Safety and efficacy of astaxanthin‐dimethyldisuccinate (Carophyll® Stay‐Pink 10%‐CWS) for salmonids, crustaceans and other fish
2019; Wiley; Volume: 17; Issue: 12 Linguagem: Inglês
10.2903/j.efsa.2019.5920
ISSN1831-4732
AutoresVasileios Bampidis, Giovanna Azimonti, Maria de Lourdes Bastos, Henrik Christensen, Birgit Dusemund, Maryline Kouba, Mojca Fašmon Durjava, Marta López‐Alonso, Secundino López Puente, Francesca Marcon, Baltasar Mayo, Alena Pechová, Mariana Petkova, Fernando Ramos, Yolanda Sanz, Roberto Edoardo Villa, Ruud Woutersen, Georges Bories, Paul Brantom, Derek Renshaw, Josef Schlatter, Reinhard Ackerl, Orsolya Holczknecht, Hans Steinkellner, Maria Vittoria Vettori, Jürgen Gropp,
Tópico(s)Agricultural safety and regulations
ResumoEFSA JournalVolume 17, Issue 12 e05920 Scientific OpinionOpen Access Safety and efficacy of astaxanthin-dimethyldisuccinate (Carophyll® Stay-Pink 10%-CWS) for salmonids, crustaceans and other fish EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), Corresponding Author EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) feedap@efsa.europa.eu Correspondence: feedap@efsa.europa.euSearch for more papers by this authorVasileios Bampidis, Vasileios BampidisSearch for more papers by this authorGiovanna Azimonti, Giovanna AzimontiSearch for more papers by this authorMaria de Lourdes Bastos, Maria de Lourdes BastosSearch for more papers by this authorHenrik Christensen, Henrik ChristensenSearch for more papers by this authorBirgit Dusemund, Birgit DusemundSearch for more papers by this authorMaryline Kouba, Maryline KoubaSearch for more papers by this authorMojca Kos Durjava, Mojca Kos DurjavaSearch for more papers by this authorMarta López-Alonso, Marta López-AlonsoSearch for more papers by this authorSecundino López Puente, Secundino López PuenteSearch for more papers by this authorFrancesca Marcon, Francesca MarconSearch for more papers by this authorBaltasar Mayo, Baltasar MayoSearch for more papers by this authorAlena Pechová, Alena PechováSearch for more papers by this authorMariana Petkova, Mariana PetkovaSearch for more papers by this authorFernando Ramos, Fernando RamosSearch for more papers by this authorYolanda Sanz, Yolanda SanzSearch for more papers by this authorRoberto Edoardo Villa, Roberto Edoardo VillaSearch for more papers by this authorRuud Woutersen, Ruud WoutersenSearch for more papers by this authorGeorges Bories, Georges BoriesSearch for more papers by this authorPaul Brantom, Paul BrantomSearch for more papers by this authorDerek Renshaw, Derek RenshawSearch for more papers by this authorJosef Rudolf Schlatter, Josef Rudolf SchlatterSearch for more papers by this authorReinhard Ackerl, Reinhard AckerlSearch for more papers by this authorOrsolya Holczknecht, Orsolya HolczknechtSearch for more papers by this authorHans Steinkellner, Hans SteinkellnerSearch for more papers by this authorMaria Vittoria Vettori, Maria Vittoria VettoriSearch for more papers by this authorJürgen Gropp, Jürgen GroppSearch for more papers by this author EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), Corresponding Author EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) feedap@efsa.europa.eu Correspondence: feedap@efsa.europa.euSearch for more papers by this authorVasileios Bampidis, Vasileios BampidisSearch for more papers by this authorGiovanna Azimonti, Giovanna AzimontiSearch for more papers by this authorMaria de Lourdes Bastos, Maria de Lourdes BastosSearch for more papers by this authorHenrik Christensen, Henrik ChristensenSearch for more papers by this authorBirgit Dusemund, Birgit DusemundSearch for more papers by this authorMaryline Kouba, Maryline KoubaSearch for more papers by this authorMojca Kos Durjava, Mojca Kos DurjavaSearch for more papers by this authorMarta López-Alonso, Marta López-AlonsoSearch for more papers by this authorSecundino López Puente, Secundino López PuenteSearch for more papers by this authorFrancesca Marcon, Francesca MarconSearch for more papers by this authorBaltasar Mayo, Baltasar MayoSearch for more papers by this authorAlena Pechová, Alena PechováSearch for more papers by this authorMariana Petkova, Mariana PetkovaSearch for more papers by this authorFernando Ramos, Fernando RamosSearch for more papers by this authorYolanda Sanz, Yolanda SanzSearch for more papers by this authorRoberto Edoardo Villa, Roberto Edoardo VillaSearch for more papers by this authorRuud Woutersen, Ruud WoutersenSearch for more papers by this authorGeorges Bories, Georges BoriesSearch for more papers by this authorPaul Brantom, Paul BrantomSearch for more papers by this authorDerek Renshaw, Derek RenshawSearch for more papers by this authorJosef Rudolf Schlatter, Josef Rudolf SchlatterSearch for more papers by this authorReinhard Ackerl, Reinhard AckerlSearch for more papers by this authorOrsolya Holczknecht, Orsolya HolczknechtSearch for more papers by this authorHans Steinkellner, Hans SteinkellnerSearch for more papers by this authorMaria Vittoria Vettori, Maria Vittoria VettoriSearch for more papers by this authorJürgen Gropp, Jürgen GroppSearch for more papers by this author First published: 18 December 2019 https://doi.org/10.2903/j.efsa.2019.5920Citations: 4 Requestor: European Commission Question number: EFSA-Q-2017-00499 Panel members: Giovanna Azimonti, Vasileios Bampidis, Maria de Lourdes Bastos, Henrik Christensen, Birgit Dusemund, Maryline Kouba, Mojca Kos Durjava, Marta López-Alonso, Secundino López Puente, Francesca Marcon, Baltasar Mayo, Alena Pechová, Mariana Petkova, Fernando Ramos, Yolanda Sanz, Roberto Edoardo Villa and Ruud Woutersen. Legal notice: Relevant information or parts of this scientific output have been blackened in accordance with the European Commission decision on the confidentiality requests formulated by the applicant. A previous, provisional version of this output which had been made publicly available pending the adoption of the decision has been replaced by this version. The full output has been shared with the European Commission, EU Member States and the applicant. Acknowledgements: The Panel wishes to thank Bruno Dujardin, José Cortiñas Abrahantes, Jaume Galobart, Wolfgang Gelbmann and Paola Manini for the support provided to this scientific output. Adopted: 13 November 2019 Amended: 3 December 2021 AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat 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 astaxanthin-dimethyldisuccinate (ATX-DMDS) for salmonids, crustaceans and other fish. The applicant has provided evidence that ATX-DMDS currently on the market complies with the conditions of authorisation for salmon and trout. ATX and ATX-DMDS are safe for salmonids, crustaceans and fish up to 100 mg ATX/kg complete diet, corresponding to 138 mg ATX-DMDS/kg. The FEEDAP Panel re-assessed the toxicological profile of ATX based on data already considered in 2014, the literature review performed by the applicant and the data available in the context of an EFSA public call for data on ATX. The acceptable daily intake (ADI) of 0.2 mg astaxanthin/kg body weight (bw) per day obtained by applying an uncertainty factor of 200 to a lowest observed adverse effect level (LOAEL) of 40 mg/kg bw per day for the increased incidence of multinucleated hepatocytes observed in a 2-year carcinogenicity study replaces the one of 0.034 mg/kg bw established by the FEEDAP Panel in 2014. The use of ATX-DMDS in the nutrition of salmonids, other fish and crustaceans up to the maximum permitted dietary level is of no concern for the safety of the consumer. No dermal or ocular risk for the users is likely to occur under practical conditions. In the absence of inhalation toxicology study, the Panel is not in the position to establish the inhalation toxicity of the additive. The use of synthetic ATX-DMDS does not pose a significant additional risk to the environment compared with natural astaxanthin. ATX-DMDS is efficacious in colouring the flesh of salmonids and other fish. ATX-DMDS is an effective pigment for crustaceans at the proposed conditions of use. Summary 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 astaxanthin-dimethyldisuccinate (ATX-DMDS, Carophyll® Stay-Pink 10%-CWS) for salmonids, crustaceans and other fish. The applicant has provided evidence that ATX-DMDS currently on the market complies with the conditions of authorisation for salmon and trout. Orally administrated ATX-DMDS is hydrolysed and converted to free ATX in the intestine of fish, then absorbed, metabolised and distributed in the same manner as free ATX. ATX and ATX-DMDS are safe for salmonids up to 100 mg ATX/kg complete diet corresponding to 138 mg ATX-DMDS/kg. This conclusion is extrapolated to other fish. ATX at a maximum concentration of 100 mg/kg complete feed is safe for crustaceans with a margin of safety of at least 8. This conclusion covers also ATX from ATX-DMDS and therefore the safety of 138 mg ATX-DMDS/kg complete feed. The FEEDAP Panel has no reservation to the deletion of the provision that limits the age of use of ATX/ATX-DMDS in salmon and trout. The FEEDAP Panel re-assessed the toxicological profile of ATX based on data already considered in 2014, the literature review performed by the applicant and the data available in the context of an EFSA public call for data on ATX. ATX is neither mutagenic nor carcinogenic. The FEEDAP Panel established an acceptable daily intake (ADI) of 0.2 mg astaxanthin/kg body weight (bw) per day by applying an uncertainty factor of 200 to a lowest observed adverse effect level (LOAEL) of 40 mg/kg bw per day for the increased incidence of multinucleated hepatocytes observed in a 2-year carcinogenicity study. This ADI replaces the one of 0.034 mg/kg bw established by the FEEDAP Panel in 2014. The use of ATX-DMDS in the nutrition of salmonids, other fish and crustaceans up to the maximum permitted dietary level is of no concern for the safety of the consumer. The FEEDAP Panel concludes that there is no new evidence that would lead the Panel to reconsider its previous conclusions on the safety for the user. No dermal or ocular risk for the users is likely to occur under practical conditions. In the absence of inhalation toxicology study, the Panel is not in the position to establish the inhalation toxicity of the additive. Due to the susceptibility of the active substance to oxidation, the additive will be placed in the market only in the form of preparations. The FEEDAP Panel recognises that once authorised, multiple formulations of the additive can be placed in the market, and consequently, not all preparations can be directly tested for user safety. The use of synthetic ATX-DMDS does not pose a significant additional risk to the environment compared with natural astaxanthin. ATX-DMDS is efficacious in colouring the flesh of salmonids and other fish. ATX-DMDS is an effective pigment for crustaceans at the proposed conditions of use. 1 Introduction 1.1 Background and Terms of Reference Regulation (EC) No 1831/20031 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; Article 13(3) of that Regulation lays down that if the holder of an authorisation proposes changing the terms of the authorisation by submitting an application to the Commission, accompanied by the relevant data supporting the request for the change, the Authority shall transmit its opinion on the proposal to the Commission and the Member States; Article 14(1) of that Regulation lays down that an application for renewal shall be sent to the Commission at the latest one year before the expiry date of the authorisation. The European Commission received a request from DSM Nutritional Products2 for an authorisation of a new use, modification of the authorisation and renewal of the authorisation of the product astaxanthin-dimethyldisuccinate (Carophyll® Stay-Pink 10%-CWS), when used as a feed additive for salmonids, crustaceans and other fish (category: sensory additives; functional group: a) colourants: ii substances which, when fed to animals, add colours to food of animal origin). 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), under Article 13(3) (modification of the authorisation of a feed additive) and under Article 14(1) (renewal of the authorisation). The particulars and documents in support of the application were considered valid by EFSA as of 19 October 2017. 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 product astaxanthin-dimethyldisuccinate (Carophyll® Stay-Pink 10%-CWS), when used under the proposed conditions of use (see Section 3.1.5). 1.2 Additional information Astaxanthin-dimethyldisuccinate (ATX-DMDS), the additive under assessment, is authorised for use in salmon and trout.3 Synthetic astaxanthin (E 161j) is authorised for fish, crustaceans and ornamental fish.4 Astaxanthin from natural origin is also authorised in the European Union (EU) for salmon and trout: astaxanthin-rich Phaffia rhodozyma (ATCC SD-5340)5 and red carotenoid-rich Paracoccus carotinifaciens.6 ATX-DMDS, the additive under assessment, was first evaluated by the FEEDAP Panel in 2007 (EFSA, 2007a). Since 2004, the FEEDAP Panel issued several scientific opinions on the safety and efficacy of astaxanthin which include: the environmental impact of astaxanthin-rich Phaffia rhodozyma (ATCC 74219) (EFSA, 2004), the safety of use of astaxanthin in animal nutrition (EFSA, 2005); the safety and efficacy of astaxanthin-rich Phaffia rhodozyma (ATCC SD-5340) for salmon and trout (EFSA, 2006) and the safety and efficacy of red carotenoid-rich bacterium Paracoccus carotinifaciens as a feed additive for salmon and trout (EFSA, 2007b; EFSA FEEDAP Panel, 2010). The most recent ones deal with the re-evaluation of synthetic astaxanthin for salmonids, crustaceans, ornamental fish, other fish and ornamental birds under Regulation (EC) No 1831/2003 (EFSA FEEDAP Panel, 2014a,b). 2 Data and methodologies 2.1 Data The present assessment is based on data submitted by the applicant in the form of a technical dossier7 in support of the authorisation request for the use of ATX-DMDS (Carophyll® Stay-Pink 10%-CWS) 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, other scientific reports and experts' elicitation knowledge, 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 ATX-DMDS in animal feed. The Executive Summary of the EURL report can be found in Annex A.8 2.2 Methodologies The approach followed by the FEEDAP Panel to assess the safety and the efficacy of astaxanthin-dimethyldisuccinate (Carophyll® Stay-Pink 10%-CWS) is in line with the principles laid down in Regulation (EC) No 429/2008 and the relevant guidance documents: Guidance for the preparation of dossiers for sensory additives (EFSA FEEDAP Panel, 2012),9 Technical guidance: Tolerance and efficacy studies in target animals (EFSA FEEDAP Panel, 2011), Technical Guidance for assessing the safety of feed additives for the environment (EFSA, 2008), Guidance on the renewal of the authorisation of feed additives (EFSA FEEDAP Panel, 2013), Guidance for establishing the safety of additives for the consumer (EFSA FEEDAP Panel, 2012), Guidance on studies concerning the safety of use of the additive for users/workers (EFSA FEEDAP Panel, 2012) and Technical Guidance: Extrapolation of data from major species to minor species regarding the assessment of additives for use in animal nutrition (EFSA, 2008). The FEEDAP Panel performed the benchmark dose (BMD) analysis following the approach described in the Guidance on the use of the benchmark dose approach in risk assessment (EFSA Scientific Committee, 2017). 3 Assessment Astaxanthin-dimethyldisuccinate (Carophyll® Stay-Pink 10%-CWS), the additive under assessment, is authorised for salmon and trout as a sensory additive (functional group: colourant) at a maximum content of 138 mg ATX-DMDS/kg complete feed.3 The applicant asks for (i) the renewal of the current authorisation, (ii) the modification of the current authorisation by deleting the provision 'Use permitted from the age of 6 months onwards or from 50 g weight onwards' and (iii) the authorisation of a new use of the additive for crustaceans and other fish. 3.1 Characterisation 3.1.1 Characterisation of the active substance Astaxanthin-dimethyldisuccinate (C50H64O10, (3R,3′R)-(±)-3,3′-bis(4-methoxy-1,4-dioxobutoxy)-β,β-carotene-4,4′-dione), CAS number 578006-46-9, is derived by esterification of synthetic ATX. The applicant states that no change in the manufacturing process have been introduced since the FEEDAP Panel made its assessment in 2007 (EFSA, 2007a,b) (Figure 1). Figure 1Open in figure viewerPowerPoint Structural formula of astaxanthin-dimethyldisuccinate ATX-DMDS contains by specification not less than 96% ATX-DMDS (all-E, 9-Z and 13-Z isomers) and not more than 4% other carotenoids. Batch to batch consistency was confirmed by analysis of five batches (2015), all complying with the specifications: ■■■■■10■■■■■ 3.1.2 Characterisation of the additive ATX-DMDS is sensitive to oxidation, light and temperature; therefore, it is necessary to produce a stabilised preparation for its use in premixtures and in feedingstuffs. CAROPHYLL® Stay-Pink 10%-CWS is one example of such a preparation. It contains 138 g ATX-DMDS/kg (100 g ATX equivalent/kg), 512 g lignosulfonate, 180 g maize starch, 70 g dextrin yellow, 50 g beeswax and 50 g dl-alpha-tocopherol/kg. Certificate of analysis from three production batches (2018) showed product consistency with values of ATX equivalent content between 113 and 114 g/kg.11 The residual solvents concentrations measured in three batches of the formulated additive were ■■■■■12 These results are in compliance with the International Cooperation on Harmonisation of Technical requirements for Registration of Veterinary Medicinal Products (VICH) thresholds (VICH guidance GL18).13 Three batches of the formulated product CAROPHYLL® Stay-Pink 10%-CWS was analysed for arsenic, cadmium, lead and mercury, with all values below 0.1 mg/kg.14 Triphenylphosphine oxide (TPPO, a carry-over impurity of ATX synthesis) could not be found in five batches of the crystalline ATX-DMDS.15,10 This is in compliance with former opinions of the FEEDAP Panel (EFSA, 2007a; EFSA FEEDAP Panel, 2014a,b) and the current authorisation for ATX-DMDS with the maximum level of 100 mg TPPO/kg additive. 3.1.3 Physical state of the product Three batches of the newly formulated CAROPHYLL® Stay-Pink 10%-CWS were analysed for particle size distribution by laser diffraction and for dusting potential by Heubach test.16 The particle size distribution showed that all particles had a diameter < 600 μm and no particles < 100 μm were found. The mean dusting potential was 0.95 g/m3 ■■■■■. The measured ATX-DMDS in the dust was 9.7 g/kg on average ■■■■■, the active substance in the air is calculated to be 9.2 mg ATX-DMDS/m3. 3.1.4 Stability and homogeneity The composition of the formulated product is different from the one assessed in 2007 (EFSA, 2007a) and the applicant provided new stability and homogeneity data. 3.1.4.1 Shelf-life of the additive The stability of three commercial batches of CAROPHYLL® Stay-Pink 10%-CWS kept in aluminium bottles was tested in two different conditions: at 15°C and at 40°C/75% relative humidity (RH). AXT-DMDS concentration was measured at regular time intervals. Recoveries after 36 months at 15°C and after 6 months at 40°C/75% RH were in the range of 97.2–98.1% and 95.2–96.1, respectively.17 3.1.4.2 Stability in premixtures and feeding stuffs Three batches of CAROPHYLL® Stay-Pink 10%-CWS were incorporated into a vitamin premixture at a level of 10 g ATX-equivalent/kg premix. Samples were stored for 6 months at 25°C. The mean recoveries of ATX-DMDS were in the range of 93.9–96.7% and 92.9–95.3% after 3 and 6 months, respectively.18 The stability of CAROPHYLL® Stay-Pink 10%-CWS (three batches) was studied in an extruded feed for trout, during processing and during subsequent storage for three months at 25°C. The target content of ATX-DMDS was 40 mg ATX-equivalent/kg feed. Recovery after processing was 95% and a monthly loss of 5.4% was calculated for a 3 months storage period.18 3.1.4.3 Homogeneity The homogeneous distribution of ATX-DMDS in three batches of trout feed (mash19 and extruded) each with target concentration of 40 mg ATX-equivalent/kg feed was examined in three samples of each feed. The coefficient of variation obtained varied between 3.2–6.8% (mash feed) and between 0.7–1.5% (extruded feed).18 3.1.5 Conditions of use The additive is currently authorised as a sensory additive, (functional group: colourant) for salmon and trout up to a maximum content of 138 mg ATX-DMDS/kg complete feed (corresponding to 100 mg ATX equivalent/kg feed). The applicant proposes to keep the same conditions. In addition, the applicant is proposing the extension of use to crustaceans and other fish with the same maximum content. The modification of the current authorisation by deleting the provision 'Use permitted from the age of 6 months onwards or from 50 g weight onwards' is also requested. 3.2 Safety 3.2.1 Absorption, distribution, metabolism, excretion and residues No new information has been provided on absorption, distribution, metabolism, excretion (ADME) and reference to previously adopted opinions have been made (EFSA, 2005, 2007a; EFSA FEEDAP Panel, 2014a). The applicant provided information on residues of ATX in the new target species (crustaceans). 3.2.1.1 Absorption, distribution, metabolism and excretion The FEEDAP Panel assessed the ADME of ATX-DMDS in salmonids in its opinion in 2007 (EFSA, 2007a) and concluded that 'Orally administered ATX-DMDS from C®SP is hydrolysed and converted to free ATX in the intestine of fish, then absorbed, metabolised and distributed in the same manner as free ATX'. The ADME of free ATX has been extensively evaluated by the FEEDAP Panel in 2005 and 2014 (EFSA, 2005; EFSA FEEDAP Panel, 2014a) and can be summarised as follows: (i) ATX apparent absorption varies mostly between 50% and 70% and is determined by several factors such as target species, dietary lipid levels and ATX stereochemistry, the all-E isomer being absorbed more efficiently than the Z isomers (ii) ATX is metabolised in fish through oxidative and reductive pathways. However, no oxidation occurs in «sea bream type» of fish like salmonids. A double step reduction at the 4 and 4′-oxo groups initiates a metabolic process leading to idoxanthin then to adonixanthin and zeaxanthin. ATX has been shown to be a vitamin A precursor for fish, which implies the cleavage of the polyene chain at C15,C15 (iii) after repeated administration of ATX, the pigment deposited in the flesh of trout and Chinook salmon (Oncorhynchus tshawytscha) is predominantly ATX (about 95%); in Arctic char (Salvelinus alpinus), idoxanthin is also deposited in the flesh (20–35%), the corresponding figures for the skin being 85% ATX and 10% idoxanthin, both esterified. No information on ADME of ATX in crustaceans has been provided. In the dossier, the applicant assumes that orally administrated ATX-DMDS is hydrolysed and converted to free astaxanthin and succinate in the intestine of crustaceans before or during absorption, but no evidence on this has been provided. The literature searches performed by the applicant (see below Sections 3.2.2.1 and 3.3.2.1) did not reveal any information specific to the metabolism of ATX in crustaceans, but only data on ATX deposition. 3.2.1.2 Residues No new data for ATX residues in salmonids and other fish were supplied by the applicant, which made reference to the former FEEDAP Panel conclusions as follows (EFSA, 2005; EFSA FEEDAP Panel, 2014a): '(i) a dose-related increase in ATX in the flesh of trout and salmon was observed with graded ATX levels in the diet. Since absorption capacity is limited, a plateau is reached in Atlantic salmon at about 10 mg ATX/kg flesh and in trout at a higher level of about 20–25 mg ATX/kg flesh, (ii) the composition of carotenoids deposited in the flesh reflects that of the dietary prey organisms or added carotenoids in terms of ATX stereoisomers; all-E isomers are deposited mainly in flesh, whereas Z isomers are preferentially stored in the liver and kidney'. Two published studies provided for the present assessment (see Section 3.2.2.2) some data on ATX deposition in shrimp muscle.20 In the first one (Paibulkichakul et al., 2008), groups of shrimps were fed for 120 days a feed supplemented with 46 or 281 mg ATX/kg, respectively (analytical values). At the end of the study (number of animals and time after the last feeding not indicated), the shrimps were killed and ATX measured in the muscle. ATX contents amounted to about (evaluated from a graph) 2 mg and 4.5 mg/kg muscle, respectively. In the second study (Niu et al., 2014), shrimps were fed for 74 days a complete feed supplemented with 100 mg ATX/kg. Six shrimps were randomly collected 2 hours after the last feeding and dissected. ATX content in muscle amounted to 3.1 ± 0.04 mg/kg. 3.2.2 Safety for the target species 3.2.2.1 Safety for salmonids and other fish In 2007, the FEEDAP Panel concluded that ATX-DMDS from C®SP is safe for salmon and trout (EFSA, 2007a) at the highest authorised ATX level in feed. The applicant performed a structured literature search for the period 2013 up to April 2017 aiming to demonstrate that, in the light of the current knowledge, the additive and its active principle ATX remain safe for the target species (salmon and trout).21 For ATX-DMDS, no one of the documents retrieved by the structured literature database searches (12 hits found) was considered relevant. For ATX two published papers out of 64 hits were considered relevant (Rama and Manjabhat, 2014; Brizio et al., 2013). Concerning the publication by Brizio et al. (2013), the FEEDAP Panel noted that the dietary level of ATX (75 mg/kg feed) in rainbow trout was below the currently authorised maximum level. Consequently, this study was not considered relevant for the assessment of target animal safety of ATX. Rama and Manjabhat (2014) fed fingerling carps (Cyprinus carpio) diets with 100 and 200 mg ATX+ATX-ester/kg extracted from shrimp exoskeleton for 14 weeks. The aim of the study was to evaluate a potential protective effect of these carotenoids against ammonia induced stress. After a single sublethal dose of ammonia, total antioxidant status (TAS), activities of superoxide dismutase (SOD) and catalase (CAT) as well as aspartate transaminase (AST) and alanine transaminase (ALT) were measured in plasma, liver, kidney and gills. TAS in tissues was reduced after exposure to ammonia. However, TAS was still higher in tissues from fish fed ATX+ATX-ester compared with control tissues. ALT and AST increased after ammonia exposure in all samples of all groups; however, feeding ATX+ATX-ester resulted in lower activities and prevented tissue damage by lipid peroxidation in the tissues. However, no performance parameters and viability/gross pathology of carps were reported in this study. Overall, there were no recent studies (published between 2013 and April 2017) which would indicate concerns on the safety of ATX-DMDS or ATX for salmonids and other fish at the highest dose currently authorised. Therefore, the FEEDAP Panel confirms its previous conclusion that ATX and ATX-DMDS are safe for salmonids up to 100 mg ATX and 138 mg ATX-DMDS/kg complete diet. This conclusion is extrapolated to other fish at the same dose. The applicant requested to delete the provision 'Use permitted from the age of 6 months onwards or from 50 g weight onwards'. In 2005, the Panel concluded that 'Considering safety of astaxanthin for the target animal and fish physiology there is no serious reason to restrict the use of astaxanthin to a particular developmental stage' (EFSA, 2005). Therefore, the FEEDAP Panel has no reservation to the deletion of the provision that limits the age of use of ATX/ATX-DMDS. 3.2.2.2 Safety for crustaceans To support safety of the additive for crustaceans, the applicant performed a structured literature search covering the period 2007–201822 to provide information on the safety of ATX-DMDS, including ATX ester(s), synthetic ATX, and esterified ATX from Haematococcus pluvialis. The outcome of the literature search, that retrieved 25 papers were relevant for the safety assessment, is reported in Appendix A and Table A.1. The FEEDAP Panel assessed the relevant hits and considered those in which crustaceans were fed with overdoses of AXT (between 200 and 1,600 mg/kg feed). In the absence of specific papers with ATX-DMDS, the FEEDAP Panel assumes that the results of studies performed with
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