Repeated Dose 90-day Oral Toxicity Research Articles

Safety evaluation of the food enzyme glucan-1,4-α-glucosidase from the non-genetically modified Aspergillus niger strain DP-Azh100.

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.

Comprehensive Safety Assessment of Lacticaseibacillus paracasei subsp. paracasei NTU 101 Through Integrated Genotypic and Phenotypic Analysis

Probiotics, as defined by the World Health Organization, are live microorganisms that, when consumed in sufficient quantities, provide health benefits to the host. Although some countries have approved specific probiotic species for use in food, safety concerns may still arise with individual strains. Lacticaseibacillus paracasei subsp. paracasei NTU 101 (NTU 101), isolated from the gut of healthy infants, has demonstrated various probiotic effects and shown safety in a prior 28-day animal feeding study. To further verify its safety and mitigate potential risks, we performed a comprehensive genotypic and phenotypic safety evaluation in accordance with the European Food Safety Authority guidelines for safety assessment through whole genome sequencing and related literature. In this research, minimum inhibitory concentration testing identified NTU 101’s resistance to chloramphenicol; however, subsequent gene analysis confirmed no associated risk of resistance. Assessments of safety, including biogenic amine content, hemolytic activity, mucin degradation, and D-lactic acid production, indicated a low level of risk. Additionally, a repeated-dose 90-day oral toxicity study in Sprague-Dawley rats revealed no toxicity at a dose of 2000 mg/kg body weight, further supporting the strain’s safety for consumption. Based on these comprehensive analyses, NTU 101 is considered safe for regular consumption as a health supplement.

Safety evaluation of the food enzyme endo-1,3(4)-β-glucanase from the non-genetically modified Talaromyces versatilis strain PF8.

The food enzyme endo-1,3(4)-β-glucanase (3-(1-3;1-4)-β-d-glucan 3(4)-glucanohydrolase; EC 3.2.1.6) is produced with the non-genetically modified Talaromyces versatilis strain PF8 by Erbslöh Geisenheim AG. The food enzyme was free from viable cells of the production organism. It is intended to be used in four food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was calculated to be up to 0.110 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 2229 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 20,264. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and four matches with respiratory or contact allergens were 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 endonuclease from the non-genetically modified Penicillium citrinum strain NP 11-15.

The food enzyme endonuclease (Aspergillus nuclease S1; EC 3.1.30.1) is produced with the non-genetically modified Penicillium citrinum strain NP 11-15 by Shin Nihon Chemical Co., Ltd. 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 0.006 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 1010 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 168,333. 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 by dietary exposure cannot be excluded, especially for individuals allergic to Penicillium. However, the likelihood of such reactions will not exceed the likelihood of allergic reactions to Penicillium. 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 carboxypeptidase C from the genetically modified Aspergillus niger strain PEG.

The food enzyme carboxypeptidase C (EC 3.4.16.5) is produced with the genetically modified Aspergillus niger strain PEG by DSM Food Specialties B.V. 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 nine food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 2.053 mg TOS/kg body weight (bw) per day in European populations. The toxicity studies were carried out with a xylanase obtained from A. niger strain XEA. The Panel considered this food enzyme as a suitable substitute for the carboxypeptidase to be used in the toxicological studies, because both strains were derived from the same recipient strain, the location of the inserts was comparable, no partial inserts were present and the production methods were essentially the same. 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 1850 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 901. A homology search for the amino acid sequence of the food enzyme to known allergens was made and one match with a wheat allergen was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, especially in wheat-allergic individuals, 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 effect of aging on the repeated-dose liver micronucleus assay using N-nitrosodipropylamine, quinoline, and carbendazim

The repeated dose liver micronucleus (RDLMN) assay has been sufficiently validated in terms of the numbers and types of chemicals studied. However, it remains unclear whether aging affects assay results. The OECD Test Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents) indicates that dosing should begin as soon as feasible after weaning and in any event before 9 weeks of age. Therefore, it is particularly important to determine whether there are age-related differences between 6 and 8 weeks of age at the start of dosing when considering the possibility of integrating this assay into a 4-week repeated dose general toxicity study. We evaluated the impact of the rats’ age on the RDLMN assay with three chemicals: N-nitrosodipropylamine, quinoline, and carbendazim. There were no significant age-related differences for the first two chemicals, whereas a markedly higher frequency of micronucleated hepatocytes (MNHEPs) was observed in younger rats for carbendazim. However, regardless of the age of animals, micronucleus induction was detected in all three chemicals. Combined with the previous reports on clofibrate and diethylnitrosamine, we concluded that animals of any age from 6 to 8 weeks could be used in the RDLMN assay.

Safety evaluation of the food enzyme asparaginase from the genetically modified Aspergillus niger strain ASP.

The food enzyme asparaginase (l-asparagine amidohydrolase; EC 3.5.1.1) is produced with the genetically modified Aspergillus niger strain ASP by DSM Food Specialties B.V. 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 the prevention of acrylamide formation in foods and in the processing of yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.792 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 1038 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1311. 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 triacylglycerol lipase from the non-genetically modified Penicillium caseifulvum strain AE-LRF.

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the non-genetically modified Penicillium caseifulvum strain AE-LRF by Amano Enzyme Inc. The food enzyme was free from viable cells of the production organism. It is intended to be used in four food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.013 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 69 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 5308. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. However, the Panel noted that traces of ■■■■■, used in the manufacture of the triacylglycerol lipase, may be found in the food enzyme. The Panel considered that the risk of allergic reactions upon dietary exposure could not be excluded, particularly in individuals sensitised to fish. 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.

Repeated dose, 28-day oral toxicity study of curcumin, anthocyanins, and sodium nitrite in Wistar rats

ObjectiveThe goal of the present study was to examine the repeated dose 28-day oral toxicity of curcumin, anthocyanins, and sodium nitrite in Wistar rats. MethodsFor this purpose, forty-eight male Wistar rats were randomly divided into 8 groups (n = 6 each), encompassing untreated controls and experimental groups treated with curcumin, anthocyanins, and sodium nitrite. Three rats from each group were sacrificed by cervical dislocation under di-ethyl ether anesthesia after 2 and 4 weeks of therapy, respectively. Blood samples were collected for serum chemistry. All of the animals' livers, hearts, and kidneys were removed and sent for histopathological examination. ResultsAfter two weeks of inquiry, certain groups displayed higher hematological values, while others had lower values compared to the control group. AST, CK, and LDH enzyme activity were higher in groups 2–8, but urea concentrations were higher in groups 6 and 8. After four weeks, the Hb, MCH, and MCHC values in group 4 were greater, as were the WBC levels in groups 4 and 6, whereas other groups had lower MCV and WBC values. The weekly body weight gain was insignificantly different between treatment groups. Throughout the experiment, none of the animals perished. Male rats' liver, kidney, and heart underwent histopathological changes after ingesting curcumin, sodium nitrite, and anthocyanin. ConclusionBased on the findings, rats were more detrimental when curcumin, sodium nitrite, and anthocyanin were ingested together than when they were consumed individually, as evidenced by histopathological abnormalities in the liver, kidneys, and heart.

Safety evaluation of the food enzyme β-glucosidase from the non-genetically modified Penicillium guanacastense strain AE-GLY.

The food enzyme β-glucosidase (β-D-glucoside glucohydrolase; EC 3.2.1.21) is produced with the non-genetically modified Penicillium guanacastense strain AE-GLY by Amano Enzyme Inc. The food enzyme is intended to be used in four food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 4.054 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 943 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 233. 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 3-phytase from the non-genetically modified Aspergillus niger strain PHY93-08.

The food enzyme 3-phytase (myo-inositol-hexakisphosphate 3-phosphohydrolase EC 3.1.3.8) is produced with the non-genetically modified Aspergillus niger strain PHY93-08 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. It is intended to be used in nine food manufacturing processes. Since residual amounts of food enzyme-total organic solids (TOS) are removed in two of the food manufacturing processes, dietary exposure was calculated only for the remaining seven processes. It was estimated to be up to 0.763 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise safety concerns. 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 2560 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 3355. 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 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 an extension of use of the food enzyme pullulanase from the non-genetically modified Pullulanibacillus naganoensis strain AE-PL.

The food enzyme pullulanase (pullulan 6-α-glucanohydrolase; EC 3.2.1.41) is produced with the non-genetically modified Pullulanibacillus naganoensis strain AE-PL by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in one food manufacturing process. Subsequently, the applicant has requested to extend its use to include seven additional processes and to revise the previous use level. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of eight food manufacturing processes. As the food enzyme-total organic solids (TOS) are not carried into the final foods in two food manufacturing processes, the dietary exposure was estimated only for the remaining six processes. The dietary exposure was calculated to be up to 0.004 mg TOS/kg body weight (bw) per day in European populations. The Panel evaluated the repeated dose 90-day oral toxicity study in rats submitted in the previous application and identified a no observed adverse effect level of 643 mg TOS/kg bw per day, the highest dose tested. When compared with the calculated dietary exposure, this resulted in a margin of exposure of at least 160,750. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Toxicological evaluation of Alpinia oxyphylla-derived molecule (PD-00105): In vitro genotoxicity studies and 90-day oral toxicity study in rats

PD-00105 corresponds to a compound initially identified in the fruit of Alpinia oxyphylla Miq., obtained by chemical synthesis and proposed to be use in dietary supplements for its potential neuroprotective properties. The aim of this study was to perform a toxicological evaluation of PD-00105 in accordance with the testing strategy recommended by food regulatory authorities. All studies were conducted in accordance with Good Laboratory Practice (GLP), and followed the Organization for Economic Co-operation and Development (OECD) test guidelines for chemicals. Studies included a bacterial reverse mutation test, one in vitro micronucleus test in mammalian cells, and a repeated dose 90-day oral toxicity study. No sign of toxicity was observed in the two genotoxicity tests. The test item induced a significant liver and kidney toxicity at high doses (50 and 100 mg/kg BW/day), highlighted by significant increases in liver and kidney absolute and relative weights, associated with histopathological findings and concomitant changes in hematology and clinical chemistry. Increases in alanine aminotransferase, alkaline phosphatase, total protein, albumin, globulin, cholesterol, LDL, and HDL have been measured in these two groups. However, findings observed in the low-dose group (10 mg/kg BW/day) were considered as minimal and non-adverse, and were limited to an increase in liver weight in males and in kidneys weight in females, without concomitant changes in blood chemistry. The No Observed Adverse Effect Level (NOAEL) of PD-00105 was established as 10 mg/kg BW/day under the conditions of this study. This study substantiates the use of PD-00105 in dietary supplements at doses of 10 mg/day, taking into account a safety margin factor for dose conversion to humans.

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 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 assessment of the substance 'phosphorous acid, triphenyl ester, polymer with 1,4-cyclohexanedimethanol and polypropylene glycol, C10-16 alkyl esters', for use in food contact materials.

The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) assessed the safety of the substance 'phosphorous acid, triphenyl ester, polymer with 1,4-cyclohexanedimethanol and polypropylene glycol, C10-16 alkyl esters', when used as an additive in all types of polyolefins. The substance is a polymer containing ≤ 13% w/w of a low molecular weight fraction (LMWF, < 1000 Da). A polyethylene sample with 0.15% w/w of the substance was used in a comprehensive set of migration tests with food simulants. The specific migration was up to 0.014 and 0.023 mg/kg in 4% acetic acid and 10% ethanol, respectively. Migration into olive oil was estimated by the Panel to be up to 5.3 mg/kg under worst-case conditions of use. The migrating LMWF species were comprehensively identified. Those without phosphorous were either without alerts for genotoxicity or listed in Regulation (EU) 10/2011 with worst-case migrations well below their respective specific migration limits. Toxicological studies were performed using phosphite and phosphate versions of the substance enriched in its LMWF. The substance does not raise a concern for genotoxicity. From a repeated dose 90-day oral toxicity study in rats with a 50:50 phosphite:phosphate blend, the Panel identified a NOAEL of 250 mg/kg bw per day for each component of the blend. No delayed neurotoxicity in hens was observed. The CEP Panel concluded that the substance does not raise a safety concern for the consumer if its LMWF is not higher than 13% w/w, if it is used at up to 0.15% w/w in polyolefin materials and articles intended for contact with all food types, except for infant formula and human milk, for long-term storage at room temperature and below, after hot-fill and/or heating up to 100°C for up to 2 h, and if its migration does not exceed 5 mg/kg food.

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 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 vitamin K2 (menaquinone-7) via toxicological tests

This study aims to evaluate the safety of MK-7 produced by fermentation process using a Bacillus subtilis var. natto strain for human ingestion via acute oral toxicity, repeated dose 90-day oral toxicity, 28-day recovery test, and genotoxicity tests. The acute oral toxicity test results indicated that all subjects survived at the dose of 5000 mg/kg with no toxic effects. For the repeated dose 90-day oral toxicity test, MK-7 was administered to rats at 500, 1500, and 4500 mg/kg for 90 d. No abnormal findings were detected in clinical observations or in clinical pathological and histopathological examinations. The no-observed-adverse-effect level(NOAEL) was determined to be 4500 mg/kg/d, the maximum dose tested. For the evaluation of genotoxicity, reverse mutation, chromosomal aberration, and micronucleus tests were performed. In the reversion mutation test, vitamin K2 did not induce reversion in bacterial strains, and no chromosomal abnormality was observed in the chromosomal abnormality test using Chinese hamster lung cells. In the micronucleus test, micronuclei were not induced using ICR mouse bone marrow cells. All the toxicity test results suggest that vitamin K2 produced by fermentation processes using Bacillus subtilis var. natto induced no toxicological changes under the experimental conditions.