{'fraction_non_alphanumeric': 0.03198851024937981, 'fraction_numerical': 0.027810419114766942, 'mean_word_length': 4.756012024048096, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 2, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Safety and efficacy of the feed additive consisting of l‐tryptophan produced by Escherichia coli KCCM 80210 for all animal species (Daesang Europe BV)

Abstract Following a request from the European Commission, the Panel on Additives and Products or substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of the feed additive consisting of l‐tryptophan produced by fermentation with Escherichia coli KCCM 80210 when used as a nutritional additive in feed for all animal species and categories. The production strain E. coli KCCM 80210 is safe for the production of l‐tryptophan and it was not detected in the final product. The Panel notes that two out of five batches of the additive do not comply with the minimum specification of 98% l‐tryptophan on a dry matter basis proposed by the applicant. The use of l‐tryptophan (≥ 98%) produced by E. coli KCCM 80210 in supplementing feed to compensate for l‐tryptophan deficiency in feedingstuffs is safe for non‐ruminant target species. There may be a risk for an increased production of toxic metabolites when unprotected l‐tryptophan is used in ruminants. The use of l‐tryptophan produced by E. coli KCCM 80210 in animal nutrition raises no safety concerns to consumers of animal products and to the environment. The additive under assessment is considered a mild eye irritant. The endotoxin activity of the additive and its dusting potential indicate a risk by inhalation for the users. The additive is not a skin irritant and is not a skin sensitiser. The additive l‐tryptophan is regarded as an effective source of the amino acid l‐tryptophan for all non‐ruminant species. In order to be as efficacious in ruminants as in non‐ruminants, it should be protected from ruminal degradation.

Background and Terms of Reference as provided by the requestor Regulation (EC) No 1831/2003 1 establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7.
The European Commission received a request from Daesang Europe BV 2 for authorisation of the feed additive consisting of L-tryptophan produced by fermentation using Escherichia coli KCCM 80210, when used as a feed additive for all animal species (category: nutritional additives; functional group: amino acids, their salts and analogues).
According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 4(1) (authorisation of a feed additive or new use of a feed additive). The particulars and documents in support of the application were considered valid by EFSA as of 15 September 2020.
According to Article 8 of Regulation (EC) No 1831/2003, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the safety for the target animals, consumer, user and the environment and on the efficacy of the feed additive consisting of L-tryptophan (≥ 98%) produced by fermentation with a genetically modified strain of E. coli (KCCM 80210), when used under the proposed conditions of use (see Section 3.1.4).

Additional information
The subject of the present assessment is the product consisting of L-tryptophan (minimum 98%) produced by a genetically modified strain of E. coli (KCCM 80210). L-Tryptophan produced by this bacterial strain had not been previously authorised as feed additive in the European Union.
L-Tryptophan (≥ 98%) produced by fermentation with specific strains of E. coli is currently authorised for use as a nutritional additive in the European Union, under the functional group 'amino acids, their salts and analogues'. 3 L-Tryptophan is authorised for use in food for nutritional purposes, 4 and for use in cosmetics. 5 It is authorised for use as a veterinary medical product without maximum residue limits. 6 The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) published several opinions on the safety and efficacy of L-tryptophan produced by different strains of E. coli for all animal species (EFSA FEEDAP Panel, 2013, 2014a,b, 2015a,b, 2016a,b, 2017a,b, 2019a-e, 2020a. The EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) issued a scientific opinion on the substantiation of health claims related to L-tryptophan (EFSA NDA Panel, 2011). L-Tryptophan is described in the European Pharmacopoeia, 9th Edition (2017), so that this citation matches the Reference List. Please confirm that this is correct. monograph 01/2017:1272.
The Norwegian Scientific Committee for Food Safety assessed the safety of L-tryptophan in food (VKM, 2013) supplements and energy drinks (VKM, 2016

2.
Data and methodologies

Data
The present assessment is based on data submitted by the applicant in the form of a technical dossier 7 in support of the authorisation request for the use of the product consisting of L-tryptophan (minimum 98.0%) produced by fermentation using E. coli KCCM 80210 as a feed additive.
The FEEDAP Panel used the data provided by the applicant together with data from other sources, such as previous risk assessments by EFSA or other expert bodies, peer-reviewed scientific papers and other scientific reports to deliver the present output.
EFSA has verified the European Union Reference Laboratory (EURL) report as it relates to the methods used for the control of the product consisting of L-tryptophan produced by fermentation using E. coli KCCM 80210 in animal feed. The Executive Summary of the EURL report can be found in Annex A. 8

Methodologies
The approach followed by the FEEDAP Panel to assess the safety and the efficacy of the additive under assessment is in line with the principles laid down in Regulation (

Assessment
The product subject of this application is an additive consisting of L-tryptophan (≥ 98%) produced by fermentation with a genetically modified strain of Escherichia coli (KCCM 80210). It is proposed to be used as nutritional additive (functional group: amino acids, their salts and analogues) in feed for all animal species and categories.

Characterisation of the production organism
The additive is produced by a genetically modified derivative of E. coli K-12 which is deposited in the Korean Culture Collection of Microorganisms (KCCM) with accession number KCCM 80210. 10 A bioinformatic analysis of the whole genome sequence (WGS) of the production strain confirmed its identity as an E. coli K-12 derivative. 11 This was based E. coli K-12 is well characterised and its safety (nonpathogenicity) has been documented (Gorbach, 1978). The strain has been shown to be ineffective in colonising the human gut (Smith, 1975) and its genome (MG1655 and W3110) has been fully sequenced (Hayashi et al., 2006). 12 The production strain was tested for its susceptibility to all the antimicrobials listed for 'Enterobacteriaceae' in the Guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018a,b). 13 All minimum inhibitory concentration (MIC) values were below or equal to the cut-off values set in the Guidance and, therefore, the strain is considered susceptible to those antibiotics.
The WGS of the production strain was interrogated for the presence of antimicrobial resistance (AMR) genes 12 This, and the fact that the production strain was not phenotypically resistant to any of the antibiotics tested, suggests that the production strain does not carry antibiotic resistance genes of concern.
The WGS of the production strain was also interrogated for the presence of toxin and virulence factor genes The chemical formula is C 11 H 12 N 2 O 2, the molecular weight is 204.23 g/mol. The structural formula is given in Figure 1.
According to the specification, the product contains ≥ 98% L-tryptophan on a DM basis and ≤ 1% moisture.
The analysis of five batches of the additive showed an average content of tryptophan of 98.4% on 'as is' basis (range 97.3-99.7%), 17 moisture was 0.1% and ash ranged 0.02-0.06%. 18 On a dry matter basis, the tryptophan content was on average 98.5% (97.4-99.8%), corresponding to the amount of identified material. Two out of the five analysed batches did not reach the specification of minimum 98% tryptophan on a DM basis.
The specific optical rotation was measured in five batches of the additive and was on average -32.3°(range -32.0 to -32.4°), 18 which is within the range described in the European Pharmacopoeia (-30 to -33°) for this amino acid and confirms the identity of the L-enantiomer. 19

Undesirable substances
Five batches of the additive were analysed for heavy metals (cadmium, lead and mercury) and arsenic. All values were below the respective limit of detection (LOD) except for lead which ranged from 0.03 to 0.06 mg/kg. 20 Polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar dioxin-like polychlorinated biphenyls (Co-planar PCBs) were analysed in three batches and found below the corresponding limit of quantification (LOQ). 21 The calculated (upper bound) levels of dioxins and the sum of dioxins and dioxin-like-PCBs were calculated to be 0.137 ng WHO-PCDD/F-TEQ/kg and 0.269 ng WHO-PCDD/F-PCB-TEQ/kg, respectively (in all three batches). In relation to mycotoxins, aflatoxins (not specified), ochratoxin A, zearalenone, fumonisins (B1, B2 and B3), deoxynivalenol and citrinin were analysed in three batches of the additive. All analytical values  were found below the LOD except for citrinin in one batch (15.6 lg/kg). 22 The detected amounts of these undesirable substances do not raise safety concerns. Microbial contamination was analysed in three batches. Salmonella spp. was absent in 25 g samples. In 1 g samples, E. coli and Enterobacteriaceae were not detected; and yeast and moulds ranged 510-700 colony-forming units (CFU)/g. 23 The concentration of 1,1 0 -ethylidene-bis-L-tryptophan (EBT) was < 10 mg/kg in all three batches analysed. The concentration of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA) was measured in three batches and was 1 mg/kg in all cases. 24 Endotoxin activity was measured in three batches (Limulus amebocyte lysate assay) and ranged from 1,720 to 5,180 EU/g. 25 The presence of viable cells of the production strain in the final product was tested in three batches of the additive. 26 No growth was detected.
The presence of DNA from the production strain was tested in three batches of the additive in triplicate. 27 No DNA of the production strain was detected.

Physical-chemical properties
The additive is described as a white to yellowish white odourless crystalline powder. It has a solubility in water of 10.6 g/L (20°C) 28 and a density of 550-650 kg/m 3 .
Dusting potential was analysed in three batches (Stauber-Heubach method) and the values ranged from 0.6 to 0.8 g/m 3 . 29 Particle size distribution of the final product was measured in three batches and the fractions of particles below 11, 52 and 105 lm diameter ranged 21-25, 64-74 and 76-86%, respectively. 30

Stability and homogeneity
The shelf-life of the additive (three batches) was tested at 25°C and 40°C when stored in bags protecting from light and air exchange for 6 months. 31 Losses observed at both temperatures ranged from 0% to 2% depending on the batch considered.
The stability of the additive (three batches) in a vitamin mineral premixture containing choline chloride (30,000 mg/kg) was studied when supplemented at an inclusion rate of 4%. 32 The samples were stored at room temperature in bags protecting from light and air exchange for 6 months. Losses observed ranged from 3% to 10%.
The stability of the additive (three batches) was studied in a complete feed for chickens for fattening, when supplemented at 0.2%. The basal diet consisted of wheat, maize and soybean meal. 33 Mash and pelleted feed were tested after storage at room temperature in bags protecting from light and air exchange for 3 months. Pelleting temperature was about 70°C. Pelleting the mash feeds produced loses up to 2%. After 3 months storage, only a loss of 2% was observed in one of the batches for each mash and pelleted feed.
One of the pelleted feeds for chicken for fattening described above was used to study the capacity of the additive to distribute homogeneously in feed. 34 Total tryptophan was analysed in 10 subsamples. The coefficient of variation was 2%.

Conditions of use
L-Tryptophan is intended to be used in feeds to achieve an adequate amino acid profile and to meet the L-tryptophan requirements for all animal species. It can be added directly to complete/complementary feedingstuffs or via premixtures. No inclusion levels have been proposed as the requirements, in quantitative terms, depend on the species, the physiological state of the animal, the performance level, the environmental conditions and the amino acid composition of the un-supplemented diet.

Safety of the production strain
The genetic modifications performed to obtain the production strain KCCM 80210 have the purpose to increase the production of L-tryptophan. None of the introduced modifications raise a safety concern. The production strain is free of all antibiotic resistance genes used during the genetic modification process. The production strain and its DNA were not detected in the final additive. The final product does not give raise to any safety concern with regard to the genetic modification of the production strain.

Safety for the target species, consumers and the environment
The L-tryptophan requirements of the target animal species and the safety of this essential amino acid in non-ruminant and ruminant nutrition were summarised in previous opinions of the EFSA FEEDAP Panel (2013, 2015a. The additive is highly purified (> 97.4% tryptophan and about 2% unidentified material on a dry matter basis) and is produced by fermentation using a strain that is considered safe. Concerns on the use of the additive would not derive from the L-tryptophan, which is considered safe but may arise from residues of the fermentation process/production strain remaining in the final product.
The endotoxin activity was up to 5,180 IU/g. These values are very low when compared with ca. 1,000,000 IU/g commonly found in feedingstuffs (Cort et al., 1990). Therefore, at the usual conditions of use of the additive in feed, the endotoxins added by the additive would be insignificant compared with the background in feed. Since the production strain was identified as an E. coli K12 derivative, the genetic modifications performed are considered safe, it was susceptible to antimicrobials of clinical human and veterinary relevance, and no viable cells and DNA of the production strain were found in the final product, L-tryptophan produced with E. coli KCCM 80210 is safe for non-ruminant target species when used to supplement the diet in appropriate amounts to satisfy the animal requirements.
The FEEDAP Panel reiterates that ruminal metabolism of unprotected L-tryptophan may result in the production of toxic quantities of 3-methylindole (skatole), which causes pulmonary disease (fog fever; emphysema) in cattle and goats (Hammond et al., 1979). Consequently, only a protected form of L-tryptophan should be used in ruminants (EFSA FEEDAP Panel, 2013).
The absorption and metabolic fate of L-tryptophan in the organism were described in a previous opinion (EFSA FEEDAP Panel, 2013). The amino acid L-tryptophan, supplemented to feed, will be incorporated into proteins of tissues and/or products of animal origin and any of its potential excess will be metabolised and excreted. Therefore, the composition of tissues and products of animal origin will not be affected by the use of L-tryptophan in animal nutrition. EBT and MTCA present in a specific brand of L-tryptophan produced by fermentation were implicated in the eosinophilia-myalgia syndrome outbreak that occurred in humans in New Mexico in 1989 (Hertzman et al., 1990). The concentrations of EBT were < 10 mg/kg additive and those of MTCA were 1 mg/kg and do not represent a safety concern according to the European Pharmacopoeia 9th edition (2017) that established a maximum permitted content of EBT (impurity A) and the sum of all other impurities (B-L, including MTCA) in Ltryptophan as 10 mg/kg and 390 mg/kg, respectively.
The amino acid L-tryptophan is a physiological and natural component of animals and plants. When given to animals, it is not excreted as such, but as urea/uric acid, indole-related compounds and carbon dioxide. The use of the product L-tryptophan in animal nutrition would not lead to any localised increase in the concentration in the environment. The use of L-tryptophan produced by E. coli KCCM 80210 as a feed additive does not represent a risk to the environment.

Conclusions on the safety for the target species, consumers and the environment
The use of L-tryptophan produced using E. coli KCCM 80210 to supplement feed to compensate for tryptophan deficiency in feedingstuffs is safe for non-ruminant species. There may be a risk for an increased production of toxic metabolites when unprotected tryptophan is used in ruminants.
The use of L-tryptophan produced by fermentation using E. coli KCCM 80210 in animal nutrition is considered safe for the consumers and for the environment.

Safety for the user
The applicant provided an in vivo acute inhalation toxicity study, a dermal irritation study, an eye irritation study and a dermal sensitisation study testing the additive under assessment.

Effects in the respiratory system
The highest measured dusting potential of the additive under assessment was 0.8 g/m 3 and the fraction of particles having a diameter < 52 lm ranged from 64% to 74% (see Section 3.1.3.2). Hence, the users can be exposed to the additive by inhalation.
In an acute inhalation toxicity study in rats performed in accordance with OECD Guideline 403 and consistent with good laboratory practices (GLP), the additive showed an inhalation median lethal concentration (LC 50 ) greater than 5.13 mg/L air and the test item required no classification. 35 Users can suffer from occupational respiratory disease depending on the level of endotoxins in air and dust (Rylander, 1999;Thorn, 2001). The bacterial endotoxin activity (analysed in three batches) ranged from 1,720 to 5,180 IU/g. The scenario used to estimate the exposure of persons handling the additive to endotoxins in the dust, based on the EFSA Guidance on user safety (EFSA FEEDAP Panel, 2012, 2014b) is described in Appendix A. The health-based recommended threshold for the quantity of inhaled endotoxins per working day is 900 IU, derived from provisional occupational exposure limits given by the Dutch Expert Committee on Occupational Safety (DECOS) (HCN, 2010) and the UK Health and Safety Executive (HSE, 2013). Based upon the calculation of the potential endotoxin content in dust, the inhalation exposure is calculated as 2,300 endotoxin IU per working day, indicating that inhalation exposure to endotoxins for persons handling the additive is above the recommended threshold.

Effects on skin and eyes
The skin irritation potential of the additive was tested in a valid study in rabbits performed according to OECD guideline 404 and consistent with GLP, which showed that it is not a skin irritant and has no corrosive effect on skin. 36 The eye irritation potential of the additive was tested in a valid study in rabbits performed according to OECD guideline 405 and consistent with GLP, which showed that according to the results obtained and under the assayed experimental conditions, the test item was classified as mild irritant to eyes. 37 In a valid skin sensitisation study following local lymph node assay (LLNA) performed in mice according OECD guideline 429 and compliant with GLP, it was found that the additive does not have to be classified as a skin sensitiser. 38 3.2.3.3. Conclusions on safety for the user L-Tryptophan produced using E. coli KCCM 80210 is not a skin irritant and it is not a skin sensitiser but is mild irritant to eyes. The endotoxin activity of the additive and its dusting potential indicate a risk by inhalation for the users.

Efficacy
Efficacy studies are not required for amino acids naturally occurring in the proteins of plants and animals. The nutritional role of the amino acid L-tryptophan is well established in the scientific literature. The additive feed grade L-tryptophan is regarded as an effective source of the amino acid Ltryptophan.
The efficacy of this essential amino acid in non-ruminant and ruminant nutrition was summarised in a previous opinion of the EFSA FEEDAP Panel (2014b). The FEEDAP Panel reiterates that, if the product L-tryptophan is used in ruminants, it should be protected from ruminal degradation.
Overdosing of supplemental L-tryptophan may increase skatole and indole in the hind gut resulting in boar taint of pork (Zamaratskaia and Squires, 2008).

3.4.
Post-market monitoring The FEEDAP Panel considers that there is no need for specific requirements for a post-market monitoring plan other than those established in the Feed Hygiene Regulation 39 and Good Manufacturing Practice.

Conclusions
The Panel notes that two out of five batches of the additive do not comply with the minimum specification of 98% L-tryptophan on a dry matter basis as proposed by the applicant.
The use of L-tryptophan (≥ 98%) produced using E. coli KCCM 80210 to supplement feed to compensate for L-tryptophan deficiency in feedingstuffs is safe for non-ruminant species. There may be a risk for an increased production of toxic metabolites when unprotected L-tryptophan is used in ruminants.
The use of L-tryptophan produced by fermentation using E. coli KCCM 80210 in animal nutrition is considered safe for the consumers and for the environment.
L-Tryptophan produced using E. coli KCCM 80210 is not a skin irritant and it is not a skin sensitiser but is mild irritant to eyes. The endotoxin activity of the additive and its dusting potential indicate a risk by inhalation for the users.
The product L-tryptophan produced by fermentation using E. coli KCCM 80210 is regarded as an effective source of the amino acid L-tryptophan for all non-ruminant species. In order to be as efficacious in ruminants as in non-ruminants, it should be protected from ruminal degradation.

Recommendation(s)
The FEEDAP Panel recommends that the specification of the additive includes limits for Ltryptophan-related impurities in compliance with the European Pharmacopeia.

6.
Documentation as provided to EFSA/Chronology