Safety evaluation of the food enzyme carboxypeptidase D from the genetically modified Aspergillus oryzae strain NZYM-MK.

The food enzyme carboxypeptidase D (EC 3.4.16.6) is produced with the genetically modified Aspergillus oryzae strain NZYM-MK by Novozymes A/S. It is free from viable cells of the production organism and its DNA. The genetic modifications do not give rise to safety concerns. The food enzyme is intended to be used in five food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.908 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 2220 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 2445. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches were found, one with a food allergen (wheat). The Panel considered that a risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to wheat, cannot be excluded, but will not exceed that of wheat consumption. 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.

Safety evaluation of a food enzyme containing bacillolysin and subtilisin activities from the non-genetically modified Bacillus amyloliquefaciens strain AR-383.

The food enzyme with two declared activities, bacillolysin (EC 3.4.24.28) and subtilisin (EC 3.4.21.62), is produced with the non-genetically modified Bacillus amyloliquefaciens strain AR-383 by AB Enzymes GmbH. The food enzyme is intended to be used in nine food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed in the production of distilled alcohol, dietary exposure was calculated only for the remaining eight food manufacturing processes. Exposure was estimated to be up to 1.958 mg TOS/kg body weight per day in European populations. As the production strain qualifies for the qualified presumption of safety approach to safety assessment and no issues of concern arising from the production process of the food enzyme were identified, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made, and 30 matches were found, including one food allergen (melon). The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure to this food enzyme cannot be excluded, but for individuals sensitised to melon, this would not exceed the risk of consuming melon. 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.

Taxonomic identity of the Bacillus licheniformis strains used to produce food enzymes evaluated in published EFSA opinions.

Bacillus paralicheniformis, a species known to produce the antimicrobial bacitracin, could be misidentified as Bacillus licheniformis, depending on the identification method used. For this reason, the European Commission requested EFSA to review the taxonomic identification of formerly assessed B. licheniformis production strains. Following this request, EFSA retrieved the raw data from 27 technical dossiers submitted and found that the taxonomic identification was established by 16S rRNA gene analyses for 15 strains and by whole genome sequence analysis for 12 strains. As a conclusion, only these 12 strains could be unambiguously identified as B. licheniformis.

Safety evaluation of the food enzyme laccase from the non-genetically modified Trametes hirsuta strain AE-OR.

The food enzyme laccase (benzenediol:oxygen oxidoreductase; EC 1.10.3.2) is produced with the non-genetically modified Trametes hirsuta strain AE-OR by Amano Enzyme Inc. The food enzyme is free from viable cells of the production organism. It is intended to be used in six food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.026 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise 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 862 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 33,154. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions by 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.

Safety evaluation of the food enzyme phosphodiesterase I from the non-genetically modified Leptographium procerum strain FDA.

The food enzyme phosphodiesterase I (oligonucleotide 5'-nucleotidohydrolase; EC 3.1.4.1) is produced with the non-genetically modified Leptographium procerum strain FDA by DSM Food Specialties B.V. The food enzyme is free from viable cells of the production organism. It is intended to be used in the processing of yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.171 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, resulted in a margin of exposure of at least 5848. A search for the similarity 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 by 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.

Safety evaluation of the food enzyme leucyl aminopeptidase from the genetically modified Aspergillus oryzae strain NZYM-BU.

The food enzyme leucyl aminopeptidase (EC 3.4.11.1) is produced with the genetically modified Aspergillus oryzae strain NZYM-BU by Novozymes A/S. 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. It is intended to be used in five food manufacturing processes. Dietary exposure to the food enzyme TOS was estimated to be up to 1.508 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 4,928 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 3,268. A search for the similarity 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 by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that the food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme oryzin from the non-genetically modified Aspergillus ochraceus strain AE-P.

The food enzyme oryzin (EC 3.4.21.63) is produced with the non-genetically modified Aspergillus ochraceus strain AE-P by Amano Enzyme Inc. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in nine food manufacturing processes. The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.1 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise 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 1862 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 18,620. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and 31 matches were found, including one food allergen (melon). The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to melon, cannot be excluded, but would not exceed the risk from consumption of this food. 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.

Safety evaluation of the food enzyme bacillolysin from the non-genetically modified Bacillus amyloliquefaciens strain AE-NP.

The food enzyme bacillolysin (EC 3.4.24.28) is produced with the non-genetically modified Bacillus amyloliquefaciens strain AE-NP by Amano Enzyme Inc. The production strain meets the requirements for the qualified presumption of safety (QPS) approach to safety assessment. The food enzyme is intended to be used in 14 food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed in three manufacturing processes, dietary exposure was calculated only for the remaining 11 food manufacturing processes in which the food enzyme-TOS is retained. It was estimated to be up to 35.251 mg TOS/kg body weight (bw) per day in European populations. As the production strain qualifies for the QPS approach and no issue of concern arising from the production process of the food enzyme were identified, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A search for the similarity 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 by 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.

Safety evaluation of the food enzyme AMP deaminase from non-genetically modified Aspergillus sp. strain DEA 56-111.

The food enzyme AMP deaminase (AMP aminohydrolase; EC 3.5.4.6) is produced with the non-genetically modified microorganism Aspergillus sp. strain DEA 56-111 by Shin Nihon Chemical Co., Ltd. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in the processing of yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.005 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 1984 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 396,800. A search for the similarity 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.

Safety evaluation of the food enzyme mucorpepsin from the non-genetically modified Rhizomucor miehei strain LP-N836.

The food enzyme mucorpepsin (EC 3.4.23.23) is produced with the non-genetically modified Rhizomucor miehei strain LP-N836 by Meito Sangyo Co., Ltd. The native enzyme can be chemically modified to produce a more thermolabile form. The food enzyme is free from viable cells of the production organism. It is intended to be used in the processing of dairy products for the production of cheese and fermented dairy products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.108 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 95 mg TOS/kg bw per day, the mid-dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 880. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and four matches with respiratory allergens and one with a food allergen (mustard) were found. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to mustard proteins, cannot be excluded. Based on the data provided, the Panel concluded that both the native and thermolabile forms of this food enzyme do not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme bacillolysin from the non-genetically modified Bacillus amyloliquefaciens strain DP-Cyb74.

The food enzyme bacillolysin (EC 3.4.24.28) is produced with the non-genetically modified Bacillus amyloliquefaciens strain DP-Cyb74 by Genencor International B.V. The production strain met all requirements for the qualified presumption of safety (QPS) approach to safety assessment. The food enzyme is intended to be used in six food manufacturing processes. Dietary exposure to the food enzyme total organic solids (TOS) was estimated to be up to 1.536 mg TOS/kg body weight per day in European populations. As the production strain qualifies for the QPS approach and no issue of concern arose from the production process of the food enzyme, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A search for the similarity 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 by 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.

Safety evaluation of the food enzyme thermolysin from the non-genetically modified Anoxybacillus caldiproteolyticus strain AE-TP.

The food enzyme thermolysin (EC. 3.4.24.27) is produced with the non-genetically modified Anoxybacillus caldiproteolyticus strain AE-TP by Amano Enzyme Inc. The food enzyme is free from viable cells of the production organism. It is intended to be used in eight food manufacturing processes. Dietary exposure was estimated to be up to 0.973 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 700 mg TOS/kg bw per day, the mid-dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 719. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that the risk of allergic reactions by 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.

Safety evaluation of the food enzyme bacillolysin from the non-genetically modified Bacillus amyloliquefaciens strain NZYM-NB.

The food enzyme bacillolysin (EC 3.4.24.28) is produced with the non-genetically modified Bacillus amyloliquefaciens strain NZYM-NB by Novozymes A/S. The production strain meets the requirements for qualified presumption of safety (QPS) approach to safety assessment. The food enzyme is intended to be used in eleven food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed during two processes, dietary exposure was estimated only for the remaining nine food manufacturing processes. Exposure was estimated to be up to 1.327 mg TOS/kg body weight per day in European populations. As the production strain qualifies for the QPS approach and no issue of concern arising from the production process of the food enzyme was identified, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A search for the similarity 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 by dietary exposure cannot be excluded (except for distilled alcohol production), 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.

Safety evaluation of the food enzyme glutaminase from the non-genetically modified Bacillus amyloliquefaciens strain AE-GT.

The food enzyme glutaminase (l-glutamine amidohydrolase; EC 3.5.1.2) is produced with the non-genetically modified Bacillus amyloliquefaciens strain AE-GT by Amano Enzyme Inc. The production strain met the requirements for the qualified presumption of safety (QPS) approach. The food enzyme is intended to be used in five food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.462 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's manufacturing process, toxicity tests were considered unnecessary by the Panel. A search for the similarity of the amino acid sequence of the food enzyme 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 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.

Safety evaluation of the food enzyme leucyl aminopeptidase from the non-genetically modified Aspergillus sp. strain AE-MB.

The food enzyme leucyl aminopeptidase (EC 3.4.11.1) is produced with the non-genetically modified Aspergillus sp. strain AE-MB by Amano Enzyme Inc. The food enzyme is considered free from viable cells of the production organism. It is intended to be used in five food manufacturing processes: processing of dairy products for the production of (1) flavouring preparations; processing of plant- and fungal-derived products for the production of (2) protein hydrolysates; processing of meat and fish products for the production of (3) protein hydrolysates, (4) modified meat and fish products and processing of (5) yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 2.273 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 183 mg TOS/kg bw per day. The calculated margin of exposure for each age group was 135 (infants), 81 (toddlers), 83 (children), 109 (adolescents), 160 (adults) and 184 (the elderly). A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. The safety of the food enzyme could not be established given the derived margins of exposure. Therefore, the Panel concluded that this food enzyme could not be considered safe under the intended conditions of use.

Safety evaluation of the food enzyme endo-1,4-ß-xylanase from the genetically modified Bacillus velezensis strain AR-112.

The food enzyme endo-1,4-ß-xylanase (4-ß-d-xylan xylanohydrolase, EC 3.2.1.8) is produced with the genetically modified Bacillus velezensis strain AR-112 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. 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 0.024 mg TOS/kg body weight (bw) per day in European populations. As the production strain B. velezensis strain AR-112 meets the requirements for the qualified presumption of safety (QPS) approach to safety assessment and no issue of concern arose from the production process, no toxicological data are required. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, 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.

Safety evaluation of the food enzyme ß-galactosidase from the non-genetically modified Papiliotrema terrestris strain AE-BLC.

The food enzyme ß-galactosidase (ß-d-galactoside galactohydrolase; EC 3.2.1.23) is produced with the non-genetically modified Papiliotrema terrestris strain AE-BLC by Amano Enzyme Inc. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in the production of galacto-oligosaccharides (GOS) from lactose. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.441 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 1800 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 4082. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that a 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.

Safety evaluation of the food enzyme 3-phytase from the genetically modified Aspergillus niger strain NPH.

The food enzyme 3-phytase (myo-inositol-hexakisphosphate 3-phosphohydrolase EC 3.1.3.8) is produced with the genetically modified Aspergillus niger strain NPH by DSM Food Specialties. 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. It is intended to be used in three food manufacturing processes: processing of cereals and other grains for the production of (1) baked products and (2) distilled alcohol, and the processing of plant- and fungal-derived products for the production of (3) plant-based analogues of milk and milk products. Since no residual amounts of total organic solids (TOS) are carried over into distilled alcohol, dietary exposure was calculated only for the remaining two food manufacturing processes. It was estimated to be up to 0.553 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 833 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1506. A search for the similarity 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 by dietary exposure cannot be excluded (except for distilled alcohol production), 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.

Safety evaluation of the food enzyme glucan 1,4-α-maltohydrolase from the genetically modified Bacillus subtilis strain BABSC.

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 Bacillus subtilis strain BABSC by Advanced Enzyme Technologies Ltd. The requirements for the qualified presumption of safety (QPS) approach have not been met. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in baking processes and starch processing for the production of glucose syrups and other starch hydrolysates. Since residual amounts of total organic solids (TOS) are removed, dietary exposure was not calculated for starch processing for the production of glucose syrups and other starch hydrolysates. For baking processes, the dietary exposure was estimated to be up to 0.101 mg TOS/kg body weight per day in European populations. No toxicological studies were provided by the applicant. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and one match with a respiratory allergen was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. In the absence of appropriate data to fully characterise the production strain, the Panel was unable to conclude on the safety of the food enzyme under the intended conditions of use.

Safety evaluation of the food enzyme catalase from the non-genetically modified Aspergillus tubingensis strain AE-CN.

The food enzyme catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) is produced with the non-genetically modified Aspergillus tubingensis strain AE-CN by Amano Enzyme Inc. The absence of viable cells of the production organism in the food enzyme was not demonstrated. The food enzyme is intended to be used in five food manufacturing processes: production of baked products, processing of egg and egg products, production of fruit and vegetable products other than juices, production of cheese and production of fish roes. The dietary exposure to the food enzyme total organic solids (TOS) was estimated to be up to 0.325 mg TOS/kg body weight (bw) per day in European populations. The results of the in vitro genotoxicity studies indicated the presence of a clastogenic agent in the food enzyme which could not be dismissed due to limitations in the in vivo studies. 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 323 mg TOS/kg bw per day, the highest dose tested. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and one match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Because of the results of the genotoxicity studies, and as the absence of viable cells from the production strain was not demonstrated, the Panel was unable to establish the safety of the food enzyme.