methods are mainly contributing to technical and economic goals. However, the safety of enzyme manufacturing might also be improved by restricting to few well-known and safe-to-use production strains which are used as hosts for genes from various sources. Industrial Enzymes Presently Marketed in the EU (Chapter 4) A list of all enzymes that are presently marketed in the EU was compiled based on information given by enzyme-producing industry (AMFEP). This list includes 186 enzymes from 47 different catalytic types and gives information on the catalytic activity, the host and donor organisms and the various fields of application. Regulation of Enzymes in Legislation Depending on their Use (Chapter 5) Enzymes are regulated in different legislation depending on their use. Within the scope of this study, the regulation of enzymes was investigated and analysed regarding legislation for chemicals, food additives and processing aids, animal feed additives, cosmetic products and medicinal products. Chemicals (Section 5.2) The analysis of the national notification systems in the EU, USA, Canada and Australia revealed that in these countries industrial enzymes are defined as chemical substances and are consequently subjected to chemicals legislation; information requirements for notification/reporting are the same as those for chemicals. In all countries, enzymes are described via their catalytic activity. Only in Canada additional information requirements for substances derived from biotechnology (including enzymes) are defined, focussing on the production organism and the enzymatic properties. In the USA, differentiation of enzymes is done on a case-by-case basis depending on the available information, but limited experience has been gained so far. In Australia, assessments on two enzymes were performed and published so far.

Executive Summary 7 In the EU, difficulties in enzyme notification were identified along with the differentiation of enzymes, the interpretation of EINECS entries, the decision on notification requirement and the need for the complete testing requirements according to Directive 67/548/EEC. Food additives and processing aids (Section 5.3) In the EU an harmonised authorisation system is only in place for food enzymes used as additives. Directive 95/2/EEC covers food additives, however, most food enzymes are applied as processing aids, which do not have a technological function in the final foodstuff. Most Member States do not have a national legislation covering these enzymes. International and national committees (SCF, JEFCA, COT), the US FDA and AMFEP issued guidelines on conditions of use, information requirements and safety evaluation of enzymes. These guidelines pertain both types of enzymes. Information and testing requirements include basic technical data on the enzyme itself, information on the source material, additives and possible contaminants (MO, heavy metals, toxins). Long-term experience in production of a particular enzyme and use in food application is explicitly taken into account. Safety concerns are mainly focussing on toxic properties of by-products and contaminants. Changes in the production process may lead to a re-evaluation on a case-by-case basis. Enzymes regarded as new are often compared to already approved enzymes to check if they are substantially different. Additional information is usually required in the case of GMM on host organism. The production of toxins resulting form unintended secondary effect is regarded as the main concern. Enzymes from GMM are often evaluated on a case-by-case basis. FDA is applying the concept of substantial equivalence to enzymes from GMM. Thus, the enzyme from GMM is compared to the conventional counterpart to evaluate if relevant properties have been affected. Additives in animal nutrition (Section 5.4) Enzymes used as feed-additives have to be authorised according to EU Directives 70/524/EEC and 87/153/EEC. According to Directive 87/153/EEC among other things the dossier has to include a characterisation of the active enzyme (e. g. identity, biological origin, genetic modification, possible toxins) as well as a description of physico-chemical, technical and biological properties of the enzyme preparation (i.e. additive). Further a safety assessment basing on relevant toxicity studies has to be made. This has to be done in order to prove the safe use of the enzyme preparation against target species, consumers, workers and the environment.the additives are defined by their IUB number together with their main activity and the production organism. Directive 87/153/EEC explicitly demands that the production organisms have to be non-pathogenic and non-toxigenic. An adaptation of Directive 87/153/EEC can be expected in the near future. So far, 61 time-limited and one unlimited authorisations on enzymes containing additives are given within the EU. It is recommended by the Scientific Committee for Animal Nutrition (SCAN) to generally regard enzymes as respiratory sensitisers (R42) unless convincing evidence to the contrary is provided. The SCAN further recognises the exposure of workers. In case of a genetic modification, both the production- and the donor-organism (source of the transferred DNA) should be described. Cosmetics products (Section 5.5) For substances used in cosmetic products, no general authorisation procedure is foreseen, but the Annexes of the Cosmetic Directive 76/768/EEC list substances that must not form part of a cosmetic product, that are restricted or designated for specified applications (e. g preservatives or UV filters). According to these Annexes only the enzyme catalase is forbidden to be used in cosmetic products, a reason therefore is not given. Prior to their listing

8 Executive Summary in one of the Annex substances have to pass a safety assessment performed by the Scientific Committee for Cosmetic and Non Food Products (SCCNFP). This is also done if concerns with regard to the safe use of cosmetic ingredients arise. No safety evaluations of the SCCNFP concerning enzymes could be revealed. According to an answer upon an request given by DG SANCO, the question of use of enzymes in cosmetic products is a rather new issue which has not been discussed and analysed in depth by the SCCNFP so far. According to AMFEP, the potential for respiratory sensitisation has to be considered as a serious concern when applying enzymes in personal care and cosmetics products. Medicinal products (Section 5.6) Medicinal products are subjected to Directive 65/65/EEC and Regulation (EEC) 2309/93. If medicinal products derive from a biotechnological process, they have to be authorised and the European Agency for Evaluation of Medicinal Products (EMEA) is therefore performing a scientific evaluation of the product. The biotechnological production process, the genetic modification of the microorganisms and the specification of the product are taken into account. Concerning toxicological properties, at present no set of safety tests can be described which are applicable to all different product groups. Parameters Applicable for the Description of Enzymes (Chapter 9) A clear distinction between the terms enzyme as active compound, enzyme concentrate and enzyme preparations is essential for the discussion of parameters applicable for a description. Within the scope of this study, the terminology used by AMFEP was applied. Most parameters used in scientific practice for the description of enzymes focus on the enzyme as the active compound. Enzymes as active compounds could be characterised by their function as well as by their molecular structure. For clearly distinguishing enzymes by function, the information on the catalytic type has to be supplemented by additional functional parameters. To unambiguously identify/distinguish enzymes, the primary structure plus information on posttranslational modification has to be specified. Functional properties of enzymes cannot reliably be deduced from enzyme structure and vice versa. Thus, functional parameters are also relevant for the description of enzymes. In industrial and regulatory contexts, the enzyme concentrate is relevant, i. e. the active component (i. e. the enzyme) plus any impurities resulting from fermentation and subsequent purification steps. A characterisation of the enzyme concentrate has to be extended to by-products or impurities resulting from fermentation and purification that may comprise 30 to 98% of a final enzyme concentrate. The enzyme concentrate is usually characterised by describing the production process and the production organism. Furthermore, the absence or level of total viable count, known pathogenic microorganisms, known toxins, as well as heavy metals are routinely estimated or verified if required by (some) legislation. Enzyme preparations, the ready-to-sell products, are described by specifying the intentionally added substances, i.e. protein, carbohydrates, fat, ash, water and diluents as well as stabilisers, standardizers, preservatives, and formulating agents. Regulatory practice for describing technical, feed, food and other enzymes largely makes use of the parameters described above, thereby focussing on parameters for enzyme identification via its catalytic activity, information required on the production organism, requirements for the production process, and additives and other ingredients used.

Executive Summary 9 Additional information often required for enzymes from GMM applied in food and feedstuff often includes a description of the genetic modification and information on functional and structural aspects of the enzyme. In the US food additive regulation, for instance, a comparative analysis of the enzyme from GMM and from wildtype is requested. For the feed sector, industry has proposed a decision tree in order to decide which data are required in case of GMM. Data requirements specified in legislation, guidelines published by industry and parameter used in scientific practice were investigated and compared and some fundamentals of the selection of parameters for a future enzyme notification system are discussed. Toxicological and ecotoxicological properties of enzymes and enzyme concentrates Irritation and Sensitisation (Chapter 6) The scientific literature investigated indicate that enzymes have the potential for sensitisation of the respiratory tract. At present, no validated test method exists to determine and to predict sensitisation via the inhalative route. No recommendations can be given regarding test methods that could be routinely used for the evaluation of sensitisation to the respiratory tract. It is proposed to apply the precautionary principle and generally label enzymes with R42 May cause sensitisation via inhalation. Based on literature review, there are no indications that enzymes are skin sensitizers. Therefore, testing the skin sensitizing properties of enzymes is not considered to be relevant. All enzymes may be potential skin irritants. Regarding irritation of the skin and the eye, test methods according to the OECD guideline 404 and 405 are suggested. Other Toxic Properties of Enzyme Concentrates (Chapter 7) The discussion of toxicological properties of enzymes and the conclusions drawn are largely based on scientific literature of enzymes used in the food and feed sector (since test data could not been obtained from the industry). A position paper from the detergent enzyme industry, testing recommendations (food sector) and testing requirements (chemical and feed regulation) are also taken into consideration. Regarding sub-chronic toxicity and mutagenicity, pure enzymes are not considered to be of concern, but this is not the case for enzyme concentrate, since a certain risk remains that toxic or mutagenic contaminants or by-products are generated in the fermentation process. Although there is no clear consensus about toxicity testing of enzyme concentrates regarding legislation, scientific literature and (detergent) enzyme industry, but a commonly applied test set exists, which comprises One repeated dose oral toxicity test on rodents. Two mutagenicity tests (one bacterial and one non-bacterial in vitro assay) In certain well defined cases (e. g. if there is a risk for generation of certain toxins, if a critical exposure is assumed) the following toxicological endpoints may become relevant for enzymes: Acute oral or dermal toxicity Immunotoxicity and toxicokinetics The following toxicological endpoints seem to be of no or little relevance for enzymes:

10 Executive Summary Acute inhalative toxicity Reproduction toxicity, chronic toxicity and carcinogenicity It is assumed, that enzymes produced by GMM demand no substantially new toxicity testing provided that the consequences of these modifications are considered prior to the testing and are used to guide the toxicity testing. No evidence could be found which indicates that the present and foreseeable improvement of certain enzyme properties (e. g. changing ph optimum or increasing thermal stability by protein engineering ) will demand new or altered toxicity testing. Therefore the following recommendations are given by the authors of the study: Prior to toxicity testing, information should be compiled on the enzyme concentrate, the production strain, the fermentation and downstream processing. Genetic modification (donor organism, vector, transferred DNA) should be taken into account. A reduction of testing requirements may be considered on a case-by-case basis, if an evaluation reveals no significant product change compared to a product already described and confirmed to be safe (this is also valid for the accompanying production process). The reduction may affect the need for acute, subchronic and mutagenicity testing. Testing requirements should, however, be extended depending on the expected exposure and application. This is also true for new types of enzymes or new production organisms. Data already available within industry should be investigated by independent institutions. Testing should generally be performed using the enzyme concentrate. Environmental Considerations (Chapter 8) The studies investigated so far revealed that enzymes seem unlikely to be dangerous to the aquatic environment due to their ready biodegradability and the low effects on aquatic organisms observed. However, enzymes derived from new technologies might have increased stability (e. g. with higher stability to temperature or ph), therefore, the ready biodegradability of such enzymes should be proved. It is suggested, only to perform a biodegradation test in the case that the enzyme has unusual stability. Decision criteria for unusual stability have to be set. Only in the case, the enzyme is not ready biodegradable, the performance of acute toxicity testing on aquatic organisms should be discussed. Regulation of Enzymes Possible Impacts of Different Approaches in Enzyme Identification (Chapter 10) Within the scope of this study, two fictitious notification/registration systems were discussed, distinguishing enzymes either by their catalytic type or by their chemical structure, and the resulting impacts in terms of regulatory and safety issues were illustrated. Enzyme identification via catalytic activity (Scenario 1) Enzymes are presently identified by their catalytic activity. Applying this system leads, especially in the context of EINECS, to several problems. If the identification of enzymes is exclusively basing on catalytic activity, no distinction is made between enzymes (e. g. between wildtype organism and GMM, between natural enzymes, extremozymes and structurally altered enzymes) and consequently no data on the toxicological and ecotoxicological properties of the enzymes would be reported.

Executive Summary 11 Enzyme identification via structural characteristics (Scenario 2) Distinguishing enzymes exclusively by structure (primary structure, posttranslational modification) in chemical legislation would provide criteria for clearly and almost unambiguously distinctions between enzyme molecules. However, this would put a considerable burden on both the manufacturer and the regulator. As neither advantages for the safety of enzymes become evident applying this approach, nor enzymes are regarded as highly dangerous substances of high priority at all (on the basis of present knowledge), these efforts do not seem to be justifiable. Furthermore, toxicological, and probably also exo-toxicological considerations are focusing on impurities in the enzyme concentrate. As the composition of by-products in enzyme concentrates in terms of quality and quantity is extremely variable only indirect evidence could be given by describing the production process and identifying and characterising the production organism. Thus, additional data would be required in any case. Consequently, the structure based concept (scenario 2) might end up in similar difficulties as the activity based concept (scenario 1). Therefore, additional criteria have to be taken into account to distinguish between enzyme concentrates. Regulation of Enzymes Outlook and recommendations (Chapter 11) In the future, enzymes could be registered under REACH but modified data requirements are needed for enzymes, given their specific properties. A framework of a registration system for technical enzyme is outlined based on possible impacts of the developments in enzyme manufacturing. EINECS is not regarded as sensible for deciding on enzyme notification. In a first step, enzyme entries in EINECS should be replaced by a database, which could be generated by means of an obligation to report all enzymes presently on the EU market. The reporting into the database should include data for the characterisation of the enzyme, the enzyme concentrate and all testing data presently available. Enzymes should be reported/registered by IUB type (catalytic activity) but equivalence of enzymes has to be justified using core and additional parameters/data. Core data/parameter are regarded as essential and have to be specified in any case. Additional data/parameter have to be specified if they are available by the applicant. The tonnage limits should be applicable for enzymes and calculated in a standardised way. Testing requirements: With the exception of the sensitising and irritating properties, enzymes used so far do not exhibit toxic or ecotoxic properties that raises serious concern. For enzyme registration, testing of the skin irritation potential should be requested. There is no validated test method to determine sensitisation of the respiratory tract, therefore it is proposed to generally label enzymes with R42 May cause sensitisation via inhalation. However, as novel enzymes become available and novel production organisms may be used, the performance of certain toxicity and ecotoxicity tests should be decided on a caseby-case basis. This testing focuses on by-products and contaminants of the enzyme concentrate (acute toxicity (oral), repeated dose toxicity (oral), mutagenicity) or on enzymes with increased stability (ready biodegradability, acute toxicity on aquatic organisms). In case of defined changes in the enzyme manufacturing process, the manufacturer should be obliged to monitor toxicity and ecotoxicity.