Safety and efficacy of Mintrex Cu (Copper chelate of hydroxy analogue of methionine) as feed additive for all species 1

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1 The EFSA Journal (2008) 693, 1-19 Safety and efficacy of Mintrex Cu (Copper chelate of hydroxy analogue of methionine) as feed additive for all species 1 Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed (Question No EFSA-Q ) Adopted on 16 April 2008 PANEL MEMBERS Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Bogdan Debski, Noël Dierick, Anders Franklin, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Joop de Knecht, Lubomir Leng, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Miklós Mézes, Carlo Nebbia, Walter Rambeck, Guido Rychen, Atte von Wright and Pieter Wester SUMMARY Following a request from the European Commission, the European Food Safety Authority was asked to deliver a scientific opinion on the safety and efficacy of a copper chelate of the hydroxy analogue of methionine (Mintrex Cu) as a feed additive for all species. Mintrex Cu contains a minimum of 17 % copper and 78 % hydroxy methionine analogue ((2- hydroxy-4-methylthio)butanoic acid, HMTBa), as shown from analyses of the product. It is intended to be used as a source of the essential trace element copper. Copper (in several forms) and HMTBa are already separately authorised as nutritional feed additives in the European Union. Mintrex Cu shows the same pattern as copper sulphate in increasing liver copper content in chickens for fattening when added to a basal diet in supplementations between 10 and 500 mg kg -1 diet. These findings are taken as a demonstration of bioavailability. Consequently, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) considers Mintrex Cu as a source of available copper for all animal species. No specific studies on tolerance in target animals to Mintrex Cu have been provided. The safety for chickens for fattening could be assessed from one of the efficacy studies. The FEEDAP Panel concludes that Mintrex Cu is safe for chickens for fattening. However, considering the marked differences in copper sensitivity existing among species and even breeds and the fact that avian species are amongst the most tolerant, the FEEDAP Panel is unable to extend its conclusion from chickens for fattening to other animal species. 1 For citation purposes: Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) on a request from the European Commission on the safety and efficacy of Mintrex Cu (Copper chelate of hydroxy analogue of methionine) as feed additive for all species. The EFSA Journal (2008) 693, 1-19 European Food Safety Authority, 2008

2 The FEEDAP Panel considers it unlikely that the use of Mintrex Cu would significantly alter tissue deposition compared to inorganic copper. However, this conclusion is based on limited evidence and does not include a direct assessment of copper concentration in edible tissues. Based on the data from acute toxicity and genotoxicity studies, the FEEDAP Panel considers that Mintrex Cu does not introduce any additional toxicity compared to other sources of dietary copper. With regards to consumer safety, the FEEDAP Panel retains copper as the component of potential toxicological significance of Mintrex Cu. A model calculation was made based on recent data on copper intake, and a higher copper deposition in tissues compared to authorised copper sources, taken as a worst case scenario. This showed that under these conditions, consumer exposure would not exceed the upper intake level for copper (5 mg day -1 ). Therefore, the FEEDAP Panel concludes that no concerns for consumer safety would result from the use of Mintrex Cu in feed at the maximum authorised copper levels. The FEEDAP Panel considers that Mintrex Cu is safe for the user provided that protective measures are taken and that it does not represent additional risks to the environment compared to other sources of copper for which it will substitute. The FEEDAP Panel made some recommendations with regards to the Register entry and highlighted the need for analytical methods specific to the determination of the chelates rather than the trace element. Key words: nutritional additive, trace element, Mintrex Cu, copper, chelate, HMTBa, hydroxy methionine, bioavailability, efficacy, safety, chickens for fattening The EFSA Journal (2008) 693, 2-19

3 TABLE OF CONTENTS Panel Members...1 Summary...1 Table of Contents...3 Background...4 Terms of reference...4 Acknowledgements...5 Assessment Introduction Characterisation of the product Description of the product Production Composition Physicochemical properties Stability Homogeneity Conditions of use Evaluation of the analytical methods by the Community Reference Laboratory (CRL) Efficacy Bioavailability Trial I Trial II Conclusions on efficacy Safety for the target species Tissue/products deposition Safety for the consumer Toxicity studies Acute toxicity Genotoxicity studies including mutagenicity Conclusions Consumer exposure Conclusions on safety for the consumers Safety for the user Safety for the environment Post-market monitoring...15 Conclusions and Recommendations...15 General Remark...16 Documentation provided to EFSA...16 References...16 Appendix...19 The EFSA Journal (2008) 693, 3-19

4 BACKGROUND Regulation (EC) No 1831/ establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lies down that any person seeking an 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 the company Novus Europe SA, 3 for authorisation of copper chelate with the hydroxy analogue of methionine (Mintrex Cu) to be used as a feed additive for all species (category: nutritional additives; functional group: compounds of trace elements) under the conditions mentioned in Table 1. 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). EFSA received directly from the applicant the technical dossier in support of this application. According to Article 8 of that Regulation, 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. The particulars and documents in support of the application were considered valid by EFSA as of 3 September The additive Mintrex Cu is a copper chelate of hydroxy analogue of methionine. Eight compounds of copper are authorised in the EU for all animal species without a time limit. 4,5 Among these, three are chelates: the cupric methionate, the cupric chelate of amino acids (derived from hydrolysed soya protein) hydrate and the cupric chelate of glycine (synthetic) hydrate. The hydroxy analogue of methionine is authorised as nutritional additive in the functional group aminoacids, their salts and derivatives. 6 The Scientific Committee on Animal Nutrition (SCAN) delivered reports on the use of copper methionate for pigs (7 October 1981), 7 and copper compounds in feedingstuffs (15 April 1982) 8 and in feedingstuffs for pigs (1 June 1983). 9 The more recent opinions were issued by the SCAN (19 February 2003) on the use of copper in feedingstuffs, 10 and by EFSA on the safety of the chelated forms of iron, copper, manganese and zinc with synthetic feed grade glycine (29 November 2005). 11 TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/2003 EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. Therefore, EFSA shall deliver an opinion on the efficacy and on the safety for the target animals, consumer, user and the environment, of copper chelate of the hydroxy analogue of methionine (Mintrex Cu), when used under the conditions described in Table OJ L 268, , p.29 Avenue Marcel Thiry 200. B-1200 Brussels OJ L 187, , p.11, and corrigendum OJ L 14, , p.54 OJ L 86, , p.4 OJ L 179, , p.2, and amendment OJ L 206, , p The EFSA Journal (2008) 693, 4-19

5 ACKNOWLEDGEMENTS The European Food Safety Authority wishes to thank the members of the Working Group on Trace Elements for the preparation of this opinion. The EFSA Journal (2008) 693, 5-19

6 Table 1. Additive Register entry as proposed by the applicant Registration number/ec No/No (if appropriate) Category of additive Functional group of additive Mintrex Cu 3b4.xx Nutritional Compounds of trace elements Description Composition, description Chemical formula Purity criteria (if appropriate) Method of analysis (if appropriate) Copper chelate of hydroxy analogue of methionine 15% Cu 78% HMTBa Cu-(HMTBa) 2 Complies with EU law on undesirable substances Atomic Absorption Spectrometry Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Mintrex Cu Novus Europe SA Species or category of animal Maximum Age Conditions of use Maximum content of the element (mg kg -1 of complete feedingstuffs) All species - Pigs - piglets up to 12 weeks 170 (total) - other pigs: 25 (total) Bovine 1. Bovine before the start of rumination - milk replacers: 15 (total) - other complete feedingstuffs: 15 (total) 2. other bovine: 35 (total) Ovine: 15 (total) Fish: 25 (total) Crustaceans: 50 (total) Other species: 25 (total) Withdrawal period (if appropriate) Not relevant Specific conditions or restrictions for use (if appropriate) Specific conditions or restrictions for handling (if appropriate) Other provisions and additional requirements for the labelling Feed formulations should be adjusted to account for the methionine activity of HMTBa For user safety: breathing protection during handling, safety glasses and gloves. Store in original closed packaging, in cool, dry place. Post market monitoring (if appropriate) Specific conditions for use in complementary feedingstuffs (if appropriate) Novus Europe SA will conduct post-marketing monitoring in compliance with EU law on feed hygiene, namely by use of HACCP and Traceability systems, and formal monitoring of customer feedback through product or service complaints. To supply Cu in final feeds within EU legal limits for each species. Marker residue Maximum Residue Limit (MRL) (if appropriate) Species or category of animal Target tissue(s) or food products Maximum content in tissues Not relevant Not relevant Not relevant Not relevant The EFSA Journal (2008) 693, 6-19

7 ASSESSMENT 1. Introduction Mintrex Cu is a chelate containing a minimum of 15 % copper and 78 % hydroxy methionine analogue ((2-hydroxy-4-methylthio)butanoic acid, HMTBa), according to the specifications provided by the applicant. It is intended to be used as a source of the essential trace element copper for all animal species. The biological role, requirements/allowances, deficiency and toxicity symptoms in farm animals have been already described in a former SCAN Opinion (EC, 2003a). Several other copper-containing compounds are authorised for all species at dietary levels between 15 and 170 mg copper kg -1 depending on the animal category. SCAN stated that no particular risks for environment were identified consecutive to the use of copper in pigs and ruminants diets at the current allowed levels. To the knowledge of the FEEDAP Panel there is no additional relevant information that may lead to modify the SCAN Opinion (EC, 2003a). 2. Characterisation of the product 2.1. Description of the product The product is composed of copper bis(-2-hydroxy-4-methylthio)butanoic acid and a maximum of 1 % mineral oil (specifications not given). The formula is Cu(CH 3 S(CH 2 ) 2 - CH(OH)-COO) 2 and the molecular weight Daltons. The CAS number of the complexed compound is The molecular structure of the copper-hmtba chelate has been examined by single crystal X-ray diffraction Production Mintrex Cu is manufactured by mixing synthetic liquid HMTBa with inorganic copper carbonate under specific heat and moisture conditions in a reactor/dryer. 13 The dry Mintrex Cu chelate is milled to a standard particle size and then blended with feed grade mineral oil Composition Based on the analyses of 13 batches, the additive contents of HMTBa and copper were 79.5±0.4 % and 17.9±0.2 %, respectively. Mintrex Cu is declared to contain not more than 1 % of mineral oil; however, quantification of the content of this component was not confirmed by analytical data. The remainder of the product (up to ca. 2 %, based on the analysed values) was not identified. In three different batches, microbial impurities, mycotoxins (aflatoxin B1), heavy metals and dioxins were determined. Microbial and aflatoxin (<0.001 mg kg-1 Mintrex Cu) contamination did not raise concern. Contents of As, Pb, Hg, Cd and F were found to be 3.1, , <0.1, and <0.15 mg kg-1 Mintrex Cu, respectively. Dioxins were found at levels of to ng WHO TEQ kg-1 Mintrex Cu. Dioxin like PCBs were not analysed Technical Dossier, Section II, Annex II Technical Dossier, Section II 14 Technical Dossier, Section II The EFSA Journal (2008) 693, 7-19

8 2.4. Physicochemical properties Mintrex Cu for all species The final product is a light greenish-blue powder composed of spherical granules with a bulk density of 0.71 g (cm 3 ) -1. Around 10 % of particles showed a diameter of <105 µm; this fraction is considered inhalable. The respirable fraction (particles 10µm) was not determined. Dusting potential was examined in one batch only and the product classified as fairly dusty by the applicant (based on dustiness measured by Dustmon L Anatec apparatus) Stability A method for direct determination of the copper complex in Mintrex Cu was not provided. The stability of the chelate has therefore not been directly analysed. Instead, the stability was derived from the calculation of changes in the free HMTBa, assuming that the copper/hmtba ratio in Mintrex Cu would remain constant and that HMTBa does not interact with any other components. 16 The applicant proposes a provisional expected shelf life of three years. At present, the shelf life of Mintrex Cu has only been demonstrated for 12 months under standard conditions (25 ºC, 60 % RH) and for six months under accelerated conditions (40 C, 75 % RH). Calculated recoveries of complexed copper from three batches at the end of the test periods showed values of 101 % for both standard and accelerated conditions. Recovery rates of total HMTBa were 100 %. Stability towards light, air oxygen and ph of Mintrex Cu is considered by the applicant to be guaranteed by packaging the additive in light-tight sealed bags when stored under normal atmospheric conditions. All data on HMTBa stability in feed and premixtures are mean values obtained from stability studies performed with Mintrex Cu, Mintrex Zn and Mintrex Mn given together. In premixtures, in mash feed, in pelleted feed and during pelleting (in each case at both standard and accelerated conditions), copper concentration, as expected, remained constant. HMTBa measurements could not be attributed to any one of the Mintrex products Homogeneity Homogeneity data were based on ten samples each of premixtures, pelleted and mash feed collected from one lot of Mintrex Cu. The coefficient of variation (CV) of copper from Mintrex Cu was 2.2 % in premixtures, 11.3 % in mash feed and 11.6 % in pellets. The data indicate that Mintrex Cu can be homogeneously distributed in premixtures and, to a lesser extent, in mash and pelleted complete feedingstuffs Conditions of use Mintrex Cu is intended to supply copper in final feed within EU legal limits for all species (25 mg kg -1 complete feed, except for the following: calves and ovine, 15 mg kg -1 complete feed; other cattle, 35 mg kg -1 complete feed; crustaceans, 50 mg kg -1 complete feed; piglets, 170 mg kg -1 complete feed). According to the applicant, feed formulations should be adjusted to account for the methionine efficacy of HMTBa in Mintrex Cu. 15 Technical Dossier, Section II 16 Technical Dossier, Section II. Supplementary information. August Technical Dossier, Section II, Annex II The EFSA Journal (2008) 693, 8-19

9 According to the current knowledge, no incompatibilities or adverse interactions with feed components, carriers, other approved additives or medical products are to be expected other than those widely recognised for copper in animal nutrition Evaluation of the analytical methods by the Community Reference Laboratory (CRL) EFSA has verified the report submitted by the Community Reference Laboratory (CRL) concerning the analytical method(s) for Mintrex Cu. The executive summary of the report can be found in the Appendix. 3. Efficacy Evidence of in vivo bioavailability is required to support efficacy for compounds of trace elements not already authorised as feed additives. A single trial in a single animal species/category, including laboratory animals, is considered sufficient. Copper is preferably stored in liver. Liver copper is one of the best response criteria to assess the biological value of copper sources in monogastrics and ruminants (Jongbloed et al., 2002). A recent field study confirms that direct analysis of copper in the liver is the best biomarker of supraoptimal copper exposure (López-Alonso et al., 2006). Copper chaperone for copper/zinc superoxide dismutase is a sensitive biomarker for copper deficiency (Iskandar et al., 2005; Denzeisen et al., 2007). Studies with Mintrex in which the diet was supplemented simultaneously with Mintrex Cu, Mintrex Mn and Mintrex Zn are not considered further in the present assessment because potential interactions could not be excluded Bioavailability Two studies were conducted in chickens to evaluate the bioavailability of copper from Mintrex Cu compared with copper sulphate (CuSO 4 5H 2 O) as a source of copper in broiler diets Trial I An experiment was conducted with 175 one day-old male chicks (unspecified commercial broiler strain) to estimate the biological availability of copper from Mintrex Cu in comparison to copper sulphate. 18 Chicks were allocated randomly to seven dietary treatments (five replicates of five birds each) and fed an unsupplemented basal corn-soybean diet or the basal diet supplemented with 75, 150 or 225 mg copper kg -1 from Mintrex Cu or copper sulphate. Total copper concentration in feed was not confirmed by analysis. Chicks were allowed to access to feed and water ad libitum. Birds were individually weighed at the beginning and at the end of the study. Feed consumption was determined at the end of the study, and mortality was recorded. In addition, birds were inspected daily and any health-related problems were recorded. At day 21, the chickens were sacrificed and subjected to necropsy. Bile and liver samples were collected and pooled per replicate. The treatments did not affect feed intake, body weight and feed conversion. Increasing dietary copper was reflected by increased copper levels in liver and bile. At corresponding copper levels of the two sources, no significant differences concerning copper storage in liver were observed. 18 Technical Dossier, Section III, Annex III.3.2.Cu1 The EFSA Journal (2008) 693, 9-19

10 Trial II Mintrex Cu for all species Two thousand four hundred commercial broiler strain (Cobb 500) male chicks were allotted to 13 dietary treatments (four replicate pens of 50 animals per treatment, except for the treatment 500 mg kg -1 which was fed to two replicate pens). 19 Nutritionally complete basal diets were supplemented with either copper sulphate or Mintrex Cu to provide diets with 0, 10, 25, 50, 125, 250, and 500 mg kg -1 of supplemental copper. Copper concentrations in the diets were confirmed by analysis. The basal diets contained 17 and 9 mg kg -1 copper for the starter and grower phase, respectively. There were two feeding phases including starter (0-14 days) and grower (14-35 days). At the end of each phase, birds were weighed by pen and two birds per pen (four birds per pen for the 500 mg kg -1 levels) were sacrificed to collect liver and tibia samples for analysis of copper concentration. Only the data at the end of the trial are reported here (Table 2). The highest copper concentrations in feed markedly decreased body weight, regardless of copper sources. This corresponds to the toxic level ( mg copper kg -1 ) that was described for chickens (NRC, 1980). Overall, there was no effect of copper source or dietary copper concentration on feed conversion or mortality. The highest dietary copper concentration significantly increased tibia ash copper and liver copper concentration for both copper sources. There were significant effects of copper source, concentration and their interaction on day 14 (data not shown; Wang et al., 2007) on dry liver copper concentration, but no significant effects between the copper sources were seen at the end of the trial. Table 2. Effect of source and level of supplemental copper on chicken performance, mortality and copper levels in tibia ash and liver (35 days) Supplemental level of copper (mg kg -1 ) C 1 Copper sulphate Mintrex Cu Intended Starter Diet Grower Diet BW (kg) 2.01 a 1.99 ab 1.94 abc 1.97 ab 1.97 ab 1.96 ab 1.41 e 2.01 a 2.01 a 1.95 ab 1.96 ab 1.93 abcd 1.78 cd F/G (g g -1 ) Mortality (%) Tibia ash copper Liver copper c 10.2 c 13.0 c 13.0 c 14.4 c 50.6 bc a 13.5 c 13.7 c 11.6 c 13.4 c 88.1 b a a,b,c,d,e: Values within rows with no common superscript differ significantly (P<0.05) (1) Control (2) analysed values (3) mg kg -1 (4) mg kg -1 DM The background copper levels (17 mg kg -1 starter, 9 mg kg -1 grower diets) may have masked effects on liver copper at lower dietary levels Conclusions on efficacy Based on the data provided in the dossier, Mintrex Cu shows the same pattern as copper sulphate in increasing liver copper in chickens for fattening when added to a basal diet in concentrations between 10 and 500 mg kg -1 diet. Copper supplementation of 250 and 500 mg kg -1 diet from both sources increased liver copper to a similar extent. The FEEDAP Panel recognises that liver storage is insensitive to dietary copper at lower concentrations in animal species with a high copper tolerance, as it was shown in trial II. However, the FEEDAP Panel considers that there is no reason to expect that the two copper sources would behave differently only at these lower concentrations. Since inorganic copper sulphate is generally recognised and 19 Technical Dossier, Section III, Annex III.3.2.Cu2 The EFSA Journal (2008) 693, 10-19

11 used as a source of available copper in animal nutrition, Mintrex Cu can be considered as a bioavailable copper source, comparable to copper sulphate, for all animal species. 4. Safety for the target species The FEEDAP Panel considers that tolerance of the target animals should be demonstrated for compounds of trace elements not previously authorised. When the application concerns all animal species, one tolerance study with the most sensitive species (or even a laboratory animal) may be sufficient. As for efficacy assessment, studies with Mintrex in which the diet contains the three trace elements copper, manganese and zinc from Mintrex given simultaneously are not further considered because potential interactions can not be excluded. The safety of Mintrex Cu for chickens for fattening at the highest copper level approved (25 mg kg -1 ) can be derived from the bioavailability study reported previously (see Section 3.1.2) which showed that concentrations up to 250 mg kg -1 complete diet were well tolerated. The FEEDAP Panel notes that marked differences exist among species and even breeds regarding tolerance to dietary copper, with avian species being amongst the most tolerant (EC, 2003a; Moeller, 2004; Thompson, 2007). Considering the maximum content of copper approved for the most sensitive species (sheep, 15 mg kg -1 diet), the FEEDAP Panel cannot conclude on the safety of the compound for target animals, other than chickens for fattening. The HTMBa contribution from Mintrex Cu needs consideration to keep total methionine at desired levels. 5. Tissue/products deposition No specific studies were made available on copper deposition in edible tissues and products from Mintrex Cu supplementation. Limited information can be derived from a study conducted in chickens for fattening a species recognised as having a high copper elimination rate using copper levels including those currently authorised in the EU (see Section and Wang et al., 2007). Copper from both copper sulphate and Mintrex Cu shows a trend to increase copper liver deposition in chickens for fattening only at supplementation levels higher than 125 mg kg - 1 feed, more than five times higher than the authorised level. Only at the highest used copper supplementations of 250 and 500 mg kg -1 feed the hepatic copper concentration was higher, but not significantly different between the two sources tested. Mintrex Cu supplementation showed a liver deposition pattern comparable to that resulting from copper sulphate. Based on the available data (trials of copper additions including the authorised maximum content in feed for chickens for fattening), there appears to be no essential differences in liver deposition of copper from Mintrex Cu compared to inorganic copper. The FEEDAP Panel does not expect a difference in tissue deposition pattern of copper in other edible tissues. This conclusion is supported by a study in dairy cows in which milk copper was examined comparing copper (manganese and zinc) from Mintrex with inorganic sources; after 103 days of feeding, the concentration of copper in milk was not significantly different between the inorganic and organic copper sources and the unsupplemented feed. 20 In a recent study, there were no differences found in eggs copper deposition when copper from inorganic or organic (excluding Mintrex ) sources was supplied to feed for laying hens within the EU authorised use levels (Huyghebaert et al., 2006). 20 Technical Dossier, Section III, Annex III.3.2.EU5 The EFSA Journal (2008) 693, 11-19

12 The FEEDAP Panel considers it unlikely that the use of Mintrex Cu would significantly alter tissue deposition compared to inorganic copper. However, this conclusion is based on limited evidence and does not include a direct assessment of copper concentration in edible tissues. 6. Safety for the consumer 6.1. Toxicity studies The applicant presented acute oral toxicity and genotoxicity studies on Mintrex Cu Acute toxicity In an acute toxic class dose study (OECD, 2001) female rats were treated by oral gavage with the limit dose of 2000 mg Mintrex Cu kg-1 bw. 21 No deaths were observed, signs of distress (e.g. tachypnoea) were observed but were reversible within two days after administration. Therefore the acute oral lethal dose of Mintrex Cu is >2000 mg kg-1 bw Genotoxicity studies including mutagenicity Mutagenicity of Mintrex Cu was assessed in a reverse mutation assay on five S. typhimurium strains (TA98, TA100, TA1535, TA1537 and TA102) with and without metabolic activation. 22 No significant increase of revertant colonies was observed up to the limit concentration of 5000 µg ml -1 and/or at inhibitory concentrations for each strain. Mintrex Cu was also tested in CHO cells for chromosomal aberrations with and without metabolic activation. 23 A significant increase of structural chromosome aberrations, exceeding the 95 th percentile of reference range, was seen in replicate experiments at concentrations >225 mg L -1 (corresponding to 34 mg copper L -1 ). The effect was observed both with and without S- 9 activation. Cytotoxicity accompanied chromosomal aberrations although the degree of cytotoxicity was very variable. Genotoxicity was assessed in vivo in an oral bone marrow micronucleus assay on adult mice which were administered the feed additive once daily at various concentrations for two consecutive days. 24 No genotoxic effects were observed following the second administration with 175, 350 or 700 (maximum tolerated dose) mg Mintrex Cu kg-1 bw day-1. It is recognised that copper compounds can induce genotoxic effects in vitro and/or in vivo (EC, 2003b); such effects may originate from indirect mechanisms, e.g. induction of oxidative stress (Corona-Rivera et al., 2007). Copper from copper sulphate was found to induce an increase of unscheduled DNA synthesis in cultured rat hepatocytes beginning at doses of 7.9 µmol L -1 (corresponding to 0.5 mg copper L -1 ) (Denizeau and Marion, 1989); chromosomal aberrations induced by Mintrex Cu in CHO cells were seen at a higher dose (34 mg copper L - 1 ). 21 Technical Dossier. Supplementary information. December Mintrex Cu. Acute oral toxicity 22 Technical Dossier. Supplementary information. December Mintrex Cu. Genotoxicity. Reverse mutation 23 Technical Dossier. Supplementary information. December Mintrex Cu. Genotoxicity. Induction of Chromosome aberration 24 Technical Dossier. Supplementary information. December Mintrex Cu. Genotoxicity. Induction of micronuclei The EFSA Journal (2008) 693, 12-19

13 Conclusions Mintrex Cu for all species Mintrex Cu shows low acute toxicity in laboratory rodents and is not mutagenic in vitro. It increases chromosomal aberrations in vitro, but it was not genotoxic in a micronucleus mouse assay. Based on comparisons with literature data, the in vitro genotoxic effects observed with Mintrex Cu do not appear different from those produced by other copper sources. Therefore the FEEDAP Panel considers that Mintrex Cu does not introduce any additional toxicity effects compared to other sources of dietary copper Consumer exposure Although no data was supplied on the metabolic fate of copper-hmtba, the FEEDAP Panel considers that the deposition pattern of copper in chicken liver and the nutritional equivalence of HMTBa from Mintrex and from free HMTBa concerning the incorporation (as methionine) into body protein (Yi et al., 2007), are sufficient indications of an extended dissociation of the molecule. As copper is the component of potential toxicological significance (see Section 6.1), its deposition in tissues and products represents the relevant issue for the appraisal of consumer safety. The SCAN assessed the consumer exposure from the use of copper in feedingstuffs, irrespective of the source (EC, 2003a). On the basis of a worst case scenario for the daily human food consumption in accordance with Commission Directive 2001/79/EC (muscle: 300g, liver: 100g, kidney: 50g, fat: 50g), 25 the consumption of products from supplemented piglets (slaughtered at 25 kg bw without withdrawal time) and calves (based on liver copper reported by Souci et al., 2000) would lead to an intake of 8.2 and 7.9 mg person -1 day -1, respectively. The calculated worst case scenarios for piglets and calves account for more than 25 % of the provisional maximum tolerable intake of 30 mg person -1 day -1 that was available at the time when the SCAN opinion was issued. However, those calculated intake figures exceed the tolerable upper intake level (UL) (5 mg day -1 ) of copper that was subsequently defined by the Scientific Committee on Food (SCF) (EC, 2003b). Moreover, the FEEDAP Panel notes that the figures of the SCAN opinion were mainly based on tissue retention data from inorganic copper sources. Based on the assessment done by the SCF and on available literature data, the FEEDAP Panel can derive an estimate of the contribution of copper from Mintrex Cu to consumer exposure. Mean dietary copper intakes by adults in different European countries have been estimated within a range of mg day -1 for males and mg day -1 for females (Van Dokkum, 1995; EC, 2003b). A recent paper provided a very similar estimate to the SCF assessment (Sadhra et al., 2007). In France the relative contribution of animal products to the total copper intake is 28 % for meat and eggs, 7 % for fish and up to 2 % for milk and dairy products; in UK meat and fish account together for more than 20 %, and milk and dairy products for not more than 10 %. Taking a worst case approach, the FEEDAP Panel assumes a maximum contribution of 40 % from foods of animal origin to total copper intake. In reaching its conclusion, the FEEDAP Panel assumes that organic copper sources will be used as substitutes for inorganic sources. Wang et al. (2007) reviewed the copper bioavailability in broilers from various organic sources and concluded on an average higher availability (in the % range) compared to copper sulphate, this conclusion being in contrast with the results on liver copper deposition and milk copper content obtained with 25 OJ L 267, , p.1 The EFSA Journal (2008) 693, 13-19

14 Mintrex Cu when doses of copper in feed within the maximum authorised levels were applied. The FEEDAP Panel assumes, as a worst case scenario, a 30 % higher tissue deposition. Based on the highest estimated average copper intake of 2.3 mg day -1, copper intake from food of animal origin would not be higher than 0.92 mg day -1. If this figure would increase by the use of organic copper compounds (e.g. Mintrex Cu), copper intake from foods of animal origin would amount to 1.2 mg day -1, thus increasing total copper intake by 0.28 to 2.6 mg day -1. According to the SCF, the 97.5 percentile in some EU countries (Austria, Germany) was estimated to be higher than 4 mg and close to the UL. The highest value reported by SCF was 4.2 mg day -1 in Austria. Following the same approach as for the mean values, the Austrian 97.5 percentile of copper intake would increase by 0.5 mg, the sum (4.7 mg) still remaining below the UL Conclusions on safety for the consumers The FEEDAP Panel retains copper as the component of potential toxicological significance of Mintrex Cu. Based on the above worst case scenario for human copper exposure, the FEEDAP Panel concludes that no concern for consumer safety would result from the use of Mintrex Cu in feed at the maximum authorised copper levels. 7. Safety for the user No specific studies on the safety of Mintrex Cu for the user were conducted. Around 10 % of the particles are <105 µm; this fraction is considered inhalable. The respirable fraction ( 10µm) was not determined. Data on occupational toxicity by copper were provided by the IPCS (1998). LD 50 values for dermal exposure were reported at >1124 and >2058 mg copper kg -1 bw in rats and rabbits, respectively. The inhalation LC 50 (exposure duration unspecified) was >1303 mg copper kg -1 bw in rabbits, and respiratory function was impaired in guinea pigs exposed to 1.3 mg copper (m 3 ) -1 for one hour. In humans, dermal exposure has not been associated with systemic toxicity but copper does induce allergic responses in sensitive individuals. Metal fume fever from inhalation of high concentrations in the air in the occupational setting has been reported. Workers apparently exposed to high air levels resulting in an estimated intake of 200 mg copper day -1 developed signs suggesting copper toxicity (e.g. elevated serum copper levels, hepatomegaly). On a precautionary basis, the FEEDAP Panel supports the recommendations made by the applicant in the Register entry (under other provisions) and makes further recommendations (see Recommendations). 8. Safety for the environment Copper is a natural element that is essential for life and is present almost everywhere in the environment. However, at high concentrations copper may be toxic and a maximum content of the element in feed has therefore been set. There is no reason to believe that chelates such as Mintrex Cu would be more harmful to the environment than the inorganic element. The FEEDAP Panel considers that the use of Mintrex Cu in feed does not represent additional risks to the environment, compared to other sources of copper for which it will substitute, as long as the maximum authorised content in feedingstuffs is not exceeded. The EFSA Journal (2008) 693, 14-19

15 9. Post-market monitoring Mintrex Cu for all species The FEEDAP Panel does not see a need for specific requirements of post-market monitoring other than the need for traceability and recall procedures established by Regulation (EC) No 183/ CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS Mintrex Cu was shown as a bioavailable copper source, comparable to an authorised inorganic source of copper in chickens for fattening. The FEEDAP Panel considers Mintrex Cu as a source of available copper for all animal species. The FEEDAP Panel concludes that Mintrex Cu is safe for chickens for fattening. However, considering the marked differences in copper sensitivity existing among species and even breeds, and the fact that avian species are amongst the most tolerant, the FEEDAP Panel is unable to extend its conclusion from chickens for fattening to other animal species. The FEEDAP Panel considers it unlikely that the use of Mintrex Cu would significantly alter tissue deposition compared to inorganic copper. However, this conclusion is based on limited evidence and does not include a direct assessment of copper concentration in edible tissues. Based on the data from acute toxicity and genotoxicity studies, the FEEDAP Panel considers that Mintrex Cu does not introduce any additional toxicity compared to other sources of dietary copper. With regards to consumer safety, the FEEDAP Panel retains copper as the component of potential toxicological significance of Mintrex Cu. A model calculation was made based on recent data on copper intake, and a higher copper deposition in tissues compared to authorised copper sources, taken as a worst case scenario. This showed that under these conditions, consumer exposure would not exceed the UL for copper. Therefore the FEEDAP Panel concludes that no concerns for consumer safety would result from the use of Mintrex Cu in feed at the maximum authorised copper levels. The FEEDAP Panel considers that Mintrex Cu is safe for the user provided that protective measures are taken. The FEEDAP Panel considers that the use of Mintrex Cu in feed does not represent additional risks to the environment compared to other sources of copper for which it will substitute. RECOMMENDATIONS The FEEDAP Panel recommends that the following modifications should be made to the Register entry as proposed by the applicant: 1. Additive In the view of the FEEDAP Panel the additive name is: Copper chelate of hydroxy analogue of methionine. 2. Description Composition, description. Copper chelate of hydroxy analogue of methionine containing a minimum of 17 % copper and 78 % (2-hydroxy-4-methylthio)butanoic acid. 26 OJ L 35, , p.1 The EFSA Journal (2008) 693, 15-19

16 Chemical formula. Cu(CH 3 S(CH 2 ) 2 -CH(OH)-COO) 2. Mintrex Cu for all species Purity Criteria. The FEEDAP Panel recommends the setting of maximum levels as follows: As 5, Pb 25, Hg 0.1, Cd 1 and F 0.2 mg kg -1 Mintrex Cu. Dioxins (sum of PCDDs and PCDFs) 0.2 ng WHO-PCDD/F-TEQ kg -1 Mintrex Cu. 3. Conditions of use Feed formulations should be adjusted to account for the methionine efficacy of HMTBa in Mintrex Cu. 4. Other provisions and additional requirements for labelling Specific conditions or restrictions for handling. In addition to the applicant s proposal, the FEEDAP Panel recommends appropriate ventilation of working rooms and reduction of the Mintrex Cu inhalable fraction as far as technically feasible. GENERAL REMARK The FEEDAP Panel wishes to draw the attention to the following. Considering: - the increasing number of applications for organic forms of trace elements to be used in animal nutrition, - the potential higher bioavailability of those compounds in feedingstuffs, and consequently (i) the potential reduction of maximum content for those trace elements in feed and (ii) the higher deposition of trace elements of organic origin in animal tissues, and - the resulting need for maximum content of organic forms of trace elements different from those already existing, the FEEDAP Panel stresses the need for analytical methods to detect those organic compounds in feed, independent from the trace element background. The availability of such methods would allow lower maximum contents in feed which would in turn reduce concerns relating to the assessment of consumer safety and reduce environmental load. DOCUMENTATION PROVIDED TO EFSA 1. Dossier Mintrex Cu as nutritional additive for all species. March Submitted by Novus Europe SA. 2. Mintrex Cu. Supplementary information. August Submitted by Novus Europe SA. 3. Mintrex Cu. Supplementary information. December Submitted by Novus Europe SA. 4. Evaluation report of the Community Reference Laboratory feed additives on the analytical methods of Mintrex Cu. December Comments from the Member States received through the EFSAnet. REFERENCES Corona-Rivera, A., Urbina-Cano, P., Bobadilla-Morales, L., Vargas-Lares, J. de J., Ramírez- Herrera, M.A., Mendoza-Magaña, M.L., Troyo-Sanromán, R., Díaz-Esquivel, P. and Corona-Rivera, J.R., Protective in vivo effect of curcumin on copper genotoxicity evaluated by comet and micronucleus assays. J. Appl. Gent. 48, The EFSA Journal (2008) 693, 16-19

17 Denizeau, F. and Marion, M., Genotoxic effects of heavy metals in rat hepatocytes. Cell Biol. Toxicol. 5, Denzeisen, R., Araya, M., Harrison, B., Keen, C., Solioz, M., Thiele, D. and McArdle, H.J., How reliable are current biomarkers for copper status. Br. J. Nutr. 98, EC (European Commission), 2003a. Opinion of the Scientific Committee for Animal Nutrition on the use of copper in feedingstuffs. < EC (European Commission), 2003b. Opinion of the Scientific Committee on Food on the Tolerable Upper Intake Level of Copper. < Huyghebaert, G., Lippens, M. and Nollet, L., Enrichment of eggs with trace minerals using inorganic or organic trace sources: a feasibility? 12 th Eur. Poultry Conf., Verona (IT). IPCS (International Programme on Chemical Safety), Environmental Health Criteria 200 on Copper. < Iskandar, M., Swist, E., Trick, K.D., Wang, B., L Abbe, M.R. and Bertinato J., Copper chaperone for Cu/Zn Superoxide Dismutase is a sensitive biomarker of mild copper deficiency induced by moderately high intakes of zinc. Nutr. J. 4, 35. Jongbloed, A., Kemme, P., De Grootte, G., Lippens, M. and Meschy, F., Bioavailability of major and trace elements. EMFEMA (International Association of the European Manufacturers of Major, Trace and Specific Feed Mineral Materials), Brussels, 118 pp. López-Alonso M., Crespo, A., Miranda, M., Castillo, C., Hernandez, J. and Benedito, J.L., Assessment of some blood parameters as potential markers of hepatic copper accumulation in cattle. J. Vet. Diagn. Invest. 18, Moeller, R.B Copper. In : Clinical Veterinary Toxicology. Plumlee K.H. (ed.) Mosby, pp NRC (National Research Council, National Academy of Science), Mineral tolerance of domestic animals. Washingthon D.C. OECD (Organisation for Economic co-operation and Development), OECD/OCDE 423. OECD Guideline for testing of chemicals. Acute Oral Toxicity Acute Toxic Class Method. < Sadhra, S.S., Wheatley, A.D. and Cross, H.J., Dietary exposure to copper in the European Union and its assessment for EU regulatory risk assessment. Sci. Total Environ. 374, Souci, S. W., Fachmann, W and Kraut, H., Food composition and nutrition tables. Scientific Publishers, Stuttgart. Thompson, L. J., Copper. In : Veterinary Toxicology. Basic and clinical principles. Gupta R.C. (ed.) Academic Press, pp Van Dokkum, W., The intake of selected minerals and trace elements in European countries. Nut. Res. Rev. 8, Wang, Z., Cerrate, S., Coto, C., Yan, F. and Waldroup, P.W., Evaluation of Mintrex Copper as a source of copper in broiler diets. Int. J. Poultry Sci. 6, The EFSA Journal (2008) 693, 17-19

18 Yi, G. F., Atwell, C. A., Hume, J. A., Dibner, J. J., Knight, C. D. and Richards, J. D. 2007: Determining the methionine activity of Mintrex organic trace minerals in broiler chicks by using radiolabel tracing or growth assay. Poult. Sci. 86, The EFSA Journal (2008) 693, 18-19

19 APPENDIX Executive Summary of the Evaluation Report of the Community Reference Laboratory Feed Additives on the Method(s) of Analysis of Mintrex Cu for all species Mintrex Cu is a product for which authorisation is sought under the category "nutritional additives", functional group 3b "compounds of trace elements", according to the classification system of Annex I, of Regulation (EC) No 1831/2003. According to the applicant, Mintrex Cu contains 15% of Copper as chelate of hydroxyl analogue of methionine, 2-hydroxy-4- methylthiobutanoic acid (HMTBa) as active substance. Mintrex Cu is also a source of methionine activity as HMTBa. In the current application authorisation is sought for use of Mintrex Cu for all animal species. Mintrex Cu is intended to be added to complete feed to supplement Cu within legal limits for each species which are: piglets up to 12 weeks 170 mg/kg, other pigs 25 mg/kg, milk replacers 15 mg/kg, other complete feed for bovine before the start of rumination 15 mg/kg, other bovine 35 mg/kg, ovine 15 mg/kg, fish 25 mg/kg, crustaceans 50 mg/kg and other species 25 mg/kg. For the determination of Cu in the feed additive, premixtures and feedingstuffs for official control the CEN standard method EN 15510:2007, as proposed by the applicant, is recommended by the CRL. The proposed method for the determination of HMTBa in the feed additive is considered suitable for the intended purpose. The suitability of the proposed method for the determination of HMTBa in feedingstuffs cannot be evaluated by the CRL. The EFSA Journal (2008) 693, 19-19