The EFSA Journal (2005) 224, 1-35

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1 The EFSA Journal (2005) 224, 1-35 Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on a request from the Commission on the evaluation of safety and efficacy of Hy D (calcifediol), based on 25-hydroxylcholecalciferol/25-hydroxy-pre-cholecalciferol, as feed additive in accordance with Council Directive 70/524/EEC. (Question N EFSA-Q ) SUMMARY Adopted on 26 May 2005 Vitamin D (calciferol) and its metabolites are essential micronutrients for the normal (skeletal) development of men and animals. They are closely associated with the calcium metabolism. Vitamin D 2 and D 3 are approved additives. Hy D consists of 12.5 g 25-hydroxylcholecalciferol (25-OH-D 3 ) kg -1, which is the first metabolite of vitamin D 3 normally hydroxylated in the liver of men and animals. The European Food Safety Authority has been requested by the European Commission to issue an opinion on the safety for target species, consumers, users and environment and on the efficacy of the product of trade name Hy D (calcifediol), when this product is used up to a maximum content of 5000 IU kg -1 complete feedingstuff for chickens and turkeys for fattening and 3000 IU kg -1 for laying hens (1µg 25-hydroxylcholecalciferol 40 IU). Sufficient information on physical and chemical properties, the method of production, on stability and dusting potential of the substance as well as on control methods is given by the applicant. No DNA from the production process is expected to be present in the final product. Despite the fact that the design of the efficacy studies submitted presents shortcomings (i.e., studies were conducted at high doses), the data supports clearly that 25-OH-D 3 is at least as effective as vitamin D 3 in optimizing performance of chickens for fattening, laying hens and turkeys. Quality of the animal products was not significantly influenced by the source of vitamin D. Since 25-OH-D 3 is more potent in its vitamin D activity than vitamin D 3, but higher potency depends on and varies with the criterion assessed and the dosage applied, reliable information to the user of the product Hy D can scientifically not be given in terms of IU of vitamin D. Therefore the FEEDAP Panel strongly recommends labelling of 25-OH-D 3 in µg. If for practical reasons this is not immediately possible then the label of the product should include (i) the potency (IU vitamin D, 1 µg of 25-OH-D 3 should be considered as 80 IU Vitamin D 3 ) and (ii) the source of the vitamin (from Vitamin D 3, Vitamin D 2 or Calcifediol). Tolerance studies were carried out in chickens for fattening, turkeys and layers. 100 µg 25-OH-D 3 kg -1 complete feed is well tolerated and could be accepted as the upper tolerated limit for chickens for fattening. As long as no more specific data on target animal safety are available and considering the above recommendation for labelling 25-OH-D 3 in µg kg -1 complete feed, the maximum 25-OH-D 3 content for chickens for fattening should be set with 100 µg 25-OH-D 3 kg -1 complete feed (a level proven as safe). A similar deduction leads to a proposal of 80 µg 25-OH- D 3 kg -1 as maximum content for laying hens. In contrast to these categories, turkeys seem to tolerate doses up to 500 µg 25-OH-D 3 kg µg 25-OH-D 3 kg -1 feed level could be applied for turkeys for fattening as maximum content.

2 Opinion on the additive Hy D (Calcifediol) 2/35 The margin of safety for chickens for fattening and laying hens could not be established, due to the shortcomings in the design of the studies. The margin of safety of the upper recommended level for turkeys could be given as about 5. In birds as well as in humans 25-OH-D 3 is the initial metabolite of vitamin D 3. It is likely that the ingested 25-OH-D 3 undergoes the same metabolic fate as the endogenous compound. No retroconversion of 25-OH-D 3 to vitamin D 3 occurs. At the highest 25-OH-D 3 dose recommended for use as feed additive in poultry, retained by the FEEDAP Panel, Hy D does not increase significantly the exposure of the consumer to 25-OH-D 3 through the consumption of turkey tissues when compared to the levels found following vitamin D 3 supplementation at a same level. The exposure resulting from turkey plus eggs consumption (3.5 µg day -1 ) estimated from theoretical and worst case consumption figures retained by the FEEDAP Panel, represents 35% and 70% of the provisional upper limit (UL) proposed by the FEEDAP Panel for the adult and children respectively. When chickens for fattening are concerned, a similar calculation leads to a consumer exposure value for chicken plus egg of 6.4 µg day -1 which complies with the provisional UL for adults (64%) but is above that for children (128%). Using more realistic consumption data, the consumer exposure appears to be below the provisional UL for both the adult (23%) and the children (46%). Therefore, should 25-OH-D 3 from Hy D be used as a substitute of vitamin D 3 for chickens for fattening and turkeys for fattening at the maximum tolerated dose of 100 µg kg -1 feed, and for laying hens at the maximum tolerated dose of 80 µg kg -1 feed, as retained by the FEEDAP Panel, no additional risk for the consumer could be expected. The substitution of vitamin D 3 by 25-OH-D 3 from Hy D should reduce considerably the vitamin D 3 contents of poultry tissues and products (eggs). As a general principle the FEEDAP Panel considers conventional toxicological studies to be inappropriate for testing pure chemically defined substances which are dietary nutrients, which is the case for 25-OH-D 3 from Hy D for which the chemical purity is established. The data submitted give some indications that 25-OH-D 3 is not genotoxic and confirm that the acute, subchronic and reproductive toxicological effects observed are entirely consistent with a physiological overload of vitamin D 3 or its metabolites. Regarding the safety for the user, the product is not an irritant to the skin or eyes. Sensitisation and respiratory effects of Hy D have not been characterised. Hy D is at such low concentrations in the final feed to be of negligible concern apart from for those groups who may already be using medication based upon Vitamin D or 25-OH-D 3. The use of protective clothing should be sufficient to avoid adverse effects in users. The FEEDAP Panel concludes that there is no necessity to perform an environmental risk assessment for this type of naturally existing compounds, under the conditions of the proposed use. The FEEDAP Panel recommends that only 25-hydroxylcholecalciferol will be specified in the annex entry including the minimum content requested (>94%). The addition of both vitamin D sources, vitamin D 3 and 25-OH-D 3, should not be permitted and this information should be included in the annex entry. Key words: Vitamin D, Vitamin D 3, 25-Hydroxycholecalciferol, Chickens for fattening, Turkeys, Laying hens.

3 Opinion on the additive Hy D (Calcifediol) 3/35 TABLE OF CONTENTS 1. Introduction Composition and characteristics of Hy D Chemical and physical characteristics of 25-OH-D Stability Shelf life of Hy D Premixes Complete feeds and treatments Control methods Efficacy of the product in target species Recommendations for vitamin D 3 in poultry Efficacy trials Chickens for fattening Growth performance and feed conversion ratio Bone mineralization Laying hens Turkeys Studies on the quality of animal produce Bioequivalence and labelling Conclusion Safety studies on target species Tolerance studies Chickens for fattening Laying hens Turkeys Conclusions on the safety for the target animals Fate of 25-OH-D 3 and body deposition Fate of 25-OH-D Deposition Chickens for fattening Laying hen eggs Turkeys Conclusions Studies on laboratory animals Conclusions Safety evaluation for the human consumer Human use of 25-OH-D Status of 25-OH-D 3 in humans Biological activity of 25-OH-D The Tolerable Upper Limit (UL) for humans Consumer exposure to 25-OH-D Conclusion User safety assessment Skin irritation Eye irritation Pure 25-OH-D Formulated product in beadlets (1.25% 25-OH-D 3 ) Hy D supplemented feed Conclusions Safety for the Environment Conclusions Documentation provided to EFSA References Scientific Panel Members... 35

4 Opinion on the additive Hy D (Calcifediol) 4/35 Acknowledgement... 35

5 Opinion on the additive Hy D (Calcifediol) 5/35 Background Council Directive 70/524/EEC 1 lays down rules governing the Community authorisation of additives for animal nutrition and, in particular, defines the conditions that substance/product should meet to be granted authorisation. The Commission received a dossier from the applicant company, Roche Vitamins Ltd, through Spain, the Rapporteur Member State, to obtain authorisation on the product Hy D (calcifediol), based on 25-hydroxylcholecalciferol/25-hydroxy-pre-cholecalciferol, when it is used as a feed additive for chickens for fattening, turkeys and laying hens, according to the conditions referred in Table 1. This additive has not been previously authorized at Community level and the company requested an authorisation in the category of vitamins feed additives. Table 1. Condition of use Hy D (calcifediol), based on 25-hydroxylcholecalciferol/25- hydroxy-pre-cholecalciferol) No. (or EC No.) Additive Chemical formula, description Species or category of animal Maximum age Minimum content Maximu m content IU 2 kg -1 of complete feedingstuff Other provisions Calcifediol 25 hydroxylchole calciferol/2 5-hydroxypre-chole calciferol Chickens for fattening Turkeys Laying Hens The mixture of Calcifediol with vit.d 3 is allowed provided that the total amount of the mixture does not exceed 5000 IU kg -1 feeding stuff for chickens for fattening and turkeys and 3000 IU -1 kg feedingstuffs for laying hens. TERMS OF REFERENCE The Commission requests the European Food Safety Authority to issue an opinion on the safety for consumer, target species, user and environment and on the efficacy of the product of trade name Hy D (calcifediol), when this product is used under the above mentioned conditions. ASSESSMENT 1. Introduction Vitamin D 3 (cholecalciferol) is one of a number of sterols that are present naturally in animals and are structurally very similar. Vitamin D and their metabolites (including 25-OH-D 3 ) are essential 1 O.J n L 270 of , p.1 2 Reference standard: cholecalciferol (IU) 1 µg 25 hydroxycholecalciferol is considered by the notifier as equivalent to 40 IU

6 Opinion on the additive Hy D (Calcifediol) 6/35 lipophilic micronutrients required for the normal development of animals. Vitamin D 3 is produced by the action of UV radiation (usually sunlight) on the skin of animals and man from the precursor 7-dehydrocholesterol or it is provided in the diet. Vitamin D 2 (ergocalciferol) is the corresponding sterol in plants. Vitamin D and its metabolites are closely associated with the absorption of calcium by animals and its deposition in the skeletal tissue. Due to this association it is known as the anti-rachitic vitamin, preventing the bone disorder, rickets. In nature vitamin D is available in the diet of man and animals, in vegetables and animal foodstuffs especially those that contain lipids. Vitamin D deficiencies can occur in animals that are reared in the absence of sunlight and supplemental vitamin D. These animals can suffer from hypocalcaemia, stunted growth, poor health, skeletal problems, and in the case of laying birds, thin shelled eggs. Since most poultry and pig production in the EU occurs in housed conditions (indoors) there is a necessity to make sure that vitamin D, calcium and phosphorus is provided in the diet in adequate quantities to maintain animal health, welfare and production. Vitamin D 2 is not allowed to be used in poultry due to its lack of efficacy. Because of the intensive rearing conditions associated with commercial poultry production, vitamin D 3 has been added to the diets of poultry, as well as other animals, for a considerable time period to ensure good health. In the normal metabolic pathway in animals vitamin D 3 is absorbed and transported to the liver where it is hydroxylated to produce the intermediate compound 25-hydroxycholecalciferol (25- OH-D 3 ) (Figure 1) which is subsequently further metabolised to participate in reactions that influence the absorption of calcium and phosphorus. Thus, it is logical to provide 25-OH-D 3 in the diet so that it is available for use without synthesis within the animal. Detailed reviews of the biochemistry, biosynthesis and of the effects of vitamin D in the diets of animals and man have been published recently (De Luca, 2004, Feldman et al., 2005, McDowell, 2000, Raiten and Picciano, 2004, Sutton and Mc Donald, 2003) Composition and characteristics of Hy D Hy D is a product manufactured in a beadlet form which contains a minimum of 12.5 g kg -1 of the active substance 25-OH-D 3. The rest of the product is food-grade cotton-seed oil (897 g kg -1 ), edible fatty acids (40 g kg -1 ), colloidal silica (30 g kg -1 ) as anti-dusting, emulsifying and anticaking agents respectively, and butylated hydroxy toluene (BHT) (20 g kg -1 ) and citric acid (0.5 g kg -1 ) as anti-oxidants The Hy D beadlets are reported to have a melting point of 60 C which is presumably due to the cotton-seed oil. Hy D is packaged in polyethylene bags which are then sealed in cardboard drums. 6 Analysis of production batches demonstrated that Hy D averaged a content of 25-OH-D 3 of 13.5 g kg -1 (variation of % of the claim on the labels). 7 Hy D is proposed to be added to poultry diets at concentrations between 3.2 to 8.0g t -1 of diet depending on poultry species and other sources of vitamin D. Prior to addition to the diet the Hy D would be premixed with a feed ingredient. Particle size is mostly (>95%) in the range µm with 3-4% of particles being <10 µm in diameter Chemical and physical characteristics of 25-OH-D 3 The structures of 25-OH-D 3 and vitamin D 3 are presented in Figure 1 and a list of some chemical and physical properties are presented in Table 2. The footnotes referred to the studies provided by the applicant in the dossier. 3 Volume 1-6. Section II.2. 4 Volume 6. Annex 5. 5 Volume 6. Annex 9. 6 Volume 1-6. Section II. 7 Volume 6. Annex 9. 8 Answers to the Questions from the EU Member States. Volume I. November, 2003.

7 Opinion on the additive Hy D (Calcifediol) 7/35 HO CH H 3 C CH H 3 C 2 CH 3 1 CH H 3 C CH H 3 C OH CH 3 HO Vitamin D 3 25-OH-D 3 Figure 1. Chemical structure of vitamin D 3 and 25-OH-D 3 25-OH-D 3 used in Hy D is a semi-synthetic product (chemically obtained from a genetic modified organism). The precursor compound 5,7,24-cholestatrienol is produced by a fermentation process using a genetically modified yeast (Saccharomyces cerevisiae). The precursor is extracted by solvent then converted chemically (hydroxylation then epoxidation/reduction) to 25- OH-pro-D 3. This intermediary compound is photochemically transformed to 25-OH-D 3 which is separated from the photo-products by crystallisation. The isomer 25-OH-D 3 previtamin D 3 is also formed but represents only a very small fraction. 9 Table 2. Chemical and physical details of 25-OH-D 3 10 Generic name 25-hydroxyvitamin D 3 Chemical name (3β,5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-3,25- diol monohydrate CAS No Empirical formula C 27 H 44 O 2.H 2 O Relative molecular mass Melting point C* Visual appearance A white to slightly pink crystalline material Solubility Insoluble in water; soluble in acetone, ethanol, DMSO and other lipophylic solvents Other names Calcifediol, calcidiol, 25-hydroxycholecalciferol, *The wide melting point range is presumably due to the water content (about 5%) and other impurities. The production organism (S. cerevisiae) contains multiple copies of yeast genes (self/autocloning) and an introduced ampicillin resistance gene from E. coli (derived from pbr322). 11 The strain (derived from ATCC 74090) has been deposited at the American Type Culture Collection with number ATCC Volume 1-6. Section II. 1 and Volume 1-6. Section II Volume 6. Annex Answers to the Questions from the EU Member States. Volume I. Annex 8. November, 2003.

8 Opinion on the additive Hy D (Calcifediol) 8/35 The preparation process including extraction, chemical then photochemical transformation and purification (crystallisation) steps ensure the complete elimination of the yeast cells. Moreover, the search for recombinant DNA in 25-OH-D 3 by detection of the ampicillin resistance gene by PCR were negative at the limit of detection of the method. In conducting the analyses for DNA the 25-OH-D 3 was dissolved (20 mg ml -1 ) in suitable solvent and thus the detection limit of 300 fg ml -1 indicated that there was less than 15 fg DNA per g of crystalline 25-OH-D Therefore, considering the absence of the yeast cells and the lack of detectable recombinant DNA, the FEEDAP Panel considers that there is no transformed DNA in the final product. The chemical purity of the 25-OH-D 3 feed-grade product used in Hy D is claimed to be >94%. The chromatographic (HPLC) analysis of two batches analysed in two laboratories indicates an average value of 97.5%. The impurities comprise a group of vitamin D 3 isomers measured together and comprising 0.23% of the 25-OH-D 3. The other compounds measured were 25-OHprevitamin D 3 (0.7%), 25-OH-provitamin D 3 (0.09%), 25-OH-tachysterol (0.16%), 25-OH-5,6 trans-vitamin D 3 and unidentified sterols (0.27%). These compounds are regarded as natural metabolites of vitamin D The only other impurity of the 25-OH-D 3 identified is erythrosine and is determined to be <5 mg kg -1 of 25-OH-D Stability The vitamers of D 3 are subject to ready decomposition in the presence of oxygen, moisture, minerals and high temperatures and thus must be packaged and stored to avoid such conditions. The stability of Hy D was assessed as the remaining product and in premixes and diets with and without pelleting. Recovery should be determined on the measured 25-OH-D 3 concentration and not on the calculated values. Recovery should be defined strictly as a proportion of what was determined in the original sample (i.e., recovery (%)=[determined concentration at time after storage/ determined concentration at start]*100) Shelf life of Hy D The stability of Hy D was determined by maintaining Hy D in commercial polyethylene containers at 25 C and 40 C for 52 weeks. 20 The applicant reported that the loss of 25-OH-D 3 from Hy D beadlets was in the range 0-8% at 25 C and 11-17% at 40 C compared to the content in the original beadlets. 21 In fact the recoveries are about 2-3% lower than presented in the dossier. The proposed shelf life for the beadlets (6 months in air and water-tight containers at temperatures of <25 C) is reasonable despite the inaccurate calculation of stability of the beadlets. 13 Volume 1-6. Section II. 14 Answers to the Questions from the EU Member States. Volume 1. November, Volume 6. Annex Volume 6. Annex 10 and Volume 6. Annex % recovery = {[concn](t=n)/[concn](t=0)}* Volume 6. Annex Volume 6. Annex 10, 11 and Volume 1-6. Section II.3.

9 Opinion on the additive Hy D (Calcifediol) 9/ Premixes Recent studies with premixes (done in small 50 g quantities and without minerals) and about 180 mg kg -1, yielded recoveries of 25-OH-D 3 of >90% when stored at 25 C and 35 C for 3 and 1 month respectively. A 70% recovery was obtained when the premix was stored at 35 C for 3 months. These calculated recoveries were based on the determined amounts of 25-OH-D 3 at the initiation of the study. The presence of minerals significantly reduces the stability and after three months at 25 C only 65% of 25-OH-D 3 could be recovered. When stored for 2 months at 35 C in the presence of trace minerals recovery averaged about 23% where the starting concentrations were about 120 mg kg Complete feeds and treatments The loss of Hy D when included in maize-soya-based diets which were pelleted and stored for 3 months amounted to about 40% compared with the original mash diet. Mash diets when stored for 3 months at 25 C also lost about 40% of the originally determined 25-OH-D 3. In this instance the recovery was based on the initially determined 25-OH-D 3 in the mash and thus accounts for the retention through pelleting and storage. As a result the recovery of 25-OH-D 3 in pellets during storage is underestimated (58.7% vs 62.2% recovery). Pelleting poultry diets containing beadlets of Hy D reduced recovery of 25-OH-D 3 by up to 15% compared to the unpelleted material. More recent studies by the applicant indicated that >96% of 25-OH-D 3 was recovered when diets were pelleted at 75 C and 85 C. In a study 24 (layer mash diets based on wheat and soyabean meal) the applicant reported average recoveries of 91, 88 and 62% after storage for 1, 2 and 3 months respectively at 25 C based on the targeted content of 25-OH-D 3. These results are overvalued since recoveries calculated on the basis of the initial, determined content of 25-OH-D 3 are 57, 60, and 39% for storage at 25 C for 1, 2 and 3 months respectively Control methods The methods used to measure the components in the active compound of Hy D, Hy D and feedingstuffs include HPLC, UV spectroscopy, IR spectroscopy, all conducted under SOP procedures. 25 Initially the compounds are extracted from the material, or dissolved, in organic solvents and then HPLC methods were applied using normal and reverse phase systems. The routine HPLC methodology utilises a USP 25-OH-D 3 standard and can, in the same run quantify 25-OH-D 3 and pre-vitamin D 3 as separate peaks. 26 RIA methods are used to quantitate 25-OH-D 3 in animal products using a test kit with 125 I-25-OH D Answers to the Questions from the EU Member States. Volume I. Annex A-9. November, Answers to the Questions from the EU Member States. Volume II. Annex A-2. July, Volume 1-6. Section II Volume 6. Annex 10 and Answers to the Questions from the EU Member States. Volume I. Annex 12 and 13. November, Volume 1-6. Section II Volume 6. Annex 23, 24 and 25.

10 Opinion on the additive Hy D (Calcifediol) 10/35 2. Efficacy of the product in target species 2.1. Recommendations for vitamin D 3 in poultry The recommendations that have been issued by three sources are reported in Table 3. It is important to highlight the fact that the NRC recommendations correspond rather to a minimal requirement to satisfy normal performance of the birds. Allowances are given by GfE and INRA. Table 3. Recommendations for Vitamin D 3 in poultry. Species/category Age Recommendation (weeks) IU kg -1 diet µg kg -1 diet Remark Reference Chickens for fattening Turkeys On DM base GfE Requirement Diet at 90% DM NRC INRA On DM base GfE2004 > On DM base GfE Requirement Male and female NRC INRA 1989 > INRA 1989 > On DM base GfE 1999 Laying hens Requirement, diet at > NRC % DM > INRA 1989 DM =Dry matter NRC: National Research Council (USA) GfE: Gesellschaft für Ernährungsphysiologie (Germany). INRA : L alimentation des animaux domestiques: porc, lapin, volaille (France). It is noteworthy that the current levels of vitamin D 3 added to the diets of domestic birds ( µg for chickens for fattening and for layers, 100 µg kg -1 for turkeys) are 2-3 fold higher than the requirement. No recommendation is presently available for 25-OH-D Efficacy trials More than 25 assays are reported by the applicant, mainly carried out in chickens for fattening. Numerous trials have been published in peer review journals (Soares et al., 1995; Yarger, et al., 1995), which are the subject of the evaluation by the applicant as well as own studies included in the dossier. 29 The trials have been carried out mainly in the United States in for chickens, for laying hens, 31 and for turkeys Volume 7A - 7E. 30 Volume 7A, 7B and 7C. 31 Volume 7D. 32 Volume 7E.

11 Opinion on the additive Hy D (Calcifediol) 11/35 The main objective of the applicant was to establish that 25-OH-D 3 was at least equivalent to vitamin D 3 as the efficacy of this vitamin is well established. This approach is acceptable due to the availability of numerous data for vitamin D 3, justifying its use as a reference. The comparison between the two forms of vitamin D 3 and its hydroxylated form was carried out at dietary levels used in practical conditions with the exception of one trial. These conditions correspond to dietary levels in excess in comparison to the requirements of the birds. This approach allows the demonstration of equivalency between the two sources in practical conditions. But under such conditions it is difficult to establish the relative biological activity of the two sources requiring comparisons at low dietary levels of vitamin D (25-OH-D 3 or vitamin D 3 ). These comparisons were not included in the majority of trials. Mostly the performance of birds fed 25-OH-D 3 and the reference vitamin D 3 was compared. The number of birds used in the experimental trials and number of replications are geared to the evaluation of growth performance and feed conversion. The experimental measurements, body weight at different intervals, feed conversion and mortality are classical parameters used for testing feed additives; however they are not specially designed for the evaluation of vitamins. The recording of bone (or egg shell) mineralization is particularly pertinent to evaluate the biological effects of vitamin D or of its derivatives. All trials were carried out with typical US diets (corn and soybean) and took into consideration the recommendation of the NRC for calcium and phosphorus. However, the applicant did not report in any of the trials carried out in chickens for fattening a dietary analysis of vitamin D 3 or 25-OH-D Chickens for fattening Growth performance and feed conversion ratio One trial has been carried out (Table 4) 33 which evaluates in the range of 0 to 20 µg kg -1 diet, the levels of 25-OH-D 3 or D 3 showing that 20 µg (800 IU) are needed at least to optimize growth and feed conversion. These results suggested a higher efficacy of 25-OH-D 3 compared with vitamin D 3 (about two fold). Table 4. at Dose response of vitamin D 3 and its metabolite 25-OH-D 3 (in the form of Hy D) low dietary supply on body weight and feed conversion of chickens for fattening. 80 birds x 10 replicates/group; duration 46 days µg vitamin kg -1 diet Vitamin D 3 (kg) Body weight 25-OH-D 3 (% of vit. D 3 ) Vitamin D 3 (kg feed kg -1 gain) Feed conversion 25-OH-D 3 (% of vit. D 3 ) 21 d 46 d 21 d 46 d 21 d 46 d 21 d 46 d 0 0,21 a 0.57 a a b 0.64 b 105* 116* b 96 97* c 0.79 c 104* 131* c 98 96* d 1.09 d 104* 115* d * e 1.37 e 104* 110* e 96 96* *Difference statistically significant at 5 % level to the corresponding vitamin D groups a, b, c Means with different letters in the same column differ significantly at p Volume 7A. Annex 12.

12 Opinion on the additive Hy D (Calcifediol) 12/35 This trial shows that the requirement for vitamin D 3 is at least 800 IU (20 µg kg -1 ), which is higher than the requirement defined by the NRC (Table 3) or from other recent data sources (5 µg for Vit D 3 or 25-OH-D 3, Bar et al., 2003; 5 µg vit D 3, Baker et al., 1998). However, Fritts and Waldroup (2003) observed optimal body weight when supplying 20 µg kg -1 of vitamin D 3 or only 5 µg kg OH-D 3. They therefore showed that the 25-OH-D 3 was about 4 fold more efficient than vitamin D 3 when supplied at levels lower than 20 µg kg -1 diet. These authors confirmed by analysis the amount of vitamin D 3 or 25-OH-D 3 in their experimental diets. Twenty trials were reported by the applicant comparing the efficacy of 25-OH-D 3 to vitamin D 3 when supplied at dietary levels higher than 20 µg kg -1. Amongst them, five trials showed a significant positive effect on performance of chickens for fattening when 25-OH-D 3 was substituted to vitamin D 3 in the diet at the same dietary level of both forms of the vitamin In all the other trials (15), vitamin D 3 and 25-OH-D 3 supplied at the same dietary levels resulted in similar performance demonstrating, in all experiments, that 25-OH-D 3 is at least as efficient as vitamin D 3 for growth performance of chickens for fattening. The comparison of the chicken body weight and that of feed conversion ratio at various dietary levels of either vitamin D 3 or 25-OH-D 3 supplemented mainly at 69 µg kg -1 diet, showed that growth performance and feed conversion ratio are quite similar, suggesting an equivalent efficacy of both sources in chickens fed at this high dietary levels. When the dietary level is lower than 20 µg, the body weight can be improved, suggesting that 25-OH-D 3 has a higher efficacy than vitamin D 3. Three field studies 38 confirm, in large numbers of birds, that the performance of chickens for fattening are similar when fed 35 to 103 µg kg -1 vitamin D 3 of 25-OH-D Bone mineralization Bone ash is the reference parameter which is used to estimate the vitamin D status of the animal. Table 5 summarizes four trials showing the effect of vitamin D 3 and its hydroxylated form on bone mineralization Table 5. (%) Dose-response of vitamin D 3 and its metabolite on tibia ash concentration Duration of experiments: 46 and 47 days. Trial No. birds X replicates Dose (µg kg -1 diet) Vitamin D 3 25-OH-D x x g 36.4 f 38.6 de 39.8 c 41.6 b 27.5 f g 38.0 e 39.5 cd 41.1 b 43.4 a 30.9 e 37.3 c 34 Volume 7A. Annex 10, 13 and Volume 7C. Annex 1, 3 and Volume 7A - 7E. 37 Answers to the Questions from the EU Member States. Volume I. November, Volume 7A. Annex 4, 5 and Volume 7A.Annex Volume 7A. Annex 17.

13 Opinion on the additive Hy D (Calcifediol) 13/ x x b 39.2 b 39.7 ab 40.4 a 28.6 f c 39.8 a bc 40.0 bc 39.4 bc Means with different letter are significantly different (comparison between all treatments) 1 µg 25-OH-D 3 is nearly equivalent to 2 µg vit. D. 30 e 37 bc 37 b 40 a 33.4 d 40.6 ab 41.8 a 39.7 bc Bone ash increased with the dietary levels of vitamin D 3 and 25-OH-D 3 from 0 to 62 µg kg -1 diet (calculated value, analytical values are not reported). In about 50% of the comparisons between vitamin D 3 and 25-OH-D 3 at similar dietary level, it was observed a higher bone ash when supplying the 25-OH-D 3. This difference was smaller or it was not observed when the dietary level of the vitamin D 3 was higher (between 60 and 100 µg kg -1 diet). The bone ash data showed that the efficacy of 25-OH-D 3 in tibia mineralization was about two fold that of vitamin D 3 for dosages up to 60 µg kg -1 diet Laying hens One trial comprising two phases was reported by the applicant. 43 The hens (initial age: 22 weeks) were supplied with 41 and 82 µg kg -1 of 25-OH-D 3 which were compared to a single level of vitamin D 3 (69 µg kg -1 ). The feed conversion ratio was decreased in hens fed the hydroxylated form of Vitamin D 3 when hens were supplied at 82 µg kg -1 diet. Egg shell thickness was increased in birds fed on 25-OH-D 3 compared to those fed on vitamin D 3 (0.17 versus 0.18 mm, non significant). A trial 44 on 288 laying hens (18-68 weeks) fed on 82 µg kg -1 diet vitamin D 3 or 25-OH-D 3 showed improved egg production after 43 weeks of age and higher egg specific gravity in one of the breeds of hens when fed on 25-OH-D 3 instead of vitamin D 3. Scientific literature published before 1980 has been included in the dossier (Soares et al., 1995), and shows that 25-OH-D 3 has a similar efficacy or even higher efficacy than vitamin D 3 for egg shell quality when supplied at low levels (<10 µg). Recently, Keshavarz (2003) compared vitamin D 3 and Hy D supplied at 69 µg active substance kg -1 in the diet. This author observed similar egg production, egg weight, feed conversion and egg shell quality in 90 hens per treatment of 45 to 65 weeks of age Turkeys Six trials have been carried out from 1993 to 1998, four in the United States (Table 6) and two in France. 45 One of the trials carried out in the United States 46 was not further considered in the assessment due to the uncertainties in vitamin D 3 and 25-OH-D 3 dietary levels during the production in the experimental diets. Furthermore no full study reports are available. 41 Volume 7B. Annex Volume 7B. Annex Volume 7D. Annex 1 and Volume 7D. Annex Volume 7E. Annex 8 and Volume 7E. Annex 2.

14 Opinion on the additive Hy D (Calcifediol) 14/35 Table 6. No. of animals x replicates 38x12 16x12 25 x 5 (f) 21 x 5 (m) 31 x 13 Summary the three acceptable turkey experiments conducted in the USA Final body Feed conversion Duration Dose level (1) weight (kg) (g feed g- 1 gain) (days) (µg kg -1 Ref feed) 84 (phase 1) 42 (phase 2) (f) 133 (m) 40/35 (phase 1/2) 138/94 (phase 1/2) (2) 99 D OH- D * * D OH- D * (1) range of dietary levels of vitamin D 3 or its metabolite, the levels of which changed at various phases of turkey rearing. (2) The concentration of Vitamin D3 were reduced tn the experiment from f=female, m=male. *significantly different from control vitamin D 3 The analytical characterisation of the feed (performed only in the second study) showed that the levels of 25-OH-D 3 were lower than expected (around 40 and 82 instead of 49 and 99 µg kg OH-D 3 ). In the third trial the analytical values in the post pellet diet were about 10% higher than expected. The mortality of the turkeys in the various trials was in the expected range and was not influenced by the dietary level of vitamin D or 25-OH-D 3. Growth performance and the feed conversion ratio were not affected by the substitution of vitamin D 3 by 25-OH-D 3 when incorporated at levels between 40 to 100 µg kg -1 (it was variable, depending on turkey age in some of the trials). In two of the trials, the body weight and feed conversion ratio (one trial) were improved when 25-0H-D 3 was used instead of vitamin D 3 (Table 6). In the two trials carried out in France, OH-D 3 was supplied in addition to a basal dietary level of vitamin D 3. In these field studies carried out on 320 male turkeys for 104 days and on 432 turkeys for 105 days (3 treatments), the addition of 25 µg 25-OH-D 3 and 62.5 µg vitamin D 3 did not improve the body weight nor the feed conversion ratio (P>0.1) in one of the trials. In the other study the feed conversion rate was slightly improved. A detailed description of these field trials is not available The report of the applicant is completed by references of publications evaluating 25-OH-D 3 relative to Vitamin D 3 in the turkey breeder hen and in young turkeys, but without clear identification of the source of 25-OH-D Studies on the quality of animal produce The carcass yield (percentage) of chickens for fattening was evaluated in 11 trials comparing 25- OH-D 3 and Vitamin D 3. It was improved in two comparisons amongst 22 comparisons. The 47 Volume 7E. Annex Volume 7E. Annex Volume 7E. Annex Volume 7E. Annex 8 and Volume 7E. Annex

15 Opinion on the additive Hy D (Calcifediol) 15/35 breast meat yield was also measured in 22 comparisons at similar levels of both compounds Four trials showed a slight improvement on the amount of the breast meat. The magnitude of the increase was low, <3% relative to the breast meat observed in vitamin D 3 fed birds. No other information than egg quality (see section 2.2), for turkeys and laying hens is available Bioequivalence and labelling Historically, the only assay to determine vitamin D was based on experiments with animals (rat). The activity of the different compounds showing vitamin D activity was compared on the basis of their antirachitic potency. One IU was the smallest dosage to prevent rickets in rats. Only later, one IU was defined as µg cholecalciferol (or ergocalciferol). In the EU, labelling of vitamin D in compound feedingstuffs is mandatory. The amount of vitamin D has to be labelled in IU (Dir 70/524/EEC). As long as only vitamin D 2 and D 3 preparations were approved (and vitamin D 2 not approved for poultry), vitamin D labelling in IU was practical. Feed manufacturers and farmers are familiar with IU as a biological standard and not as familiar with mg or µg ergocalciferol or cholecalciferol as a mass standard. The applicant is aware of this system and ask consequently for labelling purposes 25-hydroxycholecalciferol (Calcifediol) in IU. The issue arising from this request is the proper calculation of IU from 25-OH-D 3. The applicant is of the opinion that 1 µg 25-OH-D 3 is equal to 40 IU vitamin D following the international standard for (unhydroxylated) vitamin D compounds (1 IU = µg vitamin D). The company s deduction is based on a considerable number of poultry experiments with vitamin D 3 and 25-OH-D 3. But the parameters chosen for this calculation are body weight gain and feed conversion. These parameters do not reflect the primary metabolic action of vitamin D which is on the calcium metabolism of the body. The FEEDAP Panel is therefore unable to follow the proposal of the applicant company for 25- OH-D 3 labelling on the basis of 1 µg 25-OH-D 3 equal to 40 IU vitamin D. The biopotency of 25-OH-D 3, the first intermediate metabolite of cholecalciferol leading to the metabolically active di- or tri-hydroxylated metabolites, compared to cholecalciferol, varies with the parameter and the dosage used, on which the calculation is based. A comprehensive review (see Table 7) of feeding studies comes to the conclusion that 25-OH-D 3 has nearly twice the activity of vitamin D 3. From the data summarised in Table 7 it becomes evident that the main criteria for vitamin D efficacy calcium absorption and skeletal mineralization are more strongly influenced in poultry by 25-OH-D 3 than by vitamin D 3. But there is also a variation around the factor two given by the authors reaching from 1.25 until 4. In the view of the FEEDAP Panel an expression of the efficacy of 25-OH-D 3 in IU is questionable. Table 7. Relative biological activity of 25-hydroxy-cholecalciferol in domestic poultry when compared to vitamin D 3. Parameter Relative activity Reference Ca 2+ absorption 2 Myrtle and Norman, 1971 a Bone ash 1.25 Norman and Wong, 1972 a Plasma Ca 2+ 4 Haussler and Rasmussen, 1972 a Plasma Ca McNutt and Haussler, 1973 a Bone ash 2.5 Sunde, 1975 a Tibia ash 2x 1-2 Boris et al., 1977 a see Table 5 52 Volume 7A. Annex 11, 13, 14 and Volume 7B. Annex 1-6, 8 and Volume 7C. Annex 1-3, 6, 7 and 9.

16 Opinion on the additive Hy D (Calcifediol) 16/35 Bone ash low P Soares et al., 1978 a Body weight 4 Fritts and Waldroup, 2003 Body weight 2 see Table 4 a Soares et al. (1995). It seems therefore logical to give the potency of 25-OH-D 3 in µg, which is the international standard (IUPAC) anyway. But such a decision would also have the consequence that vitamin D 3 and vitamin D 2 require to be labelled in µg instead of IU. This proposal is scientifically justified. If the EU system of mandatory labelling all vitamin D active compounds in IU should be maintained, 1 µg of 25-OH-D 3 should be considered as > 40, probably 80 IU Vitamin D. To avoid misunderstanding by farmers, the labelling should consist of two parts (i) the potency (IU vitamin D) followed by (ii) the source of the vitamin (from vitamin D 3, vitamin D 2 or Calcifediol) Conclusion The efficacy of 25-OH-D 3 concerning weight gain, feed conversion and bone mineralisation for chickens for fattening is at least equivalent to that of vitamin D 3 when supplemented at dietary levels of 30 to 69 µg kg -1. At lower doses (2.5 and 25 µg kg -1 ), the efficacy concerning bone and feed conversion of 25-OH-D 3 is doubled compared to that of the vitamin D 3. The bone ash data shows that efficacy of 25-OH-D 3 is even higher than that of vitamin D 3 (about two fold). Concerning laying hens, it has been demonstrated that 25-OH-D 3, in the dose range of 41 to 82 µg kg -1, is at least equivalent to vitamin D 3 for optimizing hen performance and egg quality. In turkeys, it can be concluded that 25-OH-D 3 can be used as a substitute for vitamin D 3 in the range tested by the applicant (40 to 100 µg kg -1 ). When different levels of 25-OH-D 3 were evaluated, no significant differences were observed so it is difficult to conclude on the optimal dietary level to be used and to know if it differs from that of vitamin D 3. Quality of animal products was not significantly influenced by the source of vitamin D. The FEEDAP Panel cannot support the proposal of the applicant that 1 µg 25-OH-D 3 is equal to 1 µg vitamin D 3 or 40 IU vitamin D. The company s deduction is based on a considerable number of experiments on poultry with mostly higher dosages (>30µg) of vitamin D 3 and 25-OH- D 3, which do not allow comparable dose titration. In addition, the analysed parameters (body weight and feed conversion) do not reflect the primary metabolic action of vitamin D, which is on bone mineralization. 25-OH-D 3 has a higher potency than vitamin D 3. Considering literature and the suitable experiments submitted 1 µg of 25-OH-D 3 should be considered as > 40, probably 80 IU Vitamin D. The higher potency of 25-OH-D 3 depends on the parameter chosen. It seems therefore logical to give the potency of 25-OH-D 3 in µg, which is scientifically correct. If the EU system of mandatory labelling all vitamin D active compounds in IU should be maintained, 1 µg of 25-OH-D 3 should be considered as 80 IU Vitamin D 3. To avoid misunderstanding by farmers, the labelling should consist of two parts (i) the potency (IU vitamin D) followed by (ii) the source of the vitamin (from vitamin D 3, vitamin D 2 or Calcifediol). 3. Safety studies on target species 3.1. Tolerance studies

17 Opinion on the additive Hy D (Calcifediol) 17/35 The applicant submitted one preliminary safety study 55 and two tolerance studies on chickens for fattening 56 (both published by Yarger et al., 1995), and one each for laying hens 57 and turkeys 58. A third tolerance trial on chicken for fattening lasted only 14 days and was not considered in detail. Unfortunately analytical confirmation of the dose levels of the tolerance studies could not be found in the dossier Chickens for fattening In the preliminary study, 400 birds (five replicates with 40 males and 40 females each) per treatment were fed for 46 days diets with 6.25, 31.25, 56.25, 112.5, 225, and 450 µg vitamin D 3 and 25-OH-D 3 kg -1, respectively. Body weight and feed efficiency were not affected by dose, but the 25-OH-D 3 groups generally performed better. There was a tendency for higher mortality in the high 25-OH-D 3 groups, but not in the high vitamin D 3 groups. In experiment I, a total of 1120 broilers (280 birds, 5 replicates of 28 males and 28 females per treatment) was fed for 49 days diets (starter, grower, finisher) containing 69 µg vitamin D 3, 69, 207 and 690 µg 25-OH-D 3 kg -1, respectively. The diets did not contain a coccidiostat or other drugs, which is considered as a cause of the relatively high mortality observed (between 12 and 16.6 % without treatment interaction, however, the most common cause of death was ascites). Growth rate and feed efficiency were not influenced by dose as also serum calcium, which however showed an insignificant tendency for increased values in all 25-OH-D 3 groups. Serum 25-OH-D 3 was about three times higher in the 69 µg 25-OH-D 3 group (37 ng ml -1 ) than in the vitamin D group (13 ng ml -1 ). The three and ten fold dietary concentration of 25-OH-D 3 resulted in serum levels of 110 and 242 ng ml -1. Serum 1,25-(OH) 2 -D 3 was not different for the 69 µg vitamin D 3 and the 69 µg 25-OH-D 3 kg -1 group, it was significantly lower in the 690 µg 25-OH-D 3 kg -1 group, with the 207 µg 25-OH-D 3 kg -1 being in between. At the end of the study, all birds were killed for necropsy, organs (liver, spleen, kidney, heart, adrenal glands, bursa of Fabricius, brain, bone marrow, testes, ovary, eye, pancreas, lung, trachea, esophagus, crop, proventriculus, ventriculus, intestine -upper, middle, ceca, and rectum, skin, spinal cord, pituitary body, thymus, thyroid, parathyroid, and femoral-tibial point) from one male and one female per replicate were taken for histopathology. No treatment related abnormalities were seen by gross pathology in the tissues examined nor in blood cell counts, hematocrit, hemoglobin, or prothrombin time. Also histopathology did not reveal significant group differences. In experiment II, a total of 3500 broilers (350 birds, seven replicates of 25 males and 25 females per treatment) was fed for 49 days diets (starter, grower, finisher) containing vitamin D 3 and 25- OH-D 3 at levels of 69, 690, 3450, 6900 and µg kg -1, respectively. Because of high toxicity and morbidity, the treatments with 6900 and µg 25-OH-D 3 kg -1 had to be terminated after 22 days, treatment with 3450 µg kg -1 after 31 days. Starter and grower diets contained salinomycin (60 mg kg -1 ) a coccidiostat and bacitracin dimethyl salicylate. Average mortality of the remaining 25-OH-D 3 and of the 3 low vitamin D 3 groups at the end was 4.8 %, of the 2 high vitamin D 3 groups 9.1 and 12.6%, respectively. Other 4.3% (average of all 7 groups) were culled during the experiment. The results are summarised in Table 8. At dose level 690 µg kg -1 feed, there was a significant difference (P <0.01) in body weight between the vitamin D 3 and the 25- OH-D 3 group. Table 8. Mortality, body weight and feed efficiency after 48 days 55 Volume 8 B. Annex Volume 8A. Annex 7-9 and Volume 8B. Annex Volume 8C. Annex Volume 8D-8E. 58 Volume 8F-8G.

18 Opinion on the additive Hy D (Calcifediol) 18/35 Dietary level (µg kg -1 ) Mortality (%) a, vitamin D Mortality (%) a, 25-OH-D (16.9) b (8.88) c (17.5) c Body weight (kg), vitamin D Body weight (kg), 25-OH-D * g feed g -1 gain, vitamin D g feed g -1 gain, 25-OH-D a mortality rate includes deaths and culls b after 31 days c after 22 days At the termination of each treatment period, the birds were examined for gross pathology. The following tissues from selected birds of each group were used for histopathology: kidneys, heart, aorta, and tibia. The findings on renal calcification are given in Table 9. Table 9. Renal calcification after 48 days Dietary level (µg kg -1 ) No. of birds, examined, vitamin D No. of birds,, examined, 25-OH-D (66) b (41) c (59) c Renal calcification (%) a, vitamin D Renal calcification (%) a, 25-OH-D (100) b (100) c (100) c a includes trace, mild and moderate calcification b after 31 days c after 22 days Whereas for vitamin D 3 an increase in renal calcification could be observed at 3450 µg kg -1, such in an increase occurred for 25-OH- D 3 already at 690 µg kg -1 (from 27 renal calcification findings in this group, 16 were considered as trace and 10 as mild ). This observation is in contrast to experiment I, where renal calcification at the same doses could not be observed. Yarger et al. (1995) suggested on the basis of body weight and renal calcifications that 25-OH-D 3 is 5 to 10 times more toxic for chickens for fattening than vitamin D 3. Morrissey et al. (1977) observed renal tubular calcification after a 2 week period (14 to 28 d) at a dose level of µg vitamin D 3, but for 25-OH-D 3 already at 100 µg kg -1 feed. The authors concluded that 25-OH-D 3 may be 100 fold more toxic than vitamin D 3. The diets contained 1.2% calcium (and 0.65% phosphorus), which is about 25% higher than in the diets by Yarger et al. (1995). This may favour an earlier appearance of 25-OH-D 3 toxicity. A third trial on chickens for fattening (6 replicates with 8 birds each per treatment) with doses of 35, 70, 140, 280 and 560 µg 25-OH-D 3 kg -1 feed 59, respectively, was conducted from the age of 8 to 22 days. Body weight gain increased with higher 25-OH-D 3 amounts (up to 280 µg, significant difference to 35 µg), but 560 µg 25-OH-D 3 kg -1 feed was lower and numerically equal to 35 µg 25-OH-D 3 kg -1 feed. Feed conversion was more or less improved by all higher 25-OH-D 3 treatments. Plasma Ca and inorganic P were not dose-dependent affected by 25-OH-D 3 kg -1 feed, but 140 µg 25-OH-D 3 kg -1 feed showed lowest values (significant for Plasma Ca). Apparent Ca- and P- retention was increased up to 280 µg 25-OH-D 3 kg -1 feed. Tibia strength was numerically increased by higher 25-OH-D 3 dosages, but tibia ash significantly increases too. This experiment is not further considered due to its short duration (2 weeks) and the lack of assessment of critical parameters (e.g., renal calcification). 59 Answers to the Questions from the EU Member States. Volume II. Annex C-7. July, 2004.