Abstract
Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of the feed additive consisting of l‐arginine produced by fermentation with a genetically modified strain of Escherichia coli (CGMCC 7.401). The additive is intended to be used in feed and water for drinking for all animal species and categories. The genetic modifications introduced do not raise safety concerns. No viable cells or DNA of the production strain were detected in the final product. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive does not pose any safety concern with regard to the production strain. l‐Arginine produced with E. coli CGMCC 7.401 is safe for the target species when supplemented in appropriate amounts to the diet according to the nutritional needs of the target species. The FEEDAP Panel has concerns on the use of amino acids in water for drinking for hygienic reasons, and due to the risk of imbalances when administered simultaneously via feed and water. The use of l‐arginine produced with E. coli CGMCC 7.401 in animal nutrition is considered safe for the consumers and for the environment. The endotoxin activity of the additive does not represent a hazard for persons handling the additive. In the absence of data, the FEEDAP Panel cannot conclude on the potential of the additive to be irritant to skin and/or eyes, or to be a dermal or respiratory sensitiser. The additive l‐arginine produced with E. coli CGMCC 7.401 is regarded as an efficacious source of the essential amino acid l‐arginine for non‐ruminant species. For supplemental l‐arginine to be as efficacious in ruminants as in non‐ruminant species, it requires protection against degradation in the rumen.
Keywords: amino acid, efficacy, Escherichia coli CGMCC 7.401, l‐arginine, nutritional additive, safety
1. INTRODUCTION
1.1. Background and Terms of Reference
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 feed additive shall submit an application in accordance with Article 7.
The European Commission received a request from Eppen Europa SAS 2 for the authorisation of the additive consisting of L‐arginine produced by fermentation with Escherichia coli CGMCC 7.401, when used as a feed additive for target 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 dossier was received on 6 December 2023 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2023‐00868. The particulars and documents in support of the application were considered valid by EFSA as of 26 February 2024.
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‐arginine produced by fermentation with Escherichia coli CGMCC 7.401, when used under the proposed conditions of use (see Section 3.1.6).
1.2. Additional information
The l‐arginine produced by fermentation with E. coli CGMCC 7.401 has not been previously authorised as a feed additive in the European Union.
2. DATA AND METHODOLOGIES
2.1. Data
The present assessment is based on data submitted by the applicant in the form of a technical dossier 3 in support of the authorisation request for the use of l‐arginine produced by fermentation with E. coli CGMCC 7.401 as a feed additive.
The confidential version of the technical dossier was subject to a target consultation of the interested Member States from 27 February 2024 to 27 May 2024; the comments received were considered for the assessment.
In accordance with Article 38 of the Regulation (EC) No 178/2002 4 and taking into account the protection of confidential information and of personal data in accordance with Articles 39 to 39e of the same Regulation, and of the Decision of EFSA's Executive Director laying down practical arrangements concerning transparency and confidentiality, 5 a non‐confidential version of the dossier has been published on Open.EFSA.
According to Article 32c(2) of Regulation (EC) No 178/2002 and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 22 May to 12 June 2024 for which no comments were received.
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, 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 l‐arginine produced by fermentation with E. coli CGMCC 7.401 in animal feed. 6
2.2. Methodologies
The approach followed by the FEEDAP Panel to assess the safety and the efficacy l‐arginine produced by fermentation with E. coli CGMCC 7.401 is in line with the principles laid down in Regulation (EC) No 429/2008 7 and the relevant guidance documents: Guidance on the assessment of the safety of feed additives for the consumer (EFSA FEEDAP Panel, 2017a), Guidance on the identity, characterisation and conditions of use of feed additives (EFSA FEEDAP Panel, 2017b), Guidance on the assessment of the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017c), Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2018a), Guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018b), Guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019), EFSA statement on the requirements for whole genome sequence analysis of microorganisms intentionally used in the food chain (EFSA, 2024) and the Guidance on the assessment of the safety of feed additives for the users (EFSA FEEDAP Panel, 2023).
3. ASSESSMENT
The additive l‐arginine produced with E. coli CGMCC 7.401 is intended to be used as a nutritional additive (functional group: amino acids, their salts and analogues) in feed and water for drinking for all animal species.
3.1. Characterisation
3.1.1. Characterisation of the production organism
The production strain is a genetically modified derivative of E. coli K12 ■■■■■ and it is deposited in the China General Microbiological Culture Collection Centre with accession number CGMCC 7.401. 8
A bioinformatic analysis ■■■■■ of the production strain confirmed its identity as an E. coli K‐12 derivative. 9 This was based on ■■■■■. E. coli K‐12 is well characterised, its safety (non‐pathogenicity) has been documented (Gorbach, 1978) and its ineffectiveness in colonising the human gut has been reported (Smith, 1975).
■■■■■.
The production strain was tested for its susceptibility to all the antimicrobials relevant for the category Enterobacteriaceae (EFSA FEEDAP Panel, 2018b). 10 All minimum inhibitory concentration values were below the cut off values, therefore, the strain is considered susceptible to all the tested antibiotics.
The ■■■■■ production strain were interrogated for the presence of antimicrobial resistance genes ■■■■■ 11 ■■■■■, it can be concluded that no genes of concern were identified.
The ■■■■■ production strain were also interrogated for the presence of toxin and virulence determinant genes ■■■■■. 12 ■■■■■ These are considered of no concern.
Information related to the genetically modified microorganism 13
Description of the genetic modification
The parental strain is ■■■■■.
■■■■■
■■■■■
■■■■■
■■■■■
■■■■■
■■■■■.
3.1.2. Manufacturing process
l‐Arginine is produced by fermentation with E. coli CGMCC 7.401. ■■■■■. 14
■■■■■. 15
3.1.3. Characterisation of the active substance/additive
l‐Arginine (International Union of Pure and Applied Chemistry (IUPAC)) name: (S)‐2‐amino‐5‐guanidinopentanoic acid; synonym 2‐amino‐5‐guanidinovaleric acid, is identified with the Chemical Abstracts Service (CAS) No 74‐79‐3 and the European Inventory of Existing Commercial chemical Substances (EINECS) No 200‐811‐1. It has a molecular mass of 174.2 Da. The molecular formula of l‐arginine is C6H14N4O2. The structural formula is given in Figure 1.
FIGURE 1.
Structural formula of l‐arginine.
The specifications of the feed additive are minimum 98.5% l‐arginine on a dry matter (DM) basis, and maximum 1% water. Analytical data to confirm the specifications were provided for five batches of the additive, showing an average value of 100% arginine (99.8%–100.4%) on a DM basis. 16 Water was on average 0.4% (0.3%–0.5%).
The specific optical rotation measured in five batches of the additive ranged from +26.7 to +27.2° which falls within the reference range (+25.5 to +28.5°) set in the European Pharmacopoeia (2023) and confirms the L‐enantiomer of arginine as the additive. 17
Three batches of the additive were analysed for impurities. Cadmium lead, mercury and arsenic concentrations were below the Limit of quantification (LOQ) of the analytical methods. 18 Polychlorinated dibenzo‐p‐dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin‐like polychlorinated biphenyls (DL‐PCBs) were analysed in these three batches. All values were below the corresponding LOQ. The calculated upper bound (UB) concentration was 0.14 ng WHO‐TEQ/kg for the sum of PCDD/Fs, and 0.27 ng WHO‐TEQ/kg the sum of PCDD/Fs and DL‐PCBs. The UB for the sum of non‐DL‐PCBs was 3 μg/kg. 19 Regarding the mycotoxin content, the analysis of the same batches showed that the levels of zearalenone, ochratoxin A and fumonisins B1 + B2 + B3 were below the limit of detection (LOD) of the analytical methods. Concentrations of aflatoxins (unspecified) ranged from 4 to 5.7 μg/kg; concentrations of deoxynivalenol ranged from 272 to 308 μg/kg and concentrations of citrinin ranged from 27 to 419 μg/kg. 20
The FEEDAP Panel considers that the amounts of the detected impurities do not raise safety concerns.
The same three batches of the product were analysed for microbiological contamination: Salmonella spp., yeasts and moulds, Enterobacteriaceae and E. coli were not detected in 25 g. 21
The endotoxin activity was measured in three batches of the additive and all results were <0.03 IU/mg (LOQ of the analytical method). 22
The presence of viable cells of the production strain was investigated in three batches of the additive■■■■■. 23 ■■■■■. No viable cells of the production strain were recovered from any of the samples.
The presence of DNA from the production strain was tested in three batches of the additive ■■■■■. 24 ■■■■■. No DNA of the production strain was detected.
3.1.4. Physical properties of the additive
The additive appears as a solid white crystalline powder. The reported density is of 1460 kg/m3 and its solubility in water at 20°C is of 149 g/L. 25
The dusting potential of three batches of the additive was determined using the Stauber‐Heubach method and showed values ranging 0.4–0.9 g/m3. 26
3.1.5. Stability and homogeneity
The shelf life of the additive (three batches) was studied when stored at room temperature in closed plastic bags and protected from light for 6 months. Losses at the end of the storage period ranged 4%–10%. 27
The stability of the additive (three batches) in a premixture for chickens for fattening was studied when supplemented at 4% and stored at room temperature in sealed bags for 6 months. Losses at the end of the storage period ranged 17%–33%. 28
The stability of the additive (three batches) in a compound feed for pigs for fattening (mash and pelleted forms, consisting of wheat, soybean meal and maize) was studied when supplemented at 0.2% and stored at room temperature in paper bags protected from light for 3 months. Pelleting was performed at 76°C and after the pelleting process losses of arginine content ranged 10%–16%. Losses at the end of the storage period ranged 0%–3% in mash feed and no additional losses were observed in any of the batches of pelleted feed. 29
The stability of the additive (three batches) in water for drinking was studied when supplemented at 2%. Samples were stored at 20°C for 48 h. Losses at the end of the storage period were of 5% in all three batches. 30
The capacity for homogeneous distribution of the additive in the pelleted compound feed for pigs for fattening described above was studied in 10 subsamples. The background concentration (protein bound arginine) of valine in the compound feed was 1.3%. When total arginine was analysed, the coefficient of variation (CV) was 6%. When the background concentration of arginine was subtracted from each subsample, the CV was 56%. 31
3.1.6. Conditions of use
l‐Arginine is intended to be used in feed and water to achieve an adequate amino acid profile and to meet the l‐arginine requirements for all animal species. It can be added directly to complete feed, through complementary feed, premixtures and water. No inclusion levels have been proposed by the applicant, 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.
3.2. Safety
3.2.1. Safety of the production organism
The parental strain ■■■■■ is considered to be safe. The genetic modifications to obtain the production strain E. coli CGMCC 7.401 have the purpose of increasing the production of l‐arginine. None of the genetic modifications raise a safety concern and the production strain does not carry acquired antimicrobial resistance genes. The production strain and its DNA were not detected in the final additive. The final product does not raise any safety concern regarding to the genetic modification of the production strain.
3.2.2. Safety for the target species, consumers and the environment
The metabolism of l‐arginine, the lysine‐arginine antagonism, the arginine requirements for the different species/categories and the effects of supplementation of arginine in excess to the diets have already been reviewed in former FEEDAP Panel's opinions (EFSA, 2007; EFSA FEEDAP Panel, 2016).
The additive is highly purified (contains >98.5% l‐arginine and <1% unidentified material on DM basis) and the production strain and the manufacturing process do not raise safety concerns.
The FEEDAP Panel considers that the use of the amino acid ‘per se’ will not raise safety concerns for the target animals provided it is supplemented in appropriate amounts with respect to the nutritional needs of the target species to the diets. The endotoxin activity was < 0.30 EU/mg. These values are compared with ca. 1000 EU/mg 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 negligible compared with the background in feed. Safety concerns from the additive could derive from the residues of the fermentation process/production strain remaining in the final product. As the production strain and the manufacturing process do not raise safety concerns (see Section 3.2.1), l‐arginine produced by fermentation with E. coli CGMCC 7.401 is safe for the target species when used to supplement the diet in appropriate amounts to satisfy the animal requirements. Regarding the use in water, the FEEDAP Panel reiterates its concerns over the safety for the target species of l‐arginine administered simultaneously via water for drinking and feed owing to the risk of nutritional imbalances and hygienic reasons (EFSA FEEDAP Panel, 2010).
The amino acid l‐arginine will be incorporated into proteins of tissues and/or products of animal origin and any of its potential excess will be metabolised and excreted as urea/uric acid and carbon dioxide. Therefore, the composition of tissues and products of animal origin will not be affected by the use of l‐arginine in animal nutrition, and its use would be of no concern for the consumers of animal tissues/products of animals fed with the additive.
The amino acid l‐arginine is a physiological and natural component of the proteins of living organisms. When consumed by the animals, it will be absorbed and the non‐absorbed fraction will be incorporated into the intestinal microbial biomass or excreted as such. The use of amino acids in water for drinking, when given in addition to complete diets with a well‐balanced amino acid profile, would disturb the nitrogen balance and increase nitrogen excretion via urine. The use of the additive in animal nutrition would not lead to any localised increase in the concentration of L‐arginine or its metabolites in the environment.
3.2.3. Safety for the user
No studies were submitted to support the safety of the additive for the user.
The additive under assessment is a powder, and the highest dusting potential measured was 0.9 g/m3. Therefore, exposure of users by inhalation is possible.
Users can suffer from occupational respiratory disease depending on the level of endotoxins in air and dust (Rylander et al., 1999; Thorn & Kerekes, 2001). The endotoxin activity in the additive was determined as < 30 IU/g. The exposure of persons handling the additive to endotoxins in the dust was calculated as previously described (EFSA FEEDAP Panel, 2018c) taking the highest endotoxin value measured (in this case considering 29 IU/g) as a worst‐case scenario. 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) (Health Council of the Netherlands, 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 15 endotoxin IU per working day, indicating no risk of exceeding the recommended limit of exposure by inhalation to endotoxins for persons handling the additive.
In absence of data the FEEDAP Panel cannot conclude on the potential of the additive to be irritant to skin and/or eyes and on its potential to be a dermal or respiratory sensitiser. The endotoxin activity of the additive does not represent a risk when users are exposed via inhalation.
3.3. Efficacy
Efficacy studies are not required for amino acids that occur naturally in plant and animal proteins. The nutritional role of the amino acid l‐arginine is well established in the scientific literature.
For the supplemental l‐arginine to be as efficacious in ruminants as in non‐ruminant species, it requires protection against degradation in the rumen.
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 32 and Good Manufacturing Practice.
4. CONCLUSIONS
The production strain E. coli CGMCC 7.401 is genetically modified but raises no safety concerns. No viable cells or DNA of the production strain were detected in the final product. The FEEDAP Panel concludes that the additive does not pose any safety concern with regard to the production strain.
l‐Arginine produced by fermentation with E. coli CGMCC 7.401 is safe for the target species when supplemented in appropriate amounts to the diet according to the nutritional needs of the target species. The FEEDAP Panel has concerns on the use of amino acids in water for drinking for hygienic reasons and due to the risk of imbalances when administered simultaneously via feed and water.
The use of l‐arginine produced by fermentation with E. coli CGMCC 7.401 in animal nutrition is considered safe for the consumers and for the environment.
Regarding the user safety, in the absence of data, the FEEDAP Panel cannot conclude on the potential of the additive to be irritant to skin and/or eyes, or to be a dermal or respiratory sensitiser.
The additive l‐arginine produced by fermentation with E. coli CGMCC 7.401 is regarded as an efficacious source of the essential amino acid l‐arginine for non‐ruminant nutrition. For the supplemental l‐arginine to be as efficacious in ruminants as in non‐ruminant species, it requires protection against degradation in the rumen.
ABBREVIATIONS
- ANI
average nucleotide identity
- CAS
Chemical Abstracts Service
- CFU
colony forming unit
- CV
coefficient of variation
- DM
dry matter
- DL‐PCBs
dioxin‐like polychlorinated biphenyls
- EINECS
European Inventory of Existing Chemical Substances
- EURL
European Union Reference Laboratory
- FEEDAP
EFSA Scientific Panel on Additives and Products or Substances used in Animal Feed
- IUPAC
International Union of Pure and Applied Chemistry
- LOD
limit of detection
- LOQ
limit of quantification
- MIC
minimum inhibitory concentration
- OECD
Organisation for Economic Co‐operation and Development
- PCDDs
Polychlorinated dibenzo‐p‐dioxins
- PCDFs
polychlorinated dibenzofurans
- WGS
whole genome sequence
CONFLICT OF INTEREST
If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact interestmanagement@efsa.europa.eu.
REQUESTOR
European Commission
QUESTION NUMBER
EFSA‐Q‐2023‐00868
COPYRIGHT FOR NON‐EFSA CONTENT
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PANEL MEMBERS
Roberto Edoardo Villa, Giovanna Azimonti, Eleftherios Bonos, Henrik Christensen, Mojca Durjava, Birgit Dusemund, Ronette Gehring, Boet Glandorf, Maryline Kouba, Marta López‐Alonso, Francesca Marcon, Carlo Nebbia, Alena Pechová, Miguel Prieto‐Maradona, Ilen Röhe and Katerina Theodoridou
LEGAL NOTICE
Relevant information or parts of this scientific output have been blackened in accordance with the confidentiality requests formulated by the applicant pending a decision thereon by EFSA. The full output has been shared with the European Commission, EU Member States (if applicable) and the applicant. The blackening may be subject to review once the decision on the confidentiality requests is adopted by EFSA and in case it rejects some of the confidentiality requests.
ACKNOWLEDGEMENTS
The Panel wishes to thank the following for the support provided to this scientific output (in alphabetical order of the last name): Montserrat Anguita, Jaume Galobart, Matteo Innocenti, the members of the working group of Microbiology of the FEEDAP Panel.
EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed) , Villa, R. E. , Azimonti, G. , Bonos, E. , Christensen, H. , Durjava, M. , Dusemund, B. , Gehring, R. , Glandorf, B. , Kouba, M. , López‐Alonso, M. , Marcon, F. , Nebbia, C. , Pechová, A. , Prieto‐Maradona, M. , Röhe, I. , Theodoridou, K. , Cionci, N. B. , Pettenati, E. , & Tarrés‐Call, J. (2024). Safety and efficacy of a feed additive consisting of l‐arginine produced with Escherichia coli CGMCC 7.401 for all animal species (Eppen Europe SAS). EFSA Journal, 22 (10), e9028. 10.2903/j.efsa.2024.9028
Adopted: 18 September 2024
Notes
Regulation (EC) No 1831/2003 of the European Parliament and of the council of 22 September 2003 on the additives for use in animal nutrition. OJ L 268, 18.10.2003, p. 29.
Eppen Europe SAS. Rue de la Paix 10, Paris, France.
Dossier reference: FEED‐2023‐20002.
Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. OJ L 31, 1.2.2002, p. 1–48.
Decision https://www.efsa.europa.eu/en/corporate‐pubs/transparency‐regulation‐practical‐arrangements.
Evaluation report received on 24/5/2024 and available on the EU Science Hub https://joint‐research‐centre.ec.europa.eu/eurl‐fa‐eurl‐feed‐additives/eurl‐fa‐authorisation/eurl‐fa‐evaluation‐reports_en.
Commission Regulation (EC) No 429/2008 of 25 April 2008 on detailed rules for the implementation of Regulation (EC) No 1831/2003 of the European Parliament and of the Council as regards the preparation and the presentation of applications and the assessment and the authorisation of feed additives. OJ L 133, 22.5.2008, p. 1.
2.2.1.2.a Certificate of Deposition CGMCC 7.401.
2.2.1.2.c Bioinformatic report T1436R1356.
2.2.2.2 MIC T1436R1357.
2.2.1.2.c Bioinformatic report T1436R1356.
2.2.1.2.c Bioinformatic report T1436R1356.
22.2.1.2.c Bioinformatic report T1436R1356 and 2.2.1.2.b Genetic modifications production strain.
2.3.1.a v2 Production flow chart & medium.
2.3.1.b Statement of non‐use of antibiotics.
Annex 2.1.3 BtB impurity stability 132232 and Other supporting document – 2.1.3.a Method 1522009. Analytical method was stated to be in accordance with EU Regulation 152/2009, Appendix III, G and F method.
Annex 2.1.5.b Optical rotation.
Annex 2.1.3 BtB impurity stability 132232. LOQ in mg/kg were 0.002 for cadmium and mercury, and 0.01 for arsenic and lead.
Annex 2.1.4.a Dioxins and PCBs. Upper bound concentrations are calculated on the assumption that all values of the different congeners below the limit of quantification are equal to the limit of quantification. TEQ = toxic equivalency factors for PCDD/Fs and DL‐PCBs established by WHO in 2005 (van den Berg et al., 2006).
Annex 2.1.3 BtB impurity stability 132232. LOD in μg/kg were 2.8 for ochratoxin A, 17 for zearalenone and 25 for fumonisins B1 + B2 + B3.
Annex 2.1.3 BtB impurity stability 132232. LOD were a single colony forming unit (CFU)/25‐g samples.
Annex 2.1.4.b LAL endotoxins. Endotoxins were determined by the analytical method A of the European Pharmacopoeia.
2.1.4.c Absence viable cells T1436R1368.1_2022.
2.1.4.d Absence DNA T1436R1376.2_2022.
Other supporting document – 2.1.5.c Density MSDS – arginine.
Annex 2.1.5.a Dust v2.
Annex 2.1.3 BtB impurity stability 132232.
Annex 2.4.1.b Stability feed homogeneity.
Annex 2.4.1.b Stability feed homogeneity.
Annex 2.1.3 BtB impurity stability 132232.
Annex 2.4.1.b Stability feed homogeneity.
Regulation (EC) No 183/2005 of the European Parliament and of the Council of 12 January 2005 laying down requirements for feed hygiene. OJ L 35, 8.2.2005, p. 1.
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