Scientific Opinion on safety and efficacy of di copper chloride tri hydroxide (tribasic copper chloride, TBCC) as feed additive for all species 1

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1 SCIENTIFIC OPINION Scientific Opinion on safety and efficacy of di copper chloride tri hydroxide (tribasic copper chloride, TBCC) as feed additive for all species 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy The assessment carried out by the FEEDAP Panel is based on di copper chloride trihydroxide (tribasic copper chloride, TBCC) and TBCC formulated with starch to reduce dusting potential. Based on three studies in chickens for fattening and one in laying hens, the FEEDAP Panel concludes that TBCC is a safe source of copper for poultry with a margin of safety 10; the FEEDAP Panel extended this conclusion to the safety of TBCC for all animal species/categories up to the maximum total copper content authorised in feedingstuffs. The FEEDAP Panel does not expect substantial differences in the metabolic behaviour, particularly in the deposition of copper in edible tissues and products from copper sulphate and TBCC. Consequently the FEEDAP Panel considers that exposure of the consumer to copper from food of animal origin would not be essentially modified by the replacement of copper sulphate by TBCC in feed. In the absence of data, TBCC should be considered as a potential irritant to skin and eyes, and a potential skin sensitiser. The risk of respiratory exposure for the formulated TBCC/starch product is considered to be minimal. The FEEDAP Panel considers that the use of TBCC in feed does not represent additional risks to the environment, compared to other sources of copper which it will substitute, provided that the maximum authorised total copper content in feedingstuffs is not exceeded. Bioequivalence between TBCC and copper sulphate with regards to copper liver deposition, plasma levels of ceruloplasmin and copper, egg weight and laying hen performance, and growth of piglets was demonstrated in 11 experiments. The FEEDAP Panel concludes that TBCC is an effective source of copper for all animal species. European Food Safety Authority, 2011 KEY WORDS Nutritional additive, compounds of trace elements, di copper chloride trihydroxide, tribasic copper chloride, TBCC, safety, efficacy 1 On request from the European Commission, Question No EFSA-Q , adopted on 6 September Panel members: Gabriele Aquilina, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Albert Dierick, Mikolaj Antoni Gralak, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Reinhard Kroker, Lubomir Leng, Secundino López Puente, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Giovanna Martelli, Miklós Mézes, Derek Renshaw, Maria Saarela Kristen Sejrsen and Johannes Westendorf. Correspondence: FEEDAP@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group on Trace Elements for the preparatory work of this scientific opinion. Suggested citation: EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP); Scientific Opinion on safety and efficacy of di copper chloride tri hydroxide (tribasic copper chloride, TBCC) as feed additive for all species.. [18 pp.] doi: /j.efsa Available online: European Food Safety Authority, 2011

2 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 safety and efficacy of di copper chloride tri hydroxide (tribasic copper chloride, TBCC) as feed additive for all species. The assessment is based on TBCC and TBCC formulated with starch to reduce dusting potential. Based on three studies in chickens for fattening and one in laying hens, the FEEDAP Panel concluded that TBCC is a safe source of copper for poultry with a margin of safety 10. Considering that in the poultry studies no substantial differences were observed in the endpoints measured between copper sulphate and TBCC, the FEEDAP Panel did not expect that further essential information on the safety of TBCC would be provided by additional tolerance studies with other (more sensitive) animal species. The FEEDAP Panel concluded that TBCC is a safe source of copper for all animal species/categories up to the maximum total copper content authorised in feedingstuffs. The FEEDAP Panel did not expect substantial differences in the metabolic behaviour, particularly in the deposition of copper in edible tissues and products from copper sulphate and TBCC. Consequently the FEEDAP Panel considered that exposure of the consumer to copper from food of animal origin would not be essentially modified by the replacement of copper sulphate by TBCC in feed. In the absence of data, TBCC should be considered as a potential irritant to skin and eyes, and a potential skin sensitiser. The risk of respiratory exposure for the formulated TBCC/starch product was considered to be minimal. The FEEDAP Panel considered that the use of TBCC in feed did not represent additional risks to the environment, compared to other sources of copper which it will substitute, provided that the maximum authorised total copper content in feedingstuffs is not exceeded. Bioequivalence between TBCC and copper sulphate with regards to copper liver deposition, plasma levels of ceruloplasmin and copper, egg weight and laying hen performance, and growth of piglets was demonstrated in 11 experiments. The FEEDAP Panel concludes that TBCC is an effective source of copper for all animal species. The FEEDAP Panel made recommendations concerning the specification of the additive. 2

3 TABLE OF CONTENTS Abstract... 1 Table of contents... 3 Background... 4 Terms of reference... 4 Assessment Introduction Characterisation Qualitative and quantitative composition of the additive Purity Physical state of the product Manufacturing process Stability and homogeneity Stability Homogeneity Characteristic of the reformulated product Conditions of use Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) Safety Safety for the target species Tolerance studies Conclusions on the safety for target species Safety for the consumer Safety for the users/workers Safety for the environment Efficacy/Bioequivalence Poultry Growing cattle Beef heifers Cattle for fattening Weaned piglets Conclusions on efficacy of TBCC as source of dietary copper Post-market monitoring Conclusions and recommendations Documentation provided to EFSA References Appendix

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 lays down that any person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7. The European Commission received a request from Dr. Christian Elwert 5 for authorisation of the product di copper chloride tri hydroxide (tribasic copper chloride, TBCC), when used as a feed additive for all species (category: nutritional additives; functional group: compounds of trace elements) under the conditions mentioned in Table 1. 6 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. 7 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 9 December Di copper chloride tri hydroxide is not authorised in the European Union (EU). However, other copper compounds are authorised in the EU to be used as nutritional feed additives (trace elements) by Commission Regulation (EC) No 1334/ (cupric acetate, monohydrate; basic cupric carbonate, monohydrate; cupric chloride, dihydrate; cupric methionate; cupric oxide; cupric sulphate, pentahydrate; cupric chelate of amino acids hydrate; copperlysine sulphate), Commission Regulation (EC) No 479/ (Cupric chelate of glycine, hydrate) and Commission Regulation (EU) No 349/ (copper chelate of hydroxy analogue of methionine). 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. EFSA shall deliver an opinion on the safety for the target animal(s), consumer, user and the environment and the efficacy of the product di copper chloride tri hydroxide (tribasic copper chloride, TBCC), when used under the conditions described in Table 1. 4 OJ L 268, , p Lobejuner Strasse Nauendorf OT Merbitz. Germany 6 Original Table 1 was updated with the characteristics of the reformulated product. Technical Dossier, Supplementary information (III). 7 EFSA Dossier reference: FAD OJ L 187, , p.11 9 OJ L 86, , p.4 10 OJ L 104, , p.31 4

5 Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No (if appropriate) Category(-ies) of additive Functional group(s) of additive Di copper chloride tri hydroxide; crystal form 100 % atacamite/paratacamite in a 1:1 to 1:1.5 ratio Not available (new application) Nutritional additive Compounds of trace elements Composition, description Tribasic copper chloride; crystal form atacamite/paratacamite in a 1: 1 to 1:1.5 ratio Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Chemical formula Cu 2 Cl(OH) 3 TBCC Not applicable Description Purity criteria (if appropriate) Min. 98 % pure Min. Cu content 53 % Method of analysis (if appropriate) EN ISO 15510:2007 Species or category of animal Piglets (suckling and weaned piglets) Other pigs Bovines before the start of rumination (milk replacers and other complete feedingstuffs) Bovines after the start Conditions of use Minimum Withdrawal Maximum content Maximum Age content period mg/kg of complete feedingstuffs (if appropriate) 12 weeks Not applicable 170 Total (natural plus added) Not applicable 12 weeks and over Not applicable 25 Total (natural plus added) Not applicable Not applicable Not applicable 15 Total (natural plus added) Not applicable Not applicable Not applicable 35 Total (natural plus added) Not applicable of rumination Ovines Not applicable Not applicable 15 Total (natural plus added) Not applicable Fish Not applicable Not applicable 25 Total (natural plus added) Not applicable Crustaceans Not applicable Not applicable 50 Total (natural plus added) Not applicable Other species Not applicable Not applicable 50 Total (natural plus added) Not applicable Specific conditions or restrictions for use (if appropriate) Other provisions and additional requirements for the labelling The following declarations shall be inserted in the labelling and accompanying documents: - For sheep: Where the level of copper in feedingstuffs exceeds 10 mg/kg: the level of copper in this feedingstuff may cause poisoning in certain breeds of sheep Specific conditions or restrictions for handling (if appropriate) Post-market monitoring (if appropriate) Specific conditions for use in complementary feedingstuffs (if appropriate) - For bovines after the start of rumination: Where the level of copper in feedingstuffs is less than 20 mg/kg: the level of copper in this feedingstuff may cause copper deficiencies in cattle grazing pastures with high contents of molybdenum or sulphur None Not applicable None 5

6 Not applicable Marker residue Maximum Residue Limit (MRL) (if appropriate) Species or category of Target tissue(s) or animal food products Maximum content in tissues 6

7 ASSESSMENT 1. Introduction Di copper chloride tri hydroxide, a specific inorganic copper source, is applied as a nutritional additive, functional group Compounds of trace elements. The additive, also referred to as TBCC (tribasic copper chloride; copper oxychloride), represents the α-crystal form of atacamite/paratacamite in a ratio of 1:1 to 1:1.5. TBCC has been marketed in the USA, Canada and China since In the assessment of the initial dossier submitted by the applicant, it was noted that the particle size was low and the dusting potential high. During the course of the assessment, the applicant chose to reformulate the product in order to reduce dusting potential. The resulting modifications in product composition and characteristics are described in a separate entry (section 2.6). Product characteristics not expressively noted in this entry remain in general unchanged. 2. Characterisation TBCC occurs in three forms. The α- and β-crystals and the amorphous form show different physico-chemical properties (e.g. solubility). The current assessment relates only to the α-crystal form of the TBCC Qualitative and quantitative composition of the additive The product consists of di copper chloride tri hydroxide. The formula is described by the applicant as Cu 2 Cl(OH) 3 with the molecular weight of daltons. It occurs as a mixture of copper chloride and copper oxide hydrate (CAS number ; molecular formula Cl 2 Cu.3CuH 2 O 2 ; molecular weight ). TBCC consists of a stable mixture of the essentially similar trimorphs atacamite and paratacamite in a ratio of 1:1 to 1: It is not soluble in neutral ph but it dissolves at lower ph as in the stomach of target animals. In the first dossier the mean copper content of five batches of TBCC was 59.1 % (range: %); 12 this complied with the original product specification of a minimum copper content of 58 %. By specifications, the product contains 98 % of TBCC of which 95 % is in the α-crystal form. Chloride content ranges from 17 to 19 % Purity Fluoride and cadmium (five batches) were below detection limits (1 mg/kg), as well as lead (< 5 mg/kg). 13 Arsenic content was (31.2 mg/kg, average of five batches) above 30 mg/kg authorised in additives belonging to the functional group of trace elements, but below 50 mg/kg authorised for copper sulphate and copper carbonate, and below 100 mg/kg authorised for copper oxide. 14 The analyses of four batches of TBCC showed the sum of dioxin and dioxin-like PCBs to be below 1 ng WHO-PCDD/F-PCB-TEQ/kg Technical Dossier Section II. 12 Technical Dossier, Section II, Annex g 13 Technical Dossier, Section II, Annex a 14 Directive 2002/32/EC. OJ L 140, , p. 10 ; and subsequent amendments. 15 Technical Dossier, Section II, Annex b 7

8 2.3. Physical state of the product The applicant has provided the analysis of particle size distribution in three batches of the product. Almost all particles were below 100 µm, 63 to 76 % below 50 µm, and about 2 % below 10 µm. 16 Dusting potential was measured in three batches of the product, 17 according to the Stauber- Heubach method (Stauber and Beutel, 1984). The dusting potential of TBCC was on average 1.11 % (equating 11.1 g/kg), which is calculated to correspond to 27.8 g/m 3 an extremely high value Manufacturing process The process involves a reaction between an acidic solution of cupric chloride with free hydrochloric acid and copper tetramine dichloride. The chemical reactions required to produce TBCC in the α-crystal form of the additive are described in detail in the dossier Stability and homogeneity Stability For inorganic compounds of trace elements stability studies are not required Homogeneity Homogeneity has been studied in two mineral premixtures (15 subsamples each). The coefficient of variation (CV) of copper concentration in a premixture low in copper from TBCC (intended 15 mg Cu/kg complete feed) was 2.8 %, and 2.6 % in a premixture high in copper from TBCC (intended 160 mg Cu/kg complete feed). 18 Homogeneity in a piglet starter feed (15 subsamples) was studied using the high copper premixture. The CV in feed was calculated to be 3.1 % Characteristic of the reformulated product The reformulated additive 20 contains TBCC and non-gmo starch included as a binder. By this dilution the copper content was reduced from 59 % to 53.4 % (average of five batches). The particle size analysis (five batches) showed no particles of the critical fraction (< 50 µm). The dusting potential (three batches) was 0.03 g/m 3 air. The capacity of the additive to homogeneously distribute in feed was affected by the reformulation (CV= 8.9 %, by calculation) Conditions of use TBCC is intended to supply copper in final feed for all species up to a total content of 50 mg Cu/kg complete feed, except for the following: calves and ovine, 15 mg/kg complete feed; other cattle, 35 mg/kg complete feed; piglets, 170 mg/kg complete feed; pigs for fattening, sows and fish, 25 mg/kg complete feed. 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. 16 Technical Dossier, Section II, Annex a 17 Technical Dossier, Section II, Annexes a and a 18 Technical Dossier, Section II, Annex a 19 Technical Dossier, Section II, Annex a 20 Technical Dossier, Supplementary information (II) 8

9 2.8. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) EFSA has verified the EURL report and its amendment as they relate to the methods used for the control of the tribasic copper chloride (TBCC) in animal feed. The executive summary of the EURL report, and the amendment of the report, can be found in the Appendix. 3. Safety When solubilised, minerals such as copper form complexes in the gastro intestinal tract. For copper sulphate, complexing starts already in the saliva; for TBCC in the α-crystal form it does not start before it reaches the stomach. At intestinal level differences in the solubility between copper sulphate and TBCC could not be established (Pang and Applegate, 2007) Safety for the target species Since TBCC is a compound of copper not authorised in the EU, tolerance studies are considered necessary. It is known, as well, that copper can cause negative effects, depending on the dose and the animal species/category Tolerance studies Three tolerance studies have been provided by the applicant, two in chickens for fattening (one with two experiments), and one in laying hens. In all studies TBCC was compared to equivalent amounts of copper from copper sulphate pentahydrate. The experimental data were analysed by least squares analysis of variance using the General Linear Models procedure of the SAS Institute Chickens for fattening In study 1 (Miles et al., 1998), two experiments were conducted to study copper sulphate and TBCC as sources of supplemental copper for poultry. In experiment 1, 288 chicks were fed the basal maize-soybean diet (containing 23 % crude protein (CP), 12.7 MJ ME/kg and 26 mg Cu/kg expressed on dry matter basis (DM)) supplemented with 0, 150, 300, and 450 mg/kg Cu from copper sulphate and TBCC, respectively, for 21 days. Overall mortality was 2.1 % and not treatment related. Chicks fed 450 mg/kg Cu from copper sulphate had significantly lower feed intake (695 g) than those consuming the other diets ( g); also body weight was depressed in this group (485 g) compared to the other treatments ( g). Feeding supplemental copper significantly increased liver copper concentration in similar way with increasing dietary copper, regardless of copper source. Liver copper increased from 18 mg/kg liver dry weight in the unsupplemented control to 24, 102, 185 and to 20, 72, 204 at 150, 300 and 450 mg Cu from copper sulphate and TBCC, respectively/kg feed. In experiment 2, a 42-day floor pen study was conducted with 1260 broiler chicks (both sexes) given the basal maize-soybean diet (20/11 mg Cu/kg starter/grower) supplemented with 0, 200, 400, or 600 mg/kg Cu from either copper sulphate or TBCC. Body weight (1960 g in the control group, 1878 g for the groups with 200 and 400 mg Cu from copper sulphate, 1889 g for the groups with 200 and 400 mg Cu from TBCC/kg) and feed/gain ratio (1.76 g in the control group, 1.76 for the groups with 200 and 400 mg Cu from copper sulphate, 1.79 g for the groups with 200 and 400 mg Cu from TBCC/kg) did not differ in birds fed up to 400 mg/kg Cu from either source. Birds given 600 mg/kg Cu from either copper source had significantly lower feed intake, poorer growth, and feed conversion (1469 and 1261 g body weight, 2.10 and 2.08 feed/gain for the copper sulphate and the TBCC treatments, respectively). Liver copper increased with increasing dietary copper. The liver copper increased from 14 mg/kg liver dry weight in the unsupplemented control to 18, 102, 713 and to 18, 102, 870 at 200, 400 and 600 mg Cu from copper sulphate and TBCC, respectively/kg feed. 21 Technical Dossier, Section IV, Annex 4.2.a 9

10 In study 2 (Luo et al., 2005), a total of 420 day-old male chickens for fattening (Arbor Acres) was fed in groups of six replicates with ten birds each maize-soybean diets (22.4 % CP, 12.8 MJ ME/kg, 11.5 mg Cu/kg, 18 mg vitamin E/kg) without supplemental copper, with 150, 300 and 450 mg supplemental copper from copper sulphate pentahydrate and TBCC, respectively, for 21 days. Dietary copper was confirmed by analysis. Feed and water were available ad libitum. At the end of the experiment, chicks were weighed individually and four birds were chosen from each cage according to average body weight for collection of blood and liver samples. Mortality was low (0.7 %) and not treatment related. Feed intake and average daily gain remained unaffected when as much as 450 mg/kg supplemental copper was fed in the form of TBCC. There was a decrease, however, in both feed intake and average daily gain when similar supplemental levels of copper were provided as sulphate. Liver copper changed from 6 mg/kg liver fresh weight in the unsupplemented control to 6, 11, 29 and to 6, 15, 50 at 150, 300 and 450 mg Cu from copper sulphate and TBCC, respectively/kg feed. Copper source, added copper level, or an interaction between copper source and level affected liver copper significantly Laying hens A total of 1560 laying hens (Hy-Line W-36) in the first phase of the first cycle (21-week old) was randomly divided into 520 cages with each cage housing three birds (Liu et al., 2005). Each cage was considered as an experimental unit. Five adjoining cages were considered as a replicate, and then the 104 replicates were randomly assigned to the 13 dietary treatments. The hens were fed a basal maize-soybean diet (18.9 % CP, 12.1 MJ ME/kg) or a basal diet supplemented with 65, 130, 195, 260, 325, or 390 mg/kg of copper from either copper sulphate or TBCC for 16 weeks. Analytically verified copper concentrations in diets were not reported. No significant differences in the cumulative feed intake between birds fed diets with copper sulphate or TBCC were observed among the different supplemental copper levels. Hens consumed approximately 88.5 g of feed/hen per day during the study. No significant differences among the different supplemental copper levels were obtained for average egg production, egg mass and egg weight in birds fed copper sulphate or TBCC. The average egg production, egg mass and egg weight was 84 %, 47 g/hen per day, and 53.7 g, respectively, during the study. There were also no differences of average feed/gain conversion and body weight between birds fed copper sulphate and TBCC among the different supplemental copper levels Conclusions on the safety for target species Since maximum total content of copper in complete feedingstuffs for poultry is limited to 25 mg/kg, dosages as 450 and 600 mg Cu from a copper-containing additive, as applied in the experiments on chickens for fattening, represent an 18- and 24-fold overdose, respectively. The maximum tolerated level is estimated by the NRC (2005) with 250 mg Cu/kg complete poultry diet. Although two of the three experiments in chickens for fattening were conducted for 21 days only, and thus shorter than required by Regulation (EC) No 429/2008, and no haematology or clinical biochemistry was performed in any study, the FEEDAP Panel considers these experiments together as suitable data set to assess the safety of TBCC for chickens for fattening. All TBCC data could be compared with another copper source, copper sulphate pentahydrate, the standard copper source in commercial diets. Feed intake, body weight and feed to gain ratio in the 42-day study showed that 600 mg Cu/kg feed, from either TBCC or copper sulphate, was not tolerated. In both 21-day studies, both feed intake and body weight gain indicated tolerance to 450 mg copper from TBCC/kg, but not for the same dose from copper sulphate. In chickens for fattening, critical high levels of dietary copper from TBCC are at least equally tolerated as from copper sulphate. 10

11 The 16-week laying hens study demonstrated that 390 mg Cu from TBCC/kg diet did not affect performance endpoints. The FEEDAP Panel concludes that TBCC is a safe source of copper for poultry with a margin of safety 10. Huge differences in the maximum tolerable copper concentrations between animal species exist (e.g. 500 mg/kg for rodents; 250 mg/kg for poultry, pigs and horses; 100 mg/kg for fish; 40 mg/kg for cattle, 15 mg/kg for sheep) (NRC, 2005). Therefore, it cannot be expected that all animal species would share the same margin of safety of a copper-containing additive considering the maximum total copper content in feed set by Regulation (EC) No 1334/2003. Consequently a safe copper-containing additive could theoretically have a margin of safety (maximum tolerable concentration/maximum content authorised) of 10 in poultry and pigs (250/25), of 4 for fish (100/25), of 1.1 for cattle (40/35) and of 1 for ovines (15/15). Additional studies could only provide results in the dimension of the above theoretically derived margins of safety. Since the margin of safety derived for TBCC in studies on poultry meets the dimension of the theoretically optimum values (~ 10), the FEEDAP Panel expects that TBCC would behave similarly concerning more sensitive species. In this specific case, the FEEDAP Panel does not expect that further essential information on the safety of TBCC would be provided by additional tolerance studies with other (more sensitive) animal species. The FEEDAP Panel concludes that TBCC is a safe source of copper for all animal species/categories up to the maximum total copper content authorised in feed. This conclusion covers also the reformulated additive Safety for the consumer Comparisons of copper concentration in liver from poultry, growing cattle and piglets, and from plasma copper and ceruloplasmin levels in cattle demonstrate bioequivalence between copper sulphate and TBCC (see sections 3.1 and 4). The FEEDAP Panel therefore, considers that no substantial differences in the metabolic behaviour occur, particularly in the deposition of copper in edible tissues and products, from copper sulphate and TBCC. Consequently, the FEEDAP Panel considers that exposure of the consumer to copper from food of animal origin would not be essentially modified by the replacement of copper sulphate by TBCC Safety for the users/workers Effects on the skin and eye No data are available on skin sensitisation or irritancy to skin or eyes. In the absence of such data, the additive is to be considered as a potential irritant to skin and eyes and a potential skin sensitiser Effects on the respiratory system In its reformulated form, the additive does not contain particles smaller than 50 μm. Therefore, acute inhalation toxicity studies are not required. The amount of particles below 100 µm ranged from 0.29 to 2.52 % (five batches). Significant exposure via inhalation is considered unlikely, particularly as the dusting potential of the additive is low (0.03 g/m 3 air) Conclusions on user safety The FEEDAP Panel concluded that TBCC should be considered as a potential irritant to skin and eyes, and a potential skin sensitiser. Since the additive does not contain particles smaller than 50 µm and the dusting potential is low, the risk of respiratory exposure is considered to be minimal. 11

12 3.4. Safety for the environment Up to date, there is no information suggesting that TBCC would be more harmful to the environment than other inorganic sources of copper already authorised. The FEEDAP Panel considers that the use of TBCC in feed does not represent additional risks to the environment, compared to other sources of copper for which it will substitute, provided that the maximum authorised content in feedingstuffs is not exceeded. 4. Efficacy/Bioequivalence 4.1. Poultry From the tolerance studies reported in section 3.1, some data could be considered as demonstration of bioequivalence of TBCC to copper sulphate pentahydrate. No differences could be observed concerning liver deposition in three experiments with chickens for fattening. Also the multilinear regression equations obtained for the increase in egg weight by copper, and laying hen performance were comparable for both copper sources Growing cattle Two publications with two experiments each (one in beef heifers and one in cattle for fattening) were submitted Beef heifers Two 90-day experiments were conducted using 48 non-pregnant, crossbred heifers (24 heifers/experiment; 355 and 309 kg body weight in experiment 1 and 2, respectively) (Arthington and Spears, 2007). In each experiment, three dietary treatments were randomly allocated to heifers in individual pens consisting of (1) 100 mg of Cu/day from copper sulphate, (2) 100 mg of Cu/day from TBCC, or (3) 0 mg of Cu/day. The two experiments differed on the form of the supplement used to deliver the copper treatments (maize- vs. molasses-based supplements for experiment 1 and 2, respectively). Supplements were formulated and fed to provide equivalent amounts of crude protein (CP) and total digestible nutrients daily but differed in their concentration of the copper antagonists: molybdenum (Mo) (0.70 vs mg/kg), iron (Fe) (113 vs. 189 mg/kg), and sulphur (S) (0.18 vs %) for maize- and molasses-based supplements, respectively. All heifers were provided free-choice access to ground stargrass (Cynodon spp.) hay. Jugular blood and liver biopsy samples were collected on day 0, 30, 60, and 90 of each experiment. Body weight was determined on day 0 and 90. The average daily gain (ADG) of heifers was not affected by copper treatment (average: 0.22 and 0.44 kg for experiment 1 and 2, respectively; P> 0.20). Regarding copper concentration in liver in experiment 1, heifers provided supplemental copper, independent of the source, had greater (P< 0.05) liver copper concentrations on day 60 and 90 compared with heifers provided no supplemental copper. In experiment 2, average liver copper concentrations were greater (P= 0.04) for heifers receiving supplemental copper compared with heifers receiving no copper; however, all treatments experienced a decrease in liver copper concentration over the 90-day treatment period. In both experiments, there were no differences between the two sources of copper. Plasma ceruloplasmin concentrations did not differ in experiment 1, but were greater (P= 0.04) in experiment 2 for heifers receiving supplemental copper compared with heifers receiving no copper Cattle for fattening In experiment 1, TBCC was compared to copper sulphate in terms of ability to maintain copper status when supplemented to steers fed diets high in the copper antagonists, Mo and S (Spears et al., 2004). Sixty Angus and Angus Hereford steers (257±2 kg bw) were stratified by weight and randomly assigned to treatments. Treatments consisted of 0, 5 or 10 mg supplemental Cu/kg diet 12

13 DM from either TBCC or copper sulphate. All diets were supplemented with 5mg Mo/kg and 1.5 g S/kg. The control maize silage based diet was analysed for its copper, molybdenum and sulphur content resulting in 4.9 mg Cu, and 6.9 mg Mo and 3.0 g S per kg. Plasma and liver copper concentrations and plasma ceruloplasmin activity decreased (P< 0.01) in all treatment groups during the 98-day study. Copper supplemented steers had higher (P< 0.01) plasma copper, plasma ceruloplasmin, and liver copper than controls at the end of the study. Steers supplemented with 10 mg Cu/kg had higher (P< 0.01) plasma copper, plasma ceruloplasmin, and liver copper than those receiving 5 mg Cu/kg diet. Bioavailability of copper from TBCC, relative to copper sulphate, was estimated from plasma copper and ceruloplasmin on day 84 and liver copper on day 98 using multiple linear regressions and a slope ratio technique. Regarding all endpoints, TBCC was at least as bioavailable as copper sulphate when added to diets high in the copper antagonists, Mo and S. In experiment 2, 43 Angus and Simmental steers (375±7 kg bw) that had previously been depleted of copper were used in a 21-day repletion study (Spears et al., 2004). Steers were randomly assigned within breed to treatment, and individually fed a maize silage based diet low in Mo (1.18 mg/kg). Treatments consisted of 0, 50 or 100 mg supplemental Cu/day from either TBCC or copper sulphate. Plasma copper, plasma ceruloplasmin and liver copper increased (P< 0.01) in copper supplemented, but not in control, steers. Plasma and liver copper concentrations increased (P< 0.01) to a greater extent in steers receiving 100 mg Cu/day compared to those given 50 mg Cu/day. TBCC and copper sulphate were similar (P> 0.10) in their ability to increase copper status in copper depleted steers fed a diet low in Mo. Therefore, when evaluated in copper depleted steers fed diets low in Mo, the two copper sources had a similar bioavailability Weaned piglets A publication describing three 28-day experiments involving a total of 915 weaned piglets to assess the relative efficacy of TBCC and copper sulphate was submitted (Cromwell et al., 1998). Experiments 1 and 2 were conducted at a research station while experiment 3 was conducted at a commercial swine research facility. The basal diet was a fortified maize-soybean meal-dried whey diet (1.25 % lysine) with no antimicrobials in experiment 1, or with carbadox (55 mg/kg) in experiments 2 and 3. In experiment 1, 135 pigs (Hampshire-Yorkshire; barrows/females) were weaned at day 27 to 31 (initial and final body weight: 7.9 and 17.7 kg, respectively) and fed the basal diet (16 mg/kg Cu) with or without 100 or 200 mg/kg Cu from TBCC or from copper sulphate, with five replicates of four to six piglets each. Copper concentrations in diets were analytically verified. The 200 mg/kg level of copper from copper sulphate improved ADG (P< 0.10), and both levels of copper from TBCC tended to improve feed/gain ratio. In experiment 2, 150 pigs (Hampshire-Yorkshire; barrows/females) were weaned at day 27 to 31 (initial and final body weight: 8.9 and 20.8 kg) and fed the basal diet (18 mg/kg Cu) with or without 100, 150, or 200 mg/kg Cu from TBCC, or 200 mg/kg Cu from copper sulphate, with six replicates of five piglets each. Copper concentrations in diets were analytically verified. The basal diet contained 18 mg/kg Cu. Addition of 200 mg/kg Cu improved ADG (P< 0.08) and average daily feed intake (P< 0.01), but not feed/gain. Source of copper did not affect performance. In experiment 3, 630 terminal crossbred pigs (PIC: Pig Improvement Co., Franklin, KY; mixed sexes) were weaned at day 16 to 20 and fed a common diet for 10 to 12 days until the start of the experimental period. The same experimental diets as used in experiment 2 were fed from 9.1 to 25.5 kg body weight, with six replicates of 21 piglets each. Copper concentrations in diets were analytically verified. Both copper sources improved ADG at the same copper concentration (P< 0.01). Overall, copper concentrations in liver increased in pigs fed 200 mg/kg Cu compared to the other treatments. Copper sulphate tended to increase copper concentration in liver more than did TBCC 13

14 in one experiment (experiment 1; copper sulphate: 39, 31, 127 mg/kg DM; TBCC: 39, 32, 83 mg/kg DM) but not in another experiment (experiment 2; copper sulphate: 96 mg/kg DM; TBCC: 89 mg/kg DM) Conclusions on efficacy of TBCC as source of dietary copper Three experiments in chickens for fattening demonstrated the bioequivalence of copper from TBCC and from copper sulphate based on liver copper deposition as endpoint. Further indirect evidence for bioequivalence could be drawn from the study on laying hens based on egg weight and laying hen performance. In four experiments in growing cattle, bioequivalence of copper from TBCC or copper sulphate could be shown. It was based on copper deposition in liver and plasma ceruloplasmin for heifers (two experiments). The same endpoints plus plasma copper were used to demonstrate bioequivalence in cattle for fattening fed diets high in copper antagonists (Mo and S) in one experiment, and in another depletion-repletion experiment. Three experiments in piglets indicated that TBCC is as effective as copper sulphate in improving growth in weaned piglets. Considering the results of 11 experiments in chickens for fattening, laying hens, growing cattle and piglets, the FEEDAP Panel concludes that TBCC is an effective source of copper for all animal species. This conclusion covers also the reformulated additive. 5. 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 22 and Good Manufacturing Practice. CONCLUSIONS AND RECOMMENDATIONS The conclusions and recommendations are derived from the original product (safety for the target animals, safety for consumers and efficacy) and the reformulated product (safety for the user/worker). Both products are considered bioequivalent. CONCLUSIONS Based on three studies in chickens for fattening and one in laying hens, the FEEDAP Panel concludes that TBCC is a safe source of copper for poultry with a margin of safety 10. Considering that in the poultry studies no substantial differences were observed in the endpoints measured between copper sulphate and TBCC, the FEEDAP Panel does not expect that further essential information on the safety of TBCC would be provided by additional tolerance studies with other (more sensitive) animal species. The FEEDAP Panel concludes that TBCC is a safe source of copper for all animal species/categories up to the maximum total copper content authorised in feedingstuffs. The FEEDAP Panel does not expect substantial differences in the metabolic behaviour, particularly in the deposition of copper in edible tissues and products from copper sulphate and TBCC. Consequently the FEEDAP Panel considers that exposure of the consumer to copper from food of animal origin would not be essentially modified by the replacement of copper sulphate by TBCC in feed. In the absence of data, TBCC should be considered as a potential irritant to skin and eyes, and a potential skin sensitiser. The risk of respiratory exposure is considered to be minimal. 22 OJ L 35, , p

15 The FEEDAP Panel considers that the use of TBCC in feed does not represent additional risks to the environment, compared to other sources of copper for which it will substitute, provided that the maximum authorised total copper content in feedingstuffs is not exceeded. Considering the results of 11 experiments in chickens for fattening, laying hens, growing cattle and piglets demonstrating bioequivalence between TBCC and copper sulphate with regards to copper liver deposition, plasma levels of ceruloplasmin and copper, egg weight and laying hen performance, and growth of piglets, the FEEDAP Panel concludes that TBCC is an effective source of copper for all animal species. The FEEDAP Panel sees no reason to increase the existing maximum authorised copper content in feedingstuffs for Other species as proposed by the applicant. RECOMMENDATIONS TBCC should be specified as 98 % pure, with a corresponding minimum copper content of 58 %. Since the assessment of user/worker safety has been based on a TBCC/starch product with no particles below 50 µm, the FEEDAP Panel considers essential the inclusion of the following specification in any authorisation: Particles< 50 µm below 1 %. The FEEDAP Panel recommends that Annex I of Directive 2002/32/EC is adapted to include Di copper chloride tri hydroxide, setting the same maximum content for arsenic as that for copper sulphate pentahydrate and copper carbonate (50 mg/kg additive). DOCUMENTATION PROVIDED TO EFSA 1. Di copper chloride tri hydroxide (TBCC). June Submitted by Dr. Christian Elwert. 2. Di copper chloride tri hydroxide (TBCC). Supplementary information (I). March Submitted by Dr. Christian Elwert. 3. Di copper chloride tri hydroxide (TBCC). Supplementary information (II). June Submitted by Dr. Christian Elwert. 4. Di copper chloride tri hydroxide (TBCC). Supplementary information (III). August Submitted by Dr. Christian Elwert. 5. Evaluation report of the European Union Reference Laboratory for Feed Additives on the methods(s) of analysis for Tribasic copper chloride (TBCC). Amendment to EURL Final Report on the dossier FAD (Tribasic copper chloride) 6. Comments from Member States received through the ScienceNet. REFERENCES Arias VJ and Koutsos EA Effects of Copper Source and Level on Intestinal Physiology and Growth of Broiler Chickens. Poult. Sci. 85, Arthington JD and Spears JW Effects of tribasic copper chloride versus copper sulfate provided in corn-and molasses-based supplements on forage intake and copper status of beef heifers. J. Anim. Sci. 85, Cromwell GL, Lindemann MD, Monegue HJ, Hall DD and Orr DE Jr Tribasic copper chloride and copper sulfate as copper sources for weanling pigs. J. Anim. Sci. 76, EFSA (European Food Safety Authority), Safety and efficacy of Mintrex Cu (Copper chelate of hydroxy analogue of methionine) as feed additive for all species - Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed. The EFSA Journal 693,

16 Liu Z, Bryant MM and Roland DA Sr Layer performance and phytase retention as influenced by copper sulfate pentahydrate and tribasic copper chloride. J. Appl. Poult. Res. 14: Luo XG, Ji F, Lin YX, F. Steward A, Lu L, Liu B and Yu SX Effects of dietary supplementation with copper sulfate or tribasic copper chloride on broiler performance, relative copper bioavailability, and oxidation stability of vitamin E in feed. Poult. Sci. 84, Miles RD, O Keefe SF, Henry PR, Ammerman CB and Luo XG The effect of dietary supplementation with copper sulfate or tribasic copper chloride on broiler performance, relative copper bioavailability, and dietary prooxidant activity. Poult. Sci. 77, NRC (National Research Council) Mineral Tolerance of Animals: Second Revised Edition. Natl. Acad. Press. Washington, D.C. Pang Y and Applegate TJ Effects of dietary copper supplementation and copper source on digesta ph, calcium, zinc and copper complex size in the gastrointestinal tract of broiler chickens. Poult. Sci. 86: Spears JW, Kegley EB and Mullis LA Bioavailability of copper from tribasic copper chloride and copper sulfate in growing cattle. Anim. Feed Sci. and Technol. 116, Stauber D and Beutel R Determination and control of the dusting potential of feed premixes. Fresenius Z. Anal. Chem 318,

17 APPENDIX Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for Tribasic copper chloride (TBCC) In the current application authorisation is sought under articles 4(1) for Di copper chloride tri hydroxide under the category "nutritional additives", functional group 3(b) "compounds of trace elements", according to the classification system of Annex I of Regulation (EC) No 1831/2003. Authorisation is sought for the use of the feed additive for all animal species and categories. Di copper chloride tri hydroxide (Cu 2 Cl(OH) 3, also called tribasic copper chloride TBCC) is a pure form of crystalline copper (II) chloride hydroxide containing a minimum of 95 % of a defined ratio of the polymorphs atacamite and paratacamite - equivalent to a minimum content of total copper of 58 %. The feed additive is intended to be incorporated into premixtures and feedingstuffs. The Applicant suggested the following maximum levels of total copper in the feedingstuffs ranging from 10 to 170 mg/kg depending on the species of interest. For the identification of TBCC in the feed additive, the Applicant submitted an X-ray diffraction (XRD) method to confirm the crystal forms of TBCC. For the determination of total copper in the feed additive, premixtures and feedingstuffs the Applicant submitted the internationally recognised ring trial validated method EN 15510, based on inductively coupled plasma atomic emission spectroscopy (ICP-AES). The following performance characteristics were reported: - a relative standard deviation of repeatability (RSD r ) ranging from 2.9 to 12 %; - a relative standard deviation for reproducibility (RSD R ) ranging from 8 to 22 %; and - a limit of quantification of 3 mg/kg. The EURL identified an alternative CEN ring-trial validated method (CEN/TS 15621) based on ICP-AES after pressure digestion, for the determination of total copper in the feed additive, premixtures and feedingstuffs. The total copper concentration is determined using external calibration or standard addition technique. The following performance characteristics were reported for a feed for pigs, and for sheep, a rock phosphate, a mineral premix and a mineral mix, where the total copper content ranged from 7.3 to 470 mg/kg: - RSD r ranging from 2.6 to 6.8 %; - RSD R ranging from 3.8 to 12; and - LOQ = 1 mg/kg feedingstuffs. Furthermore, a Community method is available for the determination of total copper in feedingstuffs, but no performance characteristics for the method were provided. The UK Food Standards Agency recently reported results of a ring-trial based on the above mentioned Community method, and reported precisions (RSD r and RSD R ) for feedingstuffs ranging from 2.4 to 9.2 %. Based on these acceptable method performance characteristics the EURL recommends for official control the CEN methods EN or CEN/TS to determine total copper content by ICP- AES in the feed additive and premixtures. As for the determination of total copper content in feedingstuffs, the EURL recommends for official control the Community method based on AAS and the above mentioned CEN methods (EN or CEN/TS 15621). Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by Article 10 (Commission Regulation (EC) No 378/2005) is not considered necessary. 17

18 Amendment to EURL Final Report on the dossier FAD (Tribasic copper chloride) Background Di copper chloride tri hydroxide (Cu2Cl(OH)3, also called tribasic copper chloride TBCC) is a pure form of crystalline copper (II) chloride hydroxide containing a minimum of 95 % of a defined ratio of the polymorphs atacamite and paratacamite - equivalent to a minimum content of total copper of 58 %. In the above mentioned report the EURL concluded that X-ray diffraction is suitable for the identification of di copper chloride tri hydroxide atacamite/paratacamite crystal forms in the feed additive, whereas various standard methods are available for the determination of total copper in the feed additive, premixtures and feedingstuffs. On 12/08/2011 EFSA informed the EURL that the applicant has changed the composition of the feed additive, namely by adding 5 % of starch thereby reducing the copper content in the feed additive accordingly. In consequence EFSA requests a statement from the EURL, whether the conclusions on the analytical methods previously evaluated against the former composition of the additive are still valid for the new formulation. Conclusion Based on a new evaluation the EURL concludes that the slightly modified composition of the additive does not influence the suitability of the analytical methods as previously stated in the above mentioned EURL Final Report for official control purposes. Therefore the conclusions in this report are also valid for the new formulation. 18