Safety evaluation of the food enzyme phospholipase D from the non‐genetically modified Streptomyces netropsis strain DSM 40093

The food enzyme glucan 1,4‐α‐maltotetraohydrolase (EC 3.2.1.8) is produced with the genetically modified Bacillus licheniformis strain DP‐Dzf24 by Danisco US Inc. The production strain contains multiple copies of a known antimicrobial resistance gene. However, based on the absence of viable cells and DNA in the food enzyme, this is not considered to be a risk. The food enzyme is intended to be used in baking processes and starch processing for the production of glucose syrups. The residual amounts of the Total Organic Solids (TOS) in glucose syrups are removed by filtration and purification during starch processing. Consequently, dietary exposure was not calculated for this use. Based on the maximum use levels recommended for the baking processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme–TOS was estimated to be up to 0.271 mg TOS/kg body weight per day in European populations. Toxicological tests with the food enzyme indicated that there was no concern with respect to genotoxicity or systemic toxicity. A no‐observed‐adverse‐effect level (NOAEL) was identified in rats, which, compared with the dietary exposure, results in a margin of exposure of at least 347. The allergenicity was evaluated by searching for similarity of the amino acid sequence to those of known allergens; no match was found. The Panel considers that, under the intended conditions of use, the risk for allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered low. Based on the microbial source, genetic modifications, the manufacturing process, the compositional and biochemical data, the dietary exposure assessment and the findings in the toxicological studies, the Panel concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use.

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the genetically modified Komagataella phaffii strain DSM 34125 by Chr. Hansen. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in four food manufacturing processes. Dietary exposure was estimated to be up to 0.039 mg total organic solids (TOS)/kg body weight per day in European populations. Given the qualified presumption of safety status of the production strain and the absence of concerns resulting from the food enzyme manufacturing process, toxicity tests were considered unnecessary by the Panel. A search for the homology of the amino acid sequence of the triacylglycerol lipase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure cannot be excluded, but that the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

The food enzyme endo-1,4-β-xylanase (4-β-d-xylan xylanohydrolase; EC 3.2.1.8) is produced with the genetically modified Bacillus subtilis strain AR-153 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The production strain meets the requirements for the qualified presumption of safety (QPS) approach. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in four food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed during one food manufacturing process, dietary exposure was only calculated for the remaining three processes. Exposure was estimated to be up to 0.463 mg TOS/kg body weight per day in European populations. Given the QPS status of the production strain and the absence of concerns resulting from the food enzyme manufacturing process, toxicity tests were considered unnecessary by the Panel. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

The food enzyme α‐amylase (1,4‐α‐D‐glucan glucanohydrolase; EC 3.2.1.1) is produced with the genetically modified Bacillus licheniformis strain DP‐Dzb45 by Danisco US Inc. The production strain of the food enzyme contains multiple copies of an antimicrobial resistance gene. However, based on the absence of viable cells and DNA from the production organism in the food enzyme, this was not considered to be a risk. The α‐amylase is intended to be used in brewing processes and distilled alcohol production. Since residual amounts of the food enzyme are removed by distillation, no dietary exposure was calculated for this intended use. Based on the maximum use levels recommended for the brewing processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.138 mg TOS/kg body weight per day in European populations. Toxicological tests with the food enzyme indicated that there was no concern with respect to genotoxicity or systemic toxicity. A no observed adverse effect level was identified in rats, which, compared with the dietary exposure, results in a margin of exposure of at least 484. Similarity of the amino acid sequence to those of known allergens was searched and one match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions can be excluded in distilled alcohol production but cannot be excluded when the enzyme is used in brewing. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

The food enzyme with β‐glucanase and β‐xylanase (4‐β‐d‐xylan xylanohydrolase, EC 3.2.1.8) activities is produced with the non‐genetically modified Trichoderma reesei (strain DP‐Nya67) by DuPont. The food enzyme is intended to be used in brewing processes, grain treatment for the production of starch and gluten fractions, and distilled alcohol production. Since residual amounts of the food enzyme are removed by distillation and during grain treatment, dietary exposure was only calculated for brewing processes. Based on the maximum recommended use levels for brewing processes, dietary exposure to the food enzyme–Total Organic Solids (TOS) was estimated to be up to 4.585 mg TOS/kg body weight (bw) per day. Since the compositional data provided was insufficient to characterise the food enzyme batches used for toxicological testing, their suitability for use in the toxicological tests could not be established. As result, the toxicological studies provided were not further considered by the Panel. Similarities of the amino acid sequences to those of known allergens were searched and no matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood to occur is considered to be low. In the absence of compositional data sufficient to characterise the food enzyme batches used for toxicological testing, the Panel is unable to complete its assessment of the safety of the food enzyme.

EFSA Supporting PublicationsVolume 13, Issue 4 1014E External scientific reportOpen Access Annual report on the preparation of summary reports from dossiers on food enzymes – GMM part Jan W. Pedersen, Jan W. Pedersen Technical University of Denmark, National Food InstituteSearch for more papers by this authorFolmer D. Eriksen, Folmer D. Eriksen Technical University of Denmark, National Food InstituteSearch for more papers by this author Jan W. Pedersen, Jan W. Pedersen Technical University of Denmark, National Food InstituteSearch for more papers by this authorFolmer D. Eriksen, Folmer D. Eriksen Technical University of Denmark, National Food InstituteSearch for more papers by this author First published: 01 April 2016 https://doi.org/10.2903/sp.efsa.2016.EN-1014 The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender procedure. The present document is published complying with the transparency principle to which the Authority is subject. It may not be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Published date: 1 April 2016 Question number: EFSA-Q-2016-00249 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References EFSA CEF Panel (EFSA Panel on Food Contact Material, Enzymes, Flavourings and Processing Aids), 2013. Guidance of the Scientific Panel of Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) on the Submission of a Dossier on Food Enzymes for Safety Evaluation by the Scientific Panel of Food Contact Material, Enzymes, Flavourings and Processing Aids. EFSA Journal 2009; 1305, 1– 26. doi:10.2903/j.efsa.2009.1305. Updated on May 2013 EFSA, European Food Safety Authority (2014) Explanatory Note for the Guidance of the Scientific Panel of Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) on the Submission of a Dossier on Food Enzymes. EFSA Supporting Publication 2014: EN-689 Volume13, Issue4April 20161014E ReferencesRelatedInformation

The food enzyme α‐amylase (4‐α‐d‐glucan glucanhydrolase; EC 3.2.1.1) is produced with the genetically modified strain Bacillus licheniformis DP‐Dzb25 by Danisco US Inc. It is intended to be used in distilled alcohol production, starch processing for the production of glucose syrups, and in brewing processes. Since residual amounts of the food enzyme are removed by distillation and during starch processing, no dietary exposure was calculated for these food processes. Based on the maximum use levels recommended for brewing processes and individual data from the EFSA Comprehensive European Food Database, dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.138 mg TOS/kg body weight (bw) per day. The production strain of the food enzyme contains multiple copies of a known antimicrobial resistance gene and consequently, it does not fulfil the requirements for the Qualified Presumption of Safety (QPS) approach to safety assessment. However, considering the absence of viable cells and DNA from the production organism in the food enzyme, this is not considered to be a risk. As no other concerns arising from the microbial source and its subsequent genetic modification or from the manufacturing process have been identified, the Panel considers that toxicological tests are not needed for the assessment of this food enzyme. Similarity of the amino acid sequence to those of known allergens was searched for and no match was found. The Panel notes that the food enzyme may contain a known allergen. Therefore, allergenicity cannot be excluded for uses other than distilled alcohol production. Apart from potential allergenicity, the Panel concluded that the food enzyme 4‐α‐d‐glucan glucanhydrolase produced with the genetically modified B. licheniformis strain DP‐Dzb25 does not give rise to safety concerns under the intended conditions of use.

The food enzyme glucan 1,4-α-maltohydrolase (4-α-d-glucan α-maltohydrolase, EC 3.2.1.133) is produced with the genetically modified Escherichia coli strain MLAVSC by Advanced Enzyme Technologies Ltd. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in three food manufacturing processes. Since residual amounts of food enzyme total organic solids (TOS) are removed in one food manufacturing process, dietary exposure was calculated for the remaining two processes. It was estimated to be up to 0.172 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1000 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 5814. A search for the homology of the amino acid sequence of the glucan 1,4-α-maltohydrolase to known allergens was made and matches with three respiratory allergens and one injected allergen were found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but that the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

The food enzyme containing β‐fructofuranosidase (β‐d‐fructofuranoside fructohydrolase; EC 3.2.1.26) and β‐glucosidase (β‐d‐glucoside glucohydrolase; EC 3.2.1.21) activities is produced with the non‐genetically modified Aspergillus tubingensis strain IN 319 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. The food enzyme is intended to be used in six food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed in one process, dietary exposure was calculated only for the remaining five food manufacturing processes. It was estimated to be up to 1.832 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The Panel identified a no observed adverse effect level of 1067 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 582. A search for the homology of the amino acid sequence of the β‐fructofuranosidase and β‐glucosidase to known allergens was made and matches with one food and one respiratory allergen were found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

The food enzyme endo‐polygalacturonase ((1–4)‐α‐d‐galacturonan glycanohydrolase; EC 3.2.1.15) is produced with the genetically modified Trichoderma reesei strain AR‐414 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in eight food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed in three food manufacturing processes, dietary exposure was calculated for the remaining five food processes. It was estimated to be up to 0.216 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1000 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 4630. A search for the homology of the amino acid sequence of the endo‐polygalacturonase to known allergens was made and matches with one food allergen and 12 respiratory allergens were found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

The food enzyme glucan-1,4-α-glucosidase (4-α-d-glucan glucohydrolase; EC 3.2.1.3) is produced with the non-genetically modified Aspergillus niger strain DP-Azh100 by Genencor International B.V. It was considered free from viable cells of the production organism. The food enzyme is intended to be used in four food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed in two processes, dietary exposure was calculated only for the two remaining processes. It was estimated to be up to 1.390 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level at the highest dose tested of 1000 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 719. A search for the homology of the amino acid sequence of the food enzyme to known allergens was made and one match to a respiratory allergen was found. Known sources of food allergens were used in the food enzyme manufacturing process. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

The food enzyme subtilisin (EC 3.4.21.62) is produced with the genetically modified Bacillus subtilis strain DP‐Ezx62 by Danisco US Inc. The production strain of the food enzyme contains multiple copies of a known antimicrobial resistance gene, and consequently, it does not fulfil the requirements for the qualified presumption of safety (QPS) approach to safety assessment. However, considering the absence of viable cells and DNA from the production organism in the food enzyme, this is not considered to be a risk. As no other concerns arising from the microbial source and its subsequent genetic modification or from the manufacturing process have been identified, the Panel considered that toxicological tests are not needed for the assessment of this food enzyme. The food enzyme is intended to be used in three food manufacturing processes. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 1.060 mg TOS/kg body weight per day in European populations. A search for the homology of the amino acid sequence of the subtilisin to known allergens was made and matches with 2 food, 19 respiratory and 3 contact allergens were found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

The food enzyme sucrose:sucrose fructosyltransferase (sucrose:sucrose 1′‐β‐d‐fructosyltransferase; EC 2.4.1.99) is produced with the genetically modified Yarrowia lipolytica strain E4772 by SEQENS. The production strain met the requirements for the qualified presumption of safety (QPS) approach. The food enzyme is free from viable cells of the production organism, but not free from recombinant DNA. The food enzyme is intended to be used in the processing of sugars for the production of fructo‐oligosaccharides. The dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.088 mg TOS/kg body weight per day in European populations. Given the QPS status of the production strain and the absence of concerns resulting from the food enzyme manufacturing process, toxicity tests were considered unnecessary by the Panel. A search for the homology of the amino acid sequence of the sucrose: sucrose fructosyltransferase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but that the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

The food enzyme α‐amylase (4‐α‐d‐glucan glucanohydrolase; EC 3.2.1.1) is produced with the genetically modified Bacillus subtilis strain AR‐651 by AB Enzymes. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used in baking processes. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 1.19 mg TOS/kg body weight (bw) per day in European populations. The production strain carries known antimicrobial resistance genes and consequently, it does not fully fulfil the requirements for the qualified presumption of safety (QPS) approach to safety assessment. However, considering the absence of viable cells and DNA from the production organism in the food enzyme, this is not considered to be a risk. As no other concerns arising from the microbial source and its subsequent genetic modification or from the manufacturing process have been identified, the Panel considers that toxicological tests are not needed for the assessment of this food enzyme. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and three matches with respiratory allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concludes that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

The food enzyme alternansucrase (sucrose:1,6(1,3)-α-d-glucan 6(3)-α-d-glucosyltransferase; EC 2.4.1.140) is produced with the genetically modified Escherichia coli strain EBASSC by Advanced Enzyme Technologies Ltd. The production strain of the food enzyme contains a known antimicrobial resistance gene. However, based on the absence of viable cells and DNA from the production organism in the food enzyme, this is not considered to be a risk. The food enzyme is intended to be used in the processing of sugars for the production of maltose alternan gluco-oligosaccharides. Two sets of dietary exposure estimates were made, using the default or more specific input factors. The refined dietary exposure was estimated to be up to 0.586 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 500 mg TOS/kg bw per day, the mid-dose tested, which when compared with the refined exposure estimate, results in a margin of exposure of at least 853. A search for the homology of the amino acid sequence of the alternansucrase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.