HyD EFSA

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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 Commissionon the evaluation of safety and efficacy of HyD (calcifediol), basedon 25-hydroxylcholecalciferol/25-hydroxy-pre-cholecalciferol, as feed

additive in accordance with Council Directive 70/524/EEC.

(Question N EFSA-Q-2004-065)

Adopted on 26 May 2005SUMMARY

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 D2and D3are approved additives. HyD consists of 12.5 g 25-hydroxylcholecalciferol(25-OH-D3) kg

-1, which is the first metabolite of vitamin D3normally hydroxylated in the liver ofmen and animals.

The European Food Safety Authority has been requested by the European Commission to issuean opinion on the safety for target species, consumers, users and environment and on the

efficacy of the product of trade name HyD (calcifediol), when this product is used up to amaximum content of 5000 IU kg-1complete feedingstuff for chickens and turkeys for fatteningand 3000 IU kg-1for laying hens (1g 25-hydroxylcholecalciferol 40 IU).

Sufficient information on physical and chemical properties, the method of production, on stabilityand 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-D3 is at least aseffective as vitamin D3 in optimizing performance of chickens for fattening, laying hens andturkeys. Quality of the animal products was not significantly influenced by the source of vitaminD.

Since 25-OH-D3 is more potent in its vitamin D activity than vitamin D3, but higher potencydepends on and varies with the criterion assessed and the dosage applied, reliable information to

the user of the product HyD can scientifically not be given in terms of IU of vitamin D. Therefore

the FEEDAP Panel strongly recommends labelling of 25-OH-D3 in g. If for practical reasons thisis not immediately possible then the label of the product should include (i) the potency (IU vitaminD, 1 g of 25-OH-D3should be considered as 80 IU Vitamin D3) and (ii) the source of the vitamin(from Vitamin D3, Vitamin D2or Calcifediol).

Tolerance studies were carried out in chickens for fattening, turkeys and layers. 100 g 25-OH-D3kg-1 complete feed is well tolerated and could be accepted as the upper tolerated limit forchickens for fattening. As long as no more specific data on target animal safety are available andconsidering the above recommendation for labelling 25-OH-D3 in g kg

-1 complete feed, themaximum 25-OH-D3content for chickens for fattening should be set with 100 g 25-OH-D3kg

-1complete feed (a level proven as safe). A similar deduction leads to a proposal of 80 g 25-OH-D3kg

-1as maximum content for laying hens.

In contrast to these categories, turkeys seem to tolerate doses up to 500 g 25-OH-D3kg-1

. 100g 25-OH-D3kg

-1feed level could be applied for turkeys for fattening as maximum content.

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Opinion on the additive HyD (Calcifediol) 2/35

The margin of safety for chickens for fattening and laying hens could not be established, due tothe shortcomings in the design of the studies. The margin of safety of the upper recommendedlevel for turkeys could be given as about 5.

In birds as well as in humans 25-OH-D3is the initial metabolite of vitamin D3. It is likely that theingested 25-OH-D3undergoes the same metabolic fate as the endogenous compound. No retro-

conversion of 25-OH-D3to vitamin D3occurs.At the highest 25-OH-D3dose recommended for use as feed additive in poultry, retained by theFEEDAP Panel, HyD does not increase significantly the exposure of the consumer to 25-OH-D3through the consumption of turkey tissues when compared to the levels found following vitaminD3supplementation at a same level. The exposure resulting from turkey plus eggs consumption(3.5 g day-1) estimated from theoretical and worst case consumption figures retained by theFEEDAP Panel, represents 35% and 70% of the provisional upper limit (UL) proposed by theFEEDAP 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 g 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-D3 from HyD be used as a substitute of vitamin D3 for chickens for

fattening and turkeys for fattening at the maximum tolerated dose of 100 g kg-1 feed, and for

laying hens at the maximum tolerated dose of 80 g kg-1 feed, as retained by the FEEDAP Panel,no additional risk for the consumer could be expected.

The substitution of vitamin D3by 25-OH-D3from HyD should reduce considerably the vitamin D3contents of poultry tissues and products (eggs).

As a general principle the FEEDAP Panel considers conventional toxicological studies to beinappropriate for testing pure chemically defined substances which are dietary nutrients, which is

the case for 25-OH-D3 from HyD for which the chemical purity is established. The data

submitted give some indications that 25-OH-D3is not genotoxic and confirm that the acute, sub-chronic and reproductive toxicological effects observed are entirely consistent with aphysiological overload of vitamin D3 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 HyD have not been characterised. HyD is at such low concentrationsin the final feed to be of negligible concern apart from for those groups who may already be usingmedication based upon Vitamin D or 25-OH-D3. The use of protective clothing should besufficient to avoid adverse effects in users.

The FEEDAP Panel concludes that there is no necessity to perform an environmental riskassessment for this type of naturally existing compounds, under the conditions of the proposeduse.

The FEEDAP Panel recommends that only 25-hydroxylcholecalciferol will be specified in theannex entry including the minimum content requested (>94%).

The addition of both vitamin D sources, vitamin D3 and 25-OH-D3, should not be permitted andthis information should be included in the annex entry.

Key words:Vitamin D, Vitamin D3, 25-Hydroxycholecalciferol, Chickens for fattening, Turkeys,Laying hens.

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TABLE OF CONTENTS1. Introduction ....................................................................................................................5

1.1. Composition and characteristics of HyD............................................................ 61.2. Chemical and physical characteristics of 25-OH-D3 ............................................61.3. Stability...............................................................................................................8

1.3.1. Shelf life of HyD ................................................................................................81.3.2. Premixes ............................................................................................................91.3.3. Complete feeds and treatments..........................................................................91.4. Control methods .................................................................................................9

2. Efficacy of the product in target species....................................................................... 102.1. Recommendations for vitamin D3in poultry ...................................................... 102.2. Efficacy trials ....................................................................................................102.2.1. Chickens for fattening....................................................................................... 112.2.1.1.Growth performance and feed conversion ratio ................................................ 112.2.1.2.Bone mineralization..........................................................................................122.2.2. Laying hens......................................................................................................132.2.3. Turkeys.............................................................................................................13

2.3. Studies on the quality of animal produce ..........................................................142.4. Bioequivalence and labelling. ...........................................................................152.5. Conclusion........................................................................................................ 163. Safety studies on target species ....................................................................163.1. Tolerance studies .............................................................................................163.1.1. Chickens for fattening....................................................................................... 173.1.2. Laying hens......................................................................................................193.1.3. Turkeys.............................................................................................................203.1.4. Conclusions on the safety for the target animals............................................... 203.2. Fate of 25-OH-D3and body deposition .............................................................213.2.1. Fate of 25-OH-D3..............................................................................................213.2.2. Deposition ........................................................................................................ 21

3.2.2.1.Chickens for fattening....................................................................................... 213.2.2.2.Laying hen eggs............................................................................................... 223.2.2.3.Turkeys ............................................................................................................ 233.2.3. Conclusions...................................................................................................... 24

4. Studies on laboratory animals. .....................................................................................244.1. Conclusions...................................................................................................... 25

5. Safety evaluation for the human consumer ..................................................................255.1. Human use of 25-OH-D3...................................................................................255.2. Status of 25-OH-D3in humans..........................................................................255.3. Biological activity of 25-OH-D3 .......................................................................... 255.4. The Tolerable Upper Limit (UL) for humans...................................................... 265.5. Consumer exposure to 25-OH-D3 ..................................................................... 265.6. Conclusion........................................................................................................ 28

6. User safety assessment ...............................................................................................28Skin irritation ................................................................................................................28Eye irritation.................................................................................................................286.1. Pure 25-OH-D3 .................................................................................................286.2. Formulated product in beadlets (1.25% 25-OH-D3)...........................................296.3. HyD supplemented feed..................................................................................296.4. Conclusions...................................................................................................... 29

7. Safety for the Environment...........................................................................................29Conclusions .....................................................................................................................29Documentation provided to EFSA....................................................................................32

References ......................................................................................................................33Scientific Panel Members.................................................................................................35

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Acknowledgement............................................................................................................35

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BackgroundCouncil Directive 70/524/EEC1 lays down rules governing the Community authorisation ofadditives for animal nutrition and, in particular, defines the conditions that substance/productshould meet to be granted authorisation.

The Commission received a dossier from the applicant company, Roche Vitamins Ltd, throughSpain, the Rapporteur Member State, to obtain authorisation on the product HyD (calcifediol),based on 25-hydroxylcholecalciferol/25-hydroxy-pre-cholecalciferol, when it is used as a feedadditive for chickens for fattening, turkeys and laying hens, according to the conditions referred inTable 1. This additive has not been previously authorized at Community level and the companyrequested an authorisation in the category of vitamins feed additives.

Table 1. Condition of use HyD (calcifediol), based on 25-hydroxylcholecalciferol/25-hydroxy-pre-cholecalciferol)

Minimum

content

Maximum

contentNo.(orECNo.)

AdditiveChemicalformula,

description

Speciesor

categoryof animal M

aximu

m

age

IU2kg

-1of complete

feedingstuff

Other provisions

Calcifediol

25hydroxyl-cholecalciferol/25-hydroxy-

pre-cholecalciferol

Chickensfor

fattening

Turkeys

LayingHens

- -

5000

5000

3000

The mixture ofCalcifediol withvit.D3 is allowedprovided that thetotal amount ofthe mixture doesnot exceed 5000IU kg

-1feeding

stuff for chickensfor fattening andturkeys and 3000IU

-1kg

feedingstuffs forlaying hens.

TERMS OF REFERENCEThe 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 tradename HyD (calcifediol), when this product is used under the above mentioned conditions.

ASSESSMENT

1. Introduction

Vitamin D3(cholecalciferol) is one of a number of sterols that are present naturally in animals andare structurally very similar. Vitamin D and their metabolites (including 25-OH-D3) are essential

1

O.J n L 270 of 14.12.1970, p.12 Reference standard: cholecalciferol (IU)

1 g 25 hydroxycholecalciferol is considered by the notifier as equivalent to 40 IU

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lipophilic micronutrients required for the normal development of animals. Vitamin D3 is producedby the action of UV radiation (usually sunlight) on the skin of animals and man from the precursor7-dehydrocholesterol or it is provided in the diet. Vitamin D2(ergocalciferol) is the correspondingsterol in plants. Vitamin D and its metabolites are closely associated with the absorption ofcalcium by animals and its deposition in the skeletal tissue. Due to this association it is known asthe anti-rachitic vitamin, preventing the bone disorder, rickets.

In nature vitamin D is available in the diet of man and animals, in vegetables and animalfoodstuffs especially those that contain lipids. Vitamin D deficiencies can occur in animals thatare reared in the absence of sunlight and supplemental vitamin D. These animals can suffer fromhypocalcaemia, 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 inthe diet in adequate quantities to maintain animal health, welfare and production. Vitamin D2 isnot allowed to be used in poultry due to its lack of efficacy.

Because of the intensive rearing conditions associated with commercial poultry production,vitamin D3has been added to the diets of poultry, as well as other animals, for a considerabletime period to ensure good health.

In the normal metabolic pathway in animals vitamin D3 is absorbed and transported to the liverwhere it is hydroxylated to produce the intermediate compound 25-hydroxycholecalciferol (25-OH-D3) (Figure 1) which is subsequently further metabolised to participate in reactions thatinfluence the absorption of calcium and phosphorus. Thus, it is logical to provide 25-OH-D3in thediet 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 ofanimals and man have been published recently (De Luca, 2004, Feldman et al.,2005, McDowell,2000, Raiten and Picciano, 2004, Sutton and Mc Donald, 2003).

1.1. Composition and characteristics of HyD

HyD is a product manufactured in a beadlet form which contains a minimum of 12.5 g kg-1 of theactive substance 25-OH-D3. 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 anti-caking agents respectively, and butylated hydroxy toluene (BHT) (20 g kg-1) and citric acid (0.5 g

kg-1) as anti-oxidants.345The HyD beadlets are reported to have a melting point of 60C which

is presumably due to the cotton-seed oil. HyD is packaged in polyethylene bags which are then

sealed in cardboard drums.6Analysis of production batches demonstrated that HyD averaged acontent of 25-OH-D3of 13.5 g kg

-1(variation of 106-110% of the claim on the labels).7HyD isproposed to be added to poultry diets at concentrations between 3.2 to 8.0g t-1of diet depending

on poultry species and other sources of vitamin D. Prior to addition to the diet the HyD would be

premixed with a feed ingredient. Particle size is mostly (>95%) in the range 20-80 m with 3-4%of particles being

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Vitamin D3 25-OH-D3

Figure 1. Chemical structure of vitamin D3and 25-OH-D3

25-OH-D3used in HyD is a semi-synthetic product (chemically obtained from a genetic modifiedorganism). The precursor compound 5,7,24-cholestatrienol is produced by a fermentationprocess using a genetically modified yeast (Saccharomyces cerevisiae). The precursor isextracted by solvent then converted chemically (hydroxylation then epoxidation/reduction) to 25-OH-pro-D3. This intermediary compound is photochemically transformed to 25-OH-D3 which isseparated from the photo-products by crystallisation. Theisomer 25-OH-D3 previtamin D3 is alsoformed but represents only a very small fraction.9

Table 2. Chemical and physical details of 25-OH-D310

Generic name 25-hydroxyvitamin D3

Chemical name (3,5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-3,25-diol monohydrate

CAS No. 63283-36-3

Empirical formula C27H44O2.H2O

Relative molecular mass 418.66

Melting point 100-120C*

Visual appearance A white to slightly pink crystalline material

Solubility Insoluble in water; soluble in acetone, ethanol, DMSOand 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 frompBR322).11The strain (derived from ATCC 74090) has been deposited at the American TypeCulture Collection with number ATCC 1512.12

9Volume 1-6. Section II. 1 and 2.

10

Volume 1-6. Section II.2.11Volume 6. Annex 8.

12Answers to the Questions from the EU Member States. Volume I. Annex 8. November, 2003.

13

149

810

1712

11

15

16

75

6

20CH318

CH2191

23

22

4

CH3 21 24

2

3

OH

CH3

OH

CH3

13

149

810

171211

15

16

75

6

20CH318

CH2191

23

22

4

CH321 24

2

3

OH

CH3

CH3

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The preparation process including extraction, chemical then photochemical transformation andpurification (crystallisation) steps ensure the complete elimination of the yeast cells.13 14Moreover, the search for recombinant DNA in 25-OH-D3by detection of the ampicillin resistancegene by PCR were negative at the limit of detection of the method.

In conducting the analyses for DNA the 25-OH-D3was dissolved (20 mg mL-1) in suitable solvent

and thus the detection limit of 300 fg mL-1indicated that there was less than 15 fg DNA per g ofcrystalline 25-OH-D3.

15 Therefore, considering the absence of the yeast cells and the lack ofdetectable recombinant DNA, the FEEDAP Panel considers that there is no transformed DNA inthe final product.

The chemical purity of the 25-OH-D3feed-grade product used in HyD is claimed to be >94%.The chromatographic (HPLC) analysis of two batches analysed in two laboratories indicates anaverage value of 97.5%. The impurities comprise a group of vitamin D3 isomers measuredtogether and comprising 0.23% of the 25-OH-D3. The other compounds measured were 25-OH-previtamin D3 (0.7%), 25-OH-provitamin D3 (0.09%), 25-OH-tachysterol (0.16%), 25-OH-5,6trans-vitamin D3 and unidentified sterols (0.27%). These compounds are regarded as naturalmetabolites of vitamin D3.

16The only other impurity of the 25-OH-D3identified is erythrosine and

is determined to be

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1.3.2. Premixes

Recent studies2223with premixes (done in small 50 g quantities and without minerals) and about

180 mg kg-1, yielded recoveries of 25-OH-D3of >90% when stored at 25C and 35C for 3 and 1

month respectively. A 70% recovery was obtained when the premix was stored at 35 C for 3months. These calculated recoveries were based on the determined amounts of 25-OH-D3at the

initiation of the study. The presence of minerals significantly reduces the stability and after threemonths at 25C only 65% of 25-OH-D3could be recovered. When stored for 2 months at 35C inthe presence of trace minerals recovery averaged about 23% where the starting concentrationswere about 120 mg kg-1.

1.3.3. Complete feeds and treatments

The loss of HyD when included in maize-soya-based diets which were pelleted and stored for 3months amounted to about 40% compared with the original mash diet. Mash diets when stored

for 3 months at 25C also lost about 40% of the originally determined 25-OH-D3. In this instance

the recovery was based on the initially determined 25-OH-D3in the mash and thus accounts forthe retention through pelleting and storage. As a result the recovery of 25-OH-D3in pellets duringstorage is underestimated (58.7% vs62.2% recovery).

Pelleting poultry diets containing beadlets of HyD reduced recovery of 25-OH-D3by up to 15%compared to the unpelleted material. More recent studies by the applicant indicated that >96% of

25-OH-D3was recovered when diets were pelleted at 75C and 85C.

In a study24 (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 25Cbased on the targeted content of 25-OH-D3. These results are overvalued since recoveriescalculated on the basis of the initial, determined content of 25-OH-D3 are 57, 60, and 39% for

storage at 25C for 1, 2 and 3 months respectively.

1.4. Control methods

The methods used to measure the components in the active compound of HyD, HyD andfeedingstuffs include HPLC, UV spectroscopy, IR spectroscopy, all conducted under SOPprocedures.25 Initially the compounds are extracted from the material, or dissolved, in organicsolvents and then HPLC methods were applied using normal and reverse phase systems. Theroutine HPLC methodology utilises a USP 25-OH-D3standard and can, in the same run quantify25-OH-D3and pre-vitamin D3as separate peaks.

26

RIA methods are used to quantitate 25-OH-D3in animal products using a test kit with125I-25-OH-

D3.2728

22Answers to the Questions from the EU Member States. Volume I. Annex A-9. November, 2003.

23Answers to the Questions from the EU Member States. Volume II. Annex A-2. July, 2004.

24Volume 1-6. Section II.3.

25Volume 6. Annex 10 and 18.

26

Answers to the Questions from the EU Member States. Volume I. Annex 12 and 13. November, 2003.27Volume 1-6. Section II.5.

28Volume 6. Annex 23, 24 and 25.

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2. Efficacy of the product in target species

2.1. Recommendations for vitamin D3in 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 minimalrequirement to satisfy normal performance of the birds. Allowances are given by GfE and INRA.

Table 3.Recommendations for Vitamin D3in poultry.

RecommendationSpecies/category

Age(weeks) IU kg

-1diet g kg

-1diet

Remark Reference

0-6 450 11.25 On DM base GfE 1999

0-8 200 5Requirement

Diet at 90% DMNRC 1994

Chickens for

fattening0-6 1500 37.5 INRA 1989

1-2 1500 37.5 On DM base GfE2004

>2 1100 27.5 On DM base GfE 2004

0-24 1100 27.5Requirement

Male and femaleNRC 1994

0-8 1500 37.5 INRA 1989

Turkeys

>9 1200 30 INRA 1989

>18 450 11.25 On DM base GfE 1999

>18 300 7.5 Requirement, diet at90% DM

NRC 1994Laying hens

>18 1000 25 INRA 1989

DM =Dry matterNRC: National Research Council (USA)GfE: Gesellschaft fr Ernhrungsphysiologie (Germany).INRA : Lalimentation des animaux domestiques: porc, lapin, volaille (France).

It is noteworthy that the current levels of vitamin D3added to the diets of domestic birds (37.5-75 g for chickens for fattening and for layers, 100 g kg-1for turkeys) are 2-3 fold higher thanthe requirement. No recommendation is presently available for 25-OH-D3.

2.2. 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 inthe dossier.29 The trials have been carried out mainly in the United States in 1990-1995 forchickens,301995-1997 for laying hens,31and 1993-1998 for turkeys.32

29Volume 7A - 7E.

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Volume 7A, 7B and 7C.31Volume 7D.

32Volume 7E.

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The main objective of the applicant was to establish that 25-OH-D3 was at least equivalent tovitamin D3as the efficacy of this vitamin is well established. This approach is acceptable due tothe availability of numerous data for vitamin D3, justifying its use as a reference. The comparisonbetween the two forms of vitamin D3and its hydroxylated form was carried out at dietary levelsused in practical conditions with the exception of one trial. These conditions correspond todietary levels in excess in comparison to the requirements of the birds. This approach allows thedemonstration of equivalency between the two sources in practical conditions. But under suchconditions it is difficult to establish the relative biological activity of the two sources requiringcomparisons at low dietary levels of vitamin D (25-OH-D3 or vitamin D3). These comparisonswere not included in the majority of trials.

Mostly the performance of birds fed 25-OH-D3 and the reference vitamin D3 was compared. Thenumber of birds used in the experimental trials and number of replications are geared to theevaluation of growth performance and feed conversion. The experimental measurements, bodyweight at different intervals, feed conversion and mortality are classical parameters used fortesting 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 thebiological effects of vitamin D or of its derivatives.

All trials were carried out with typical US diets (corn and soybean) and took into considerationthe recommendation of the NRC for calcium and phosphorus. However, the applicant did notreport in any of the trials carried out in chickens for fattening a dietary analysis of vitamin D 3or25-OH-D3.

2.2.1. Chickens for fattening

2.2.1.1. Growth performance and feed conversion ratio

One trial has been carried out (Table 4)33which evaluates in the range of 0 to 20 g kg-1diet, the

levels of 25-OH-D3or D3showing that 20 g (800 IU) are needed at least to optimize growth andfeed conversion. These results suggested a higher efficacy of 25-OH-D3compared with vitaminD3(about two fold).

Table 4. Dose response of vitamin D3and its metabolite 25-OH-D3 (in the form of HyD)at

low dietary supply on body weight and feed conversion of chickens forfattening.

80 birds x 10 replicates/group; duration 46 days

Body weight Feed conversiong

vitaminkg-1

diet

Vitamin D3

(kg)25-OH-D

3

(% of vit. D3)

Vitamin D3(kg feed kg-1

gain)

25-OH-D3(% of vit. D3)

21 d 46 d 21 d 46 d 21 d 46 d 21 d 46 d

0 0,21a 0.57

a 1.61 2.28

a

2.5 0.32b 0.64

b 105* 116* 1.53 2.17

b 96 97*

5.0 0.34c 0.79

c 104* 131* 1.45 2.07

c 98 96*

10 0.35d 1.09

d 104* 115* 1.41 1.93

d 99 98 *

20 0.37e 1.37

e 104* 110* 1.40 1.88

e 96 96*

*Difference statistically significant at 5 % level to the corresponding vitamin D groupsa, b, cMeans with different letters in the same column differ significantly at p 0.05.

33Volume 7A. Annex 12.

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This trial shows that the requirement for vitamin D3is at least 800 IU (20 g kg-1), which is higher

than the requirement defined by the NRC (Table 3) or from other recent data sources (5 g forVit D3 or 25-OH-D3, Bar et al., 2003; 5 g vit D3, Baker et al., 1998). However, Fritts andWaldroup (2003) observed optimal body weight when supplying 20 g kg -1of vitamin D3or only5 g kg-125-OH-D3. They therefore showed that the 25-OH-D3was about 4 fold more efficientthan vitamin D3when supplied at levels lower than 20 g kg

-1diet. These authors confirmed byanalysis the amount of vitamin D3or 25-OH-D3in their experimental diets.

Twenty trials were reported by the applicant comparing the efficacy of 25-OH-D3 to vitamin D3when supplied at dietary levels higher than 20 g kg-1. Amongst them, five trials showed asignificant positive effect on performance of chickens for fattening when 25-OH-D3 wassubstituted to vitamin D3in the diet at the same dietary level of both forms of the vitamin.

3435Inall the other trials (15), vitamin D3and 25-OH-D3supplied at the same dietary levels resulted insimilar performance demonstrating, in all experiments, that 25-OH-D3 is at least as efficient asvitamin D3for 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 D3 or 25-OH-D3 supplemented mainly at 69 g kg-1

diet, showed thatgrowth performance and feed conversion ratio are quite similar, suggesting an equivalentefficacy of both sources in chickens fed at this high dietary levels. When the dietary level is lowerthan 20 g, the body weight can be improved, suggesting that 25-OH-D3has a higher efficacythan vitamin D3.

3637

Three field studies38 confirm, in large numbers of birds, that the performance of chickens forfattening are similar when fed 35 to 103 g kg-1vitamin D3of 25-OH-D3.

2.2.1.2. Bone mineralization

Bone ash is the reference parameter which is used to estimate the vitamin D status of theanimal. Table 5 summarizes four trials showing the effect of vitamin D3and its hydroxylated formon bone mineralization

Table5. Dose-response of vitamin D3and its metabolite on tibia ash concentration(%)

Duration of experiments: 46 and 47 days.

Trial No. birds X replicates Dose (g kg-1

diet) Vitamin D3 25-OH-D3

139

80 x 10

02.551020

33.7g

36.4f

38.6de

39.8

c

41.6b

33.7g

38.0e

39.5cd

41.1

b

43.4a

240

74 x 10 1.56

6.2527.5

f

-30.9

e

37.3c

34Volume 7A. Annex 10, 13 and 17.

35Volume 7C. Annex 1, 3 and 4.

36Volume 7A - 7E.

37Answers to the Questions from the EU Member States. Volume I. November, 2003.

38

Volume 7A. Annex 4, 5 and 6.39Volume 7A.Annex 12.

40Volume 7A. Annex 17.

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31.262.5

38.8b

39.7ab

39.2

b

40.4a

341

74 x 10

1.566.253162

28.6f

-36.2

c

39.8a

30e

37bc

37

b

40a

442

74 x 10

31627894

-39.5

bc

40.0bc

39.4

bc

33.4d

40.6ab

41.8

a

39.7bc

Means with different letter are significantly different (comparison between all treatments)1 g 25-OH-D3is nearly equivalent to 2 g vit. D.

Bone ash increased with the dietary levels of vitamin D3and 25-OH-D3from 0 to 62 g kg-1diet

(calculated value, analytical values are not reported). In about 50% of the comparisons betweenvitamin D3 and 25-OH-D3 at similar dietary level, it was observed a higher bone ash when

supplying the 25-OH-D3. This difference was smaller or it was not observed when the dietarylevel of the vitamin D3was higher (between 60 and 100 g kg

-1diet). The bone ash data showedthat the efficacy of 25-OH-D3 in tibia mineralization was about two fold that of vitamin D3 fordosages up to 60 g kg-1diet.

2.2.2. Laying hens

One trial comprising two phases was reported by the applicant.43 The hens (initial age: 22weeks) were supplied with 41 and 82 g kg-1of 25-OH-D3which were compared to a single levelof vitamin D3(69 g kg

-1). The feed conversion ratio was decreased in hens fed the hydroxylatedform of Vitamin D3 when hens were supplied at 82 g kg

-1 diet. Egg shell thickness wasincreased in birds fed on 25-OH-D3compared to those fed on vitamin D3(0.17 versus 0.18 mm,non significant).

A trial44on 288 laying hens (18-68 weeks) fed on 82 g kg-1diet vitamin D3or 25-OH-D3showedimproved egg production after 43 weeks of age and higher egg specific gravity in one of thebreeds of hens when fed on 25-OH-D3instead of vitamin D3.

Scientific literature published before 1980 has been included in the dossier (Soares et al., 1995),and shows that 25-OH-D3has a similar efficacy or even higher efficacy than vitamin D3for eggshell quality when supplied at low levels (

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Table 6. Summary the three acceptable turkey experiments conducted in the USAFinal bodyweight (kg)

Feed conversion(g feed g-

1gain)

No. ofanimals xreplicates

Duration(days)

Dose level(1)

(g kg

-1feed)

D325-OH-

D3D3

25-OH-D3

Ref

38x12

16x12

84 (phase1)

42 (phase2)

40/35 (phase 1/2)138/94 (phase 1/2)

-14.6

15.114.8

-3.02

2.962.97

47

25 x 5 (f)21 x 5 (m)

11249.56999

-11.0

11.2-

11.3*

-2.46

2.43

2.41*

48

31 x 13101 (f)133 (m)

49.596-69

(2)

99

-8.6-

8.7

8.9*

-2.45

-

2.35

2.26

49

(1) range of dietary levels of vitamin D3 or its metabolite, the levels of which changed at variousphases of turkey rearing.

(2) The concentration of Vitamin D3 were reduced tn the experiment from 96-69.f=female, m=male.*significantly different from control vitamin D3

The analytical characterisation of the feed (performed only in the second study) showed that thelevels of 25-OH-D3were lower than expected (around 40 and 82 instead of 49 and 99 g kg

-1 25-OH-D3). In the third trial the analytical values in the post pellet diet were about 10% higher thanexpected.

The mortality of the turkeys in the various trials was in the expected range and was notinfluenced by the dietary level of vitamin D or 25-OH-D3. Growth performance and the feed

conversion ratio were not affected by the substitution of vitamin D3 by 25-OH-D3 whenincorporated at levels between 40 to 100 g kg-1(it was variable, depending on turkey age insome of the trials). In two of the trials, the body weight and feed conversion ratio (one trial) wereimproved when 25-0H-D3was used instead of vitamin D3(Table 6).

In the two trials carried out in France,5025-OH-D3was supplied in addition to a basal dietarylevel of vitamin D3. In these field studies carried out on 320 male turkeys for 104 days and on432 turkeys for 105 days (3 treatments), the addition of 25 g 25-OH-D3and 62.5 g vitamin D3did not improve the body weight nor the feed conversion ratio (P>0.1) in one of the trials. In theother study the feed conversion rate was slightly improved. A detailed description of these fieldtrials is not available.

The report of the applicant is completed by references of publications evaluating 25-OH-D3relative to Vitamin D3 in the turkey breeder hen and in young turkeys, but without clearidentification of the source of 25-OH-D3.

51

2.3. Studies on the quality of animal produce

The carcass yield (percentage) of chickens for fattening was evaluated in 11 trials comparing 25-OH-D3 and Vitamin D3. It was improved in two comparisons amongst 22 comparisons. The

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50Volume 7E. Annex 8 and 9.

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breast meat yield was also measured in 22 comparisons at similar levels of both compounds.525354Four trials showed a slight improvement on the amount of the breast meat. The magnitudeof the increase was low,

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Bone ash low P 2 - 2.5 Soares et al.,1978aBody weight 4 Fritts and Waldroup, 2003

Body weight 2 see Table 4aSoares et al.(1995).

It seems therefore logical to give the potency of 25-OH-D3 in g, which is the internationalstandard (IUPAC) anyway. But such a decision would also have the consequence that vitamin D3and vitamin D2require to be labelled in g instead of IU. This proposal is scientifically justified.

If the EU system of mandatory labelling all vitamin D active compounds in IU should bemaintained, 1 g of 25-OH-D3should be considered as > 40, probably 80 IU Vitamin D. To avoidmisunderstanding by farmers, the labelling should consist of two parts (i) the potency (IU vitaminD) followed by (ii) the source of the vitamin (from vitamin D3, vitamin D2or Calcifediol).

2.5. Conclusion

The efficacy of 25-OH-D3 concerning weight gain, feed conversion and bone mineralisation for

chickens for fattening is at least equivalent to that of vitamin D3when supplemented at dietarylevels of 30 to 69 g kg-1. At lower doses (2.5 and 25 g kg-1), the efficacy concerning bone andfeed conversion of 25-OH-D3is doubled compared to that of the vitamin D3. The bone ash datashows that efficacy of 25-OH-D3is even higher than that of vitamin D3(about two fold).

Concerning laying hens, it has been demonstrated that 25-OH-D3, in the dose range of 41 to 82g kg-1, is at least equivalent to vitamin D3for optimizing hen performance and egg quality. Inturkeys, it can be concluded that 25-OH-D3can be used as a substitute for vitamin D3 in therange tested by the applicant (40 to 100 g kg-1).

When different levels of 25-OH-D3were evaluated, no significant differences were observed so itis difficult to conclude on the optimal dietary level to be used and to know if it differs from that ofvitamin D3.

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 g 25-OH-D3 is equal to1 g vitamin D3 or 40 IU vitamin D. The companys deduction is based on a considerablenumber of experiments on poultry with mostly higher dosages (>30g) of vitamin D3and 25-OH-D3, which do not allow comparable dose titration. In addition, the analysed parameters (bodyweight and feed conversion) do not reflect the primary metabolic action of vitamin D, which is onbone mineralization.

25-OH-D3 has a higher potency than vitamin D3. Considering literature and the suitableexperiments submitted 1 g of 25-OH-D3should be considered as > 40, probably 80 IU VitaminD. The higher potency of 25-OH-D3 depends on the parameter chosen. It seems thereforelogical to give the potency of 25-OH-D3in g, which is scientifically correct.

If the EU system of mandatory labelling all vitamin D active compounds in IU should bemaintained, 1 g of 25-OH-D3 should be considered as 80 IU Vitamin D3. To avoidmisunderstanding by farmers, the labelling should consist of two parts (i) the potency (IU vitaminD) followed by (ii) the source of the vitamin (from vitamin D3, vitamin D2or Calcifediol).

3. Safety studies on target species

3.1. Tolerance studies

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The applicant submitted one preliminary safety study55and two tolerance studies on chickens forfattening56(both published by Yarger et al., 1995), and one each for laying hens57and turkeys58.A third tolerance trial on chicken for fattening lasted only 14 days and was not considered indetail. Unfortunately analytical confirmation of the dose levels of the tolerance studies could notbe found in the dossier.

3.1.1. Chickens for fattening

In the preliminary study, 400 birds (five replicates with 40 males and 40 females each) pertreatment were fed for 46 days diets with 6.25, 31.25, 56.25, 112.5, 225, and 450 g vitamin D 3and 25-OH-D3kg

-1, respectively. Body weight and feed efficiency were not affected by dose, butthe 25-OH-D3groups generally performed better. There was a tendency for higher mortality in thehigh 25-OH-D3groups, but not in the high vitamin D3groups.

In experiment I, a total of 1120 broilers (280 birds, 5 replicates of 28 males and 28 females pertreatment) was fed for 49 days diets (starter, grower, finisher) containing 69 g vitamin D3, 69,207 and 690 g 25-OH-D3 kg

-1, respectively. The diets did not contain a coccidiostat or otherdrugs, which is considered as a cause of the relatively high mortality observed (between 12 and16.6 % 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, whichhowever showed an insignificant tendency for increased values in all 25-OH-D3groups. Serum25-OH-D3 was about three times higher in the 69 g 25-OH-D3group (37 ng mL

-1) than in thevitamin D group (13 ng mL-1). The three and ten fold dietary concentration of 25-OH-D3resultedin serum levels of 110 and 242 ng mL-1. Serum 1,25-(OH)2-D3was not different for the 69 gvitamin D3and the 69 g 25-OH-D3kg

-1 group, it was significantly lower in the 690 g 25-OH-D3kg-1 group, with the 207 g 25-OH-D3kg

-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 onemale and one female per replicate were taken for histopathology.

No treatment related abnormalities were seen by gross pathology in the tissues examined nor inblood cell counts, hematocrit, hemoglobin, or prothrombin time. Also histopathology did notreveal significant group differences.

In experiment II, a total of 3500 broilers (350 birds, seven replicates of 25 males and 25 femalesper treatment) was fed for 49 days diets (starter, grower, finisher) containing vitamin D3and 25-OH-D3at levels of 69, 690, 3450, 6900 and 13800 g kg

-1, respectively. Because of high toxicityand morbidity, the treatments with 6900 and 13800 g 25-OH-D3kg

-1 had to be terminated after

22 days, treatment with 3450 g kg-1

after 31 days. Starter and grower diets containedsalinomycin (60 mg kg-1) a coccidiostat and bacitracin dimethyl salicylate. Average mortality ofthe remaining 25-OH-D3and of the 3 low vitamin D3groups at the end was 4.8 %, of the 2 highvitamin D3groups 9.1 and 12.6%, respectively. Other 4.3% (average of all 7 groups) were culledduring the experiment. The results are summarised in Table 8. At dose level 690 g kg -1feed,there was a significant difference (P

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Dietary level (g kg-1) 69 690 3450 6900 13800

Mortality (%)a, vitamin D3 5.43 4.29 4.84 9.14 13.14

Mortality (%)a, 25-OH-D3 4.57 6.29 (16.9)b (8.88)c (17.5)c

Body weight (kg), vitamin D3 2.49 2.50 2.19 1.23 0.87Body weight (kg), 25-OH-D3 2.51 2.22*

g feed g-1gain, vitamin D3 1.87 1.89 1.92 2.30 2.72

g feed g-1gain, 25-OH-D3 1.86 1.87

amortality rate includes deaths and culls

bafter 31 days

cafter 22 days

At the termination of each treatment period, the birds were examined for gross pathology. Thefollowing 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 (g kg-1) 69 690 3450 6900 13800

No. of birds, examined, vitamin D3 28 28 28 28 59

No. of birds, , examined, 25-OH-D3 28 28 (66)b (41)c (59)c

Renal calcification (%) a, vitamin D3 17.9 7.1 55.2 89.3 100

Renal calcification (%) a, 25-OH-D3 7.1 96.4 (100)b (100)c (100)c

aincludes trace, mild and moderate calcification

bafter 31 days

cafter 22 days

Whereas for vitamin D3 an increase in renal calcification could be observed at 3450 g kg-1, such

in an increase occurred for 25-OH- D3already at 690 g kg-1(from 27 renal calcification findingsin this group, 16 were considered as trace and 10 as mild). This observation is in contrast toexperiment 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-D3is 5 to 10 times more toxic for chickens for fattening than vitamin D3.

Morrissey et al. (1977) observed renal tubular calcification after a 2 week period (14 to 28 d) at adose level of 10000 g vitamin D3, but for 25-OH-D3already at 100 g kg

-1 feed. The authorsconcluded that 25-OH-D3 may be 100 fold more toxic than vitamin D3. 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-D3 toxicity.

A third trial on chickens for fattening (6 replicates with 8 birds each per treatment) with doses of35, 70, 140, 280 and 560 g 25-OH-D3kg

-1feed59, respectively, was conducted from the age of8 to 22 days. Body weight gain increased with higher 25-OH-D3 amounts (up to 280 g,significant difference to 35 g), but 560 g 25-OH-D3kg

-1feed was lower and numerically equalto 35 g 25-OH-D3kg

-1feed. Feed conversion was more or less improved by all higher 25-OH-D3treatments. Plasma Ca and inorganic P were not dose-dependent affected by 25-OH-D 3 kg

-1feed, but 140 g 25-OH-D3kg

-1feed showed lowest values (significant for Plasma Ca). ApparentCa- and P- retention was increased up to 280 g 25-OH-D3 kg

-1 feed. Tibia strength wasnumerically increased by higher 25-OH-D3dosages, but tibia ash significantly increases too. Thisexperiment is not further considered due to its short duration (2 weeks) and the lack ofassessment of critical parameters (e.g., renal calcification).

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Two more recent studies (Bar et al., 2003; Fritts and Waldroup 2003) showed that 100 g 25-OH-D3kg

-1 complete feed can be considered as safe for chickens for fattening.

3.1.2. Laying hens

After a pre-period of 4 weeks, the tolerance trial was started with laying hens of 25 weeks of age.A total of 450 layers (9 replicates with 10 hens each) were distributed to 5 treatment groups feddiets containing 69 g vitamin D3, 41.24, 82.5, 412.5, and 825.0 g 25-OH-D3 kg

-1 feed,respectively. The duration of experimental phase I was 16 weeks, 270 layers remaining (9replicates with 6 hens each -after necropsy) were fed the same diets for another 16 weeks(phase II)60. Table 10 reviews the most important criteria of phase I, Table 11 those of phase II.

Table 10. Tolerance study on laying hens, week 25 to week 41 of age, phase I

Vitamin D3 25-OH-D3Dietary level (g kg-1)

and source 69 41.25 82.5 412.5 825.0Egg production (%) 83.4 84.2 84.0 83.2 82.5*Egg weight (g) 56.8 57.3* 57.4* 55.9* 54.4*Egg shell thickness (mm) 0.17 0.18* 0.18* 0.17 0.16*Body weight change (g) 336 344 324 295 233*Feed consumption (g d-1) 108.6 108.3 108.1 109.2* 111.5*kg feed kg-1eggs 2.29 2.25 2.24 2.35* 2.48*

* figures significantly different (p

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In phase II, 825 g 25-OH-D3 kg-1negatively influenced egg production, egg weight and feed

efficiency data (feed kg-1eggs) compared to the vitamin D3group (Table 11). Also egg weightand feed consumption kg-1eggs of the 412.5 g 25-OH-D3group differed significantly from thevitamin D3 group. 82.5 g 25-OH-D3 kg

-1feed improved significantly egg production, egg weight,egg shell thickness and the feed efficiency parameters. Egg quality criteria, not dependent onegg weight, were not significantly influenced by the treatment.

No increase in mortality was observed in both phases. The pathology report indicates thepresence of granular, basophilic material in the kidneys of birds sacrificed at days 7 and 112, butdoes not state that this was a treatment or dose-related effect (full report not submitted).

3.1.3. Turkeys

A total of 1380 turkeys (6 replicates with 25 females, and 6 replicates with 21 male turkeys) werefed diets containing 68.9 g vitamin D3 and 44.5, 99, 495, and 990 g 25-OH-D3 kg

-1 for 16weeks.

Final body weight and feed efficiency was not significantly affected by the 25-OH-D3 treatment.

However, 99 g 25-OH-D3 showed a significantly higher body weight and a better feedconversion rate than the vitamin D3 control.

All mortality and observations made during the course of the trial were found to be consistent withcommercial practices. Total mortality was significantly higher for the groups with 495 (13.1%),and 990 g 25-OH-D3 kg

-1(14.8%) compared to 6.0% in the control vitamin D3group. Howeverthe increase in mortality was particularly seen in males and occurred mainly in the initial testphase due to an increased incidence of air sacculitis, omphalitis, and colibacillosis. No mortalitywas observed between day 84 and day 112. Five cases of soft bones were recorded in the 990g 25-OH-D3 group, one in the 495 g 25-OH-D3 group, and zero in the other groups.

Total disorders observed (dehydration, reduced pigmentation score -legs- and featheringcondition) finally increased with 990 g 25-OH-D3 kg

-1 to 26 cases compared to 0-3 in the other

groups.One randomly selected turkey per replicate on day 7 and two randomly selected turkeys perreplicate on day 112 from the 99, 495, and 990 g 25-OH-D3 groups were sacrificed forhematology, gross necropsy (also weights of liver, kidney, heart, bursa of fabricius, brain, spleen,thymus, bone marrow, and ovaries) and histopathology of the organs weighed (except ovaries)adrenal glands, testes, ovary, eye, pancreas, bone and bone marrow, lung, trachea, esophagus,crop, proventriculus, ventriculus, intestines, skin, spinal cord, pituitary body, thyroid, parathyroid,breast and thigh muscle, and gross lesions if applicable.

No significant differences were found among any of the blood parameters and tissue weights(based on equal body weight). The histology report did not indicate tissue alterations attributableto the highest 25-OH-D3treatment. In the kidney regenerative tubular epithelium was present in

most of the turkeys and mononuclear cell infiltrate was present in many of the males and a few ofthe females. Tubular mineralisation was present in the kidneys in four males and two females inthe groups receiving vitamin D3, in one male receiving 25-OH-D3at 99.0 g, two males and twofemales receiving 495 g 25-OH-D3, and in five males and one female receiving 990 g 25-OH-D3kg

-1feed.

3.1.4. Conclusions on the safety for the target animals

The studies on chickens for fattening clearly show that 25-OH-D3has a higher toxic potential thanvitamin D3. Because of large steps between the dosages in the crucial experiment II, thetolerance studies would not allow precise calculations of a safety factor, but it can be estimated,based on the incidence of renal calcifications that 25-OH-D3 may have a 5-10 higher toxic

potential for chickens for fattening than vitamin D3. Although a margin of safety for the upper levelrecommended by the notifier (70 g 25-OH-D3kg

-1) can not be given, it is certainly less than 10,

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because 690 g 25-OH-D3kg-1caused weight gain depression and lead to a higher occurrence

of renal calcifications.

For laying hens a comparison concerning a potentially different tolerance of vitamin D3 and 25-OH-D3 (recommended level by the notifier: 75 g 25-OH-D3 kg

-1) can not be made due to thestudy design. The study on laying hens, based on production parameters, showed that 825 g

25-OH-D3 kg-1

diet are not tolerated well by layers. Even 412.5 g 25-OH-D3 kg-1

feed resulted fortwo 16 week periods in smaller egg weight and increased feed consumption kg-1 eggs. It isconcluded that the margin of safety is smaller than 5.5 (412.5/75). A field study allowsestablishing 82.5 g 25-OH-D3 kg

-1diet as safe.

As long as no more detailed data is available it would be prudent to accept 100 g 25-OH-D3 kg-1

complete feed as the upper tolerated limit for chickens for fattening and 80 g 25-OH-D3 kg-1for

laying hens.

In contrast to chickens for fattening and laying hens, turkeys seem to tolerate higher doses of 25-OH-D3well (recommended level by the notifier: 100 g 25-OH-D3kg

-1). The significantly highermortality observed for 495 and 990 mg 25-OH-D3kg

-1feed compared to 69 g vitamin D3may beregarded as incidental, because the figures are not clearly attributable to 25-OH-D3 treatment.

However, for 990 g 25-OH-D3kg-1feed there is a weak evidence for mild intolerance. Thereforethe margin of safety of the upper recommended level could be given as about 5 (495/100).

3.2. Fate of 25-OH-D3and body deposition

3.2.1. Fate of 25-OH-D3

The comparative absorption and excretion of cholecalciferol and 25-OH-D3 in birds (Bar et al.,1980) has shown that: i) the relative net absorption of 25-OH-D3 is higher than that ofcholecalciferol in chickens (84 and 67% respectively) while cholecalciferol absorption wassignificantly higher in the turkey than in the chicken (75%) (no data given for 25-OH-D 3), ii)absorption occurs mainly at the upper jejunum level, iii) overall net excretion of cholecalciferol aspolar (chloroform-methanol extractable) metabolites was 20% and 14% in the chicken and turkeyrespectively, that of 25-OH- D3 representing only 7% for both species.

In mammals and birds, 25-OH-D3 is the initial metabolite of cholecalciferol resulting fromhydroxylation in the liver (Haussler and Rasmussen, 1972). In birds, it represents the mostsignificant metabolite in the plasma, kidney and bone. Subsequent metabolism at the kidney levelgives rise to the biologically active metabolite 1,25-(OH)2-D3 (Holick et al., 1971) which ismarkedly present in the same tissues. 25-OH-D3is metabolized also to 25,26-(OH)2-D3, 21,25--(OH)2-D3 and several other more polar and excretable compounds that are detectable in theplasma.

No retroconversion of 25-OH-D3to Vitamin D3occurs. It is very likely that the metabolic fate oforally administered 25-OH-D3 would be qualitatively the same as that of the endogenouslyproduced 25-OH-D3.

3.2.2. Deposition

3.2.2.1. Chickens for fattening

One-day old (male and female) chickens for fattening were given commercial-type feedssupplemented with the same quantity of vitamin D3or 25-OH-D3(69 g kg-

1 feed) but also higher

doses (x3 and x10) of 25-OH-D3(207 and 690 g kg-

1

feed).

61

62

After 49 days administration, a61

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longer period than usual in Europe, the birds were slaughtered and serum and tissues sampledfor 25-OH-D3but also 1,25-(OH)2-D3 determination. No gender difference was observed. Serumlevels of 25-OH-D3 were 3 times higher in birds fed 25-OH-D3when compared to a similar dose ofvitamin D3 (43 and 13 ng L

-1 respectively). As dietary 25-OH-D3increased from 207 to 690 g kg-1

feed, serum 25-OH-D3 increased from 113 to 246 ng L-1. However, serum 1,25-(OH)2-D3

concentration fell from 68 to 54 pg mL-1 despite an 18-fold increase in serum 25-OH-D3concentrations from the birds fed vitamin D3 to those fed the highest level of 25-OH-D3 whichindicates a tight regulation of this key metabolite. The results of the analysis of 25-OH-D 3 intissues (Table 12) indicate that its concentration is about 3 times higher in the animalsadministered directly this compound in comparison to a similar amount of vitamin D3. A significantpositive correlation is observed between the serum and tissue 25-OH-D3 concentrations. Theslope for skin/fat concentration is 3 times that for breast muscle.

Table 12. 25-OH-D3concentration in tissues of chicken for fattening fed vitamin D3or25-OH-D3for 49 days (g kg-

1 wet tissue)

Vitamin D source and inclusion rate (g kg-1

feed)

25-OH- D3Tissues Vitamin D369 69 690

Breast muscle 1.4 3.8 18.1*

Thigh muscle 2.1 6.4* 33.2*

Skin + fat 4.4 12.9* 118*

* figures significantly different (p 0.05) from the vitamin D3 group

3.2.2.2. Laying hen eggs

The incidence of vitamin D3supplementation of feed on the concentration of vitamin D3and 25-OH-D3 in egg yolk has been studied in laying hens

63(Mattila et al., 1999). A metabolic equilibriumwas reached after the first time point retained, i.e. 4 weeks exposure. At a dose currently used inlaying hen feed, i.e. 62 g vitamin D3kg

-1, 25-OH-D3 concentration in egg yolk was about 30 % ofthat of vitamin D3 (Table 13). The increase of the concentration of vitamin D3 in feed (x 3.5)resulted in a considerable increase (x 7) of vitamin D3 deposition in egg yolk whereas 25-OH-D3concentration was multiplied by a factor of 1.5 only.

Table 13. Vitamin D3 and 25-OH-D3 concentration in eggs of laying hens fed vitamin D3for 4 to 6 weeks (g per 100 g egg yolk) (Mattila et al.,1999)

Vitamin D3inclusion (g kg-1feed)

26.6 62.4 216

Vitamin D3 1.4 3.4 23

25-OH-D3 0.5 1.0 1.5

Laying hens 20 weeks of age were pre-conditioned for 28 days on a standard diet then assignedto 5 groups of 9 animals that received either vitamin D3at 69 g kg-

1feed (control group) or 25-OH-D3 at 41, 82, 413 and 825 g kg-

1 feed (x0.5, x1, x5 and x10 the dose proposed for use

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respectively) for 28 days measured values were close to the target dosage at the beginning ofthe study but decline by 20% at the end of the study. Eggs were collected the 56 th day. Theanimals were slaughtered and tissues sampled. 25-OH-D3concentration was determined in theeggs and tissues, using an HPLC/IRC method (see 1.4). The results (Table 14) indicate that 25-OH-D3concentrations were not significantly different in the eggs laid by either the vitamin D3 or25-OH-D3supplemented animals as long as the dosages were similar. Higher doses (x5 or x10)increased the egg contents by a factor of 2 and 4 respectively. When muscle (breast and thigh)and skin plus fat are concerned, a similar situation was observed where 25-OH-D3 levels werenot significantly different in the birds that received vitamin D3 or a similar quantity of 25-OH-D 3.However, a 5-time increase of the 25-OH-D3 dosage increased the residual levels by 2.5, 4, 2.5and 3 in the liver, skin/fat, breast muscle and thigh muscle respectively.

Table 14. 25-OH-D3 concentration in eggs and tissues of laying hens fed vitamin D3or25-OH-D3for 28 days (g kg-

1wet tissue)Vitamin D source and inclusion rate (g kg-1feed)

25-OH-D3Tissues

Vitamin D369 41 83 413 825

Whole Egg 10.5

11.9

13.2

24.2* 46.9*Breast muscle 2.5 - 3.4 8.2* -

Thigh muscle 3.7 - 4.6 14.5* -

Skin plus fat 10.7 - 13.5 44.8* -

Liver 7.6 7.5 9.2 25.6* -

Kidney - - 9.2 - -


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Liver 3.1 6.4 6.5

Kidney - 6.4 -


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4.1. Conclusions

As a general principle the FEEDAP Panel considers conventional toxicological studies to beinappropriate for testing chemically defined pure substances which are dietary nutrients, which isthe case for 25-OH-D3from HyD for which the chemical purity is established.

The data submitted give some indications that 25-OH-D3 is not genotoxic and confirm that theacute, sub-chronic and reproductive toxicological effects observed are entirely consistent with aphysiological overload of vitamin D3 or its metabolites.

5. Safety evaluation for the human consumer

5.1. Human use of 25-OH-D3

25-OH-D3is used in human medicine to prevent rickets, to manage metabolic bone disease andfor the treatment of hypocalcemia.67 No incompatibilities are known, and any-side effects aredescribed related to overdosing in susceptible individuals.68

5.2. Status of 25-OH-D3in humans

As 25-OH-D3 is a normal metabolite in vitamin D metabolism in man, it is very likely thatexogenous sources of 25-OH-D3 in the diet will be metabolised in the same way as theendogenously produced 25-OH-D3. Several studies in humans and animals have demonstratedthat while vitamin D is absorbed mainly into the lymph, the more polar metabolite 25-OH-D3, atphysiological concentrations, is also absorbed more rapidly and efficiently from the proximaljejunum into the portal vein (Thompson et al., 1966; Blomstrand and Forsgren, 1967; Sitrin et al.,1982; Maislos, and Shany, 1987). Increasing amounts (25 and 100 g) of dietary vitamin Dincrease serum 25-OH-D3concentrations in a dose dependent manner, both doses leading toplateau concentrations after 2-3 months (Vieth et al.,2001). The concentration of 25-OH-D3 inthe serum, which is dependent on the balance between supply (direct or from vitamin D

metabolism) and clearance, is maintained within a range from 75 to 200 nmol L -1across a widerange of daily supplies of vitamin D (20 to 250 g), suggesting that an homeostatic controlsystem exists and regulates serum 25-OH-D3. This metabolite is further metabolized in thekidney to the main active metabolite 1,25-(OH)2D which is strictly regulated by parathyroidhormone and calcium. The blood level of 1,25-(OH)2D is maintained within a narrow rangeindependant of normal variations of vitamin D supply and in circulating vitamin D or 25-OH-D3.Pharmacologic doses of 25-OH-D3do not change or may even decrease plasma levels of 1,25-(OH)2D unless vitamin D deficiency is present (review from Ovesenet al., 2003).

Vitamin D3 in high doses is very toxic to man and during the toxic episode the serumconcentration of 25-OH-D3 reaches very high levels. There are no systematic studies on thetoxicity of 25-OH-D3in the human.

5.3. Biological activity of 25-OH-D3

Vitamin D status in humans is determined by measuring serum 25-OH-D3 concentration. Thebiological activity of 25-OH-D3 is greater than that of vitamin D. However, there is not yetconsensus on the conversion factor that should be used for 25-OH-D3 to calculate vitamin Dequivalence. Depending on the testing system used but always referring to the rat, the factorvaries from 1.5 to 5 (Blunt et al., 1968; Reeve et al., 1982). The activity factor of 1.5 is used in theAmerican Dietary Allowances (Institute of Medicine, 1997) whereas a factor of 5 is used in theBritish Food composition tables (Chan et al.,1995). It must be noted that the half-life of 25-OH-D3(20-28 days) is much shorter than that of vitamin D3(Haddad and Rojanasathit 1976).

67Volume 6. Annex 21.

68Volume 8H. Annex 10.

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5.4. The Tolerable Upper Limit (UL) for humans

The UL for vitamin D intake in humans, based on symptoms of hypercalcaemia that appear asthe first toxicity sign, has been set to 50 g day-1 in adults and 25 g day-1in children up to the

age of 11 (EC, 2002; Institute of Medicine, 1997). The FEEDAP Panel suggests an UL for 25-OH-D3 estimated using the relative biological activity factor of 5 which represents the mostconservative approach considering either the results obtained in the rat (see 5.3.) or the chicken(see Table 7). It would represent 10 g day-1in the adult and 5 g day-1in children.

5.5. Consumer exposure to 25-OH-D3

Vitamin D supply to humans includes vitamin D3produced in the skin under the influence of UV-light and dietary vitamin D3and vitamin D2. Studies have shown that dietary vitamin D3activity ismainly composed of native vitamin D3. However, in some foodstuffs 25-OH-D3 contributes tovitamin D activity (Mattila et al., 1993; Mattila, 1995; review from Ovesen et al., 2003). 25-OH-D3

contents are typically low (

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Table 16. 25-OH-D3human intake based on consumption model from Directive2001/79/EC.

Amountconsumed

Content of 25-OH-D3 (g kg-

1)

in chicken in turkey

Intake of 25-OH-D3 (g day-1)

From chicken From

turkeyand egg and egg

300 g Muscle 7.7a 2.3d 2.31 0.69

100 g Liver 10.0b 6.4d 1.00 0.64

10 g Kidney 9.2c 6.4d 0.09 0.06

90 g Skin/fat 18.1a 8.2d 1.63 0.74

100 g Egg 13.8b 1.38 1.38

Total 6.41 3.51a)

from chickens for fattening, after interpolation corresponding to 100 g 25-OH-D3 kg-1

feed.b)

from laying hens, after interpolation corresponding to 100 g 25-OH-D3 kg

-1

feed.c)from laying hens 83 g 25-OH-D3 kg-1

feed.d)

from turkeys for fattening 99 g 25-OH-D3 kg-1

feed.

With the aim to refine this calculation, in the case of chicken meat plus egg, another estimationhas been performed taking into consideration more realistic data, based on intake data for themean adult population in the EU, derived from the SCOOP project (EC, 2004). The calculated

exposure (Table 17) indicates a value of 2.3 g day-1 which represents 23% and 46% of theprovisional UL for the adult and children respectively, considering that the childrens consumptionof meat and egg would be as high as that of the adult.

It must be noted that, due to the non reversiblity of the conversion of 25-OH-D3to vitamin D3,the

vitamin D3contents of tissues or products (eggs) from animals fed 25-OH-D3supplemented dietsshould be very low when compared to those from animals that receive vitamin D3in their diet.

Table 17. 25-OH-D3human intake based on SCOOP data (EC, 2004).

(Maximum meataintake: 175 g day-1, maximum egg intake 36 g day-1)

Amount consumed Content of 25-OH-D3 (gkg-1) in chicken tissue and

egga

25-OH-D3 intake(g day-

1)from chicken and egg

105 g Muscleb 7.7 0.81

35 g Liverb

10.0 0.353.5 g Kidneyb 9.2 0.03

31.5 g Skin/fatb 18.1 0.57

36 g Egg 13.8 0.50

Total 2.26a)

See Table 16b)

meat intake calculated with the same proportion as in Directive 2001/79/EC, i.e. 60% muscle, 20% liver, 2%kidney and 18% skin/fat.

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5.6. Conclusion

At the highest 25-OH-D3dose recommended for use as feed additive in poultry, retained by theFEEDAP Panel, HyD does not increase significantly the exposure of the consumer to 25-OH-D3through the consumption of turkey tissues when compared to the levels found following vitaminD3supplementation at a same level. The exposure resulting from turkey plus eggs consumption

(3.5 g day-1) estimated from theoretical and worst case consumption figures retained by theFEEDAP Panel, represents 35% and 70% of the provisional UL for the adult and childrenrespectively.

When chicken for fattening is concerned, a similar calculation leads to a consumer exposure

value for chicken plus egg of 6.4 g day-1 which complies with the provisional UL for adults (64%)but is above that for children (128%). Using more realistic consumption data, the consumerexposure appears to be below the provisional UL for both the adult (23%) and children (46%).

Therefore, should 25-OH-D3 from HyD be used as a substitute of vitamin D3 for chickens for

fattening and turkeys for fattening at the maximum tolerated dose of 100 g kg-1 feed, and forlaying hens at the maximum tolerated dose of 80 g kg-1 feed, as retained by the FEEDAP Panel,

no additional risk for the consumer could be expected.The substitution of vitamin D3by 25-OH-D3from HyD should reduce considerably the vitamin D3contents of poultry tissues and products (eggs).

6. User safety assessment

Skin irritation

A GLP compliant study70of skin irritation of Hy-D beadlets was conducted in 3 rabbits accordingto OECD guideline 404. The HyD beadlets were non-irritant to rabbit skin.

Eye irritation

A GLP compliant study71 of eye irritation of HyD beadlets was conducted in three rabbitsaccording to OECD guideline 405. The HyDbeadlets caused some irritation to the conjunctivawhich resolved within 72 hours and does not therefore require to be classified for eye irritation.No studies of sensitization, inhalation, or dermal toxicity are available however a full occupationalexposure assessment has been carried out by the applicant relevant to the production andhandling of the product in its various forms.

6.1. Pure 25-OH-D3The advised protective measures include use of externally supplied air when handling thisproduct, thus inhalation exposure is not considered further. Since bioavailability via the dermalroute is unknown, exposure by this route is calculated, based on an assumption of 10% ofexposed dose. Since the maximum exposed dose used in this analysis is more than 100 timeshigher than a measured atmospheric level during normal handling this approximation isconsidered by the FEEDAP Panel to be acceptable. Based on this assessment it is concludedthat exposure in the absence of protective clothing could result in a dose equivalent to 5 times

the therapeutic dose of 25-OH-D3or 125 g.

Use of externally supplied air and full protective clothing while handling the product is thusconcluded to be necessary but sufficient to avoid exposures which may result in chronic effects.

70Answers to the Questions from the EU Member States. Volume III. Annex 2. January, 2005.

71Answers to the Questions from the EU Member States. Volume III. Annex 3. January, 2005.

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Since the acute toxicity is relatively low the protective measures applied are also considered tobe sufficient to prevent any acute effects.

The notifier reports monitoring of workers72on one occasion for blood levels of 25-OH-D3 and

1,25-diOH-D3after handling product during normal operations showed no pattern of change inblood values. Atmospheric levels achieved during handling pure 25-OH-D3were 0.33 mg 25-OH-

D3m-3.

6.2. Formulated product in beadlets (1.25% 25-OH-D3)

Since HyD is a product formulated into beadlets to minimise respirable dusts this reduces thepotential exposure of users to 25-OH-D3.

7374About 95% of particles have diameters in the 20-

80m while 3-4% have diameters of

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The applicant has given sufficient information on physical and chemical properties, the method

of production, stability and dusting potential of HyD as the hydroxylated form of vitamin D3(25-OH-D3). No DNA from the production process is expected to be present in the final product.

EFFICACY

The efficacy of 25-OH-D3 concerning weight gain, feed conversion and bone mineralisation forchickens for fattening is at least equivalent to that of vitamin D3when supplemented at dietarylevels of 30 to 69 g kg-1. At lower doses (2.5 and 25 g kg-1), the efficacy concerning bone andfeed conversion of 25-OH-D3is doubled compared to that of the vitamin D3. The bone ash datashows that efficacy of 25-OH-D3is even higher than that of vitamin D3(about two fold).

Concerning laying hens, it has been demonstrated that 25-OH-D3, in the dose range of 41 to 82g kg-1, is at least equivalent to vitamin D3for optimizing hen performance and egg quality. Inturkeys, it can be concluded that 25-OH-D3can be used as a substitute for vitamin D3 in therange tested by the applicant (40 to 100 g kg-1).

When different levels of 25-OH-D3were evaluated, no significant differences were observed so itis difficult to conclude on the optimal dietary level to be used and to know if it differs from that of

vitamin D3.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 g 25-OH-D3 is equal to1 g vitamin D3 or 40 IU vitamin D. The companys deduction is based on a considerablenumber of experiments on poultry with mostly higher dosages (>30g) of vitamin D3and 25-OH-D3, which do not allow comparable dose titration. In addition, the analysed parameters (bodyweight and feed conversion) do not reflect the primary metabolic action of vitamin D, which is onbone mineralization.

25-OH-D3 has a higher potency than vitamin D3. Considering literature and the suitableexperiments submitted 1 g of 25-OH-D3should be considered as > 40, probably 80 IU VitaminD. The higher potency of 25-OH-D3 depends on the parameter chosen. It seems therefore

logical to give the potency of 25-OH-D3in g, which is scientifically correct.

If the EU system of mandatory labelling all vitamin D active compounds in IU should bemaintained, 1 g of 25-OH-D3 should be considered as 80 IU Vitamin D3. To avoidmisunderstanding by farmers, the labelling should consist of two parts (i) the potency (IU vitaminD) followed by (ii) the source of the vitamin (from vitamin D3, vitamin D2or Calcifediol).

SAFETY FOR THE TARGET SPECIES

The studies on chickens for fattening clearly show that 25-OH-D3has a higher toxic potential thanvitamin D3. Because of large steps between the dosages in the crucial experiment II, thetolerance studies would not allow precise calculations of a safety factor, but it can be estimated,

based on the incidence of renal calcifications that 25-OH-D3 may have a 5-10 higher toxicpotential for chickens for fattening than vitamin D3. Although a margin of safety for the upper levelrecommended by the notifier (70 g 25-OH-D3kg

-1) can not be given, it is certainly less than 10,because 690 g 25-OH-D3kg

-1caused weight gain depression and lead to a higher occurrenceof renal calcifications.

For laying hens a comparison concerning a potentially different tolerance of vitamin D3 and 25-OH-D3 (recommended level by the notifier: 75 g 25-OH-D3 kg

-1) can not be made due to thestudy design. The study on laying hens, based on production parameters, showed that 825 g25-OH-D3 kg

-1diet are not tolerated well by layers. Even 412.5 g 25-OH-D3 kg-1feed resulted for

two 16 week periods in smaller egg weight and increased feed consumption kg-1 eggs. It isconcluded that the margin of safety is smaller than 5.5 (412.5/75). A field study allows

establishing 82.5 g 25-OH-D3 kg-1

diet as safe.

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As long as no more detailed data is available it would be prudent to accept 100 g 25-OH-D3 kg-1

complete feed as the upper tolerated limit for chickens for fattening and 80 g 25-OH-D3 kg-1for

laying hens.

In contrast to chickens for fattening and laying hens, turkeys seem to tolerate higher doses of 25-OH-D3well (recommended level by the notifier: 100 g 25-OH-D3kg

-1). The significantly higher

mortality observed for 495 and 990 mg 25-OH-D3kg-1

feed compared to 69 g vitamin D3may beregarded as incidental, because the figures are not clearly attributable to 25-OH-D3 treatment.However, for 990 g 25-OH-D3kg

-1feed there is a weak evidence for mild intolerance. Thereforethe margin of safety of the upper recommended level could be given as about 5 (495/100).

SAFETY FOR THE CONSUMER

In birds the ingested 25-OH-D3 behaves qualitatively in the same way as the endogenousmetabolite derived from vitamin D3metabolism. No retro-conversion of 25-OH-D3 to Vitamin D3occurs.

Deposition of 25-OH-D3 in the laying hen egg or turkey tissues is not significantly different in the

birds that received either vitamin D3or a similar quantity of 25-OH-D3corresponding to the doseproposed for use. However, deposition of 25-OH-D3 in chicken tissues resulting from 25-OH-D3supplementation of the diet at the dose proposed for use is about three times higher than thatfound following the use of the same quantity of vitamin D3(69 g kg-

1).

As a general principle the FEEDAP Panel considers conventional toxicological studies to beinappropriate for testing chemically defined pure substances which are dietary nutrients, which is

the case for 25-OH-D3from HyD for which the chemical purity is established.

The data submitted give some indications that 25-OH-D3 is not genotoxic and confirm that theacute, sub-chronic and reproductive toxicological effects observed are entirely consistent with aphysiological overload of vitamin D3 or its metabolites.

At the highest 25-OH-D3dose recommended for use as feed additive in poultry, retained by theFEEDAP Panel, HyD does not increase significantly the exposure of the consumer to 25-OH-D3through the consumption of turkey tissues when compared to the levels found following vitaminD3supplementation at a same level. The exposure resulting from turkey plus eggs consumption

(3.5 g day-1) estimated from theoretical and worst case consumption figures retained by theFEEDAP Panel, represents 35% and 70% of the provisional UL for the adult and childrenrespectively.

When chicken for fattening is concerned, a similar calculation leads to a consumer exposure

value for chicken plus egg of 6.4 g day-1 which complies with the provisional UL for adults (64%)but is above that for children (128%). Using more realistic consumption data, the consumerexposure appears to be below the provisional UL for both the adult (23%) and children (46%).

Therefore, should 25-OH-D3 from HyD be used as a substitute of vitamin D3 for chickens forfattening and turkeys for fattening at the maximum tolerated dose of 100 g kg-1 feed, and forlaying hens at the maximum tolerated dose of 80 g kg-1 feed, as retained by the FEEDAP Panel,no additional risk for the consumer could be expected.

The substitution of vitamin D3by 25-OH-D3from HyD should reduce considerably the vitamin D3contents of poultry tissues and products (eggs).

SAFETY FOR THE USER

The product is not an irritant to the skin or eyes. Sensitisation and respiratory effects of HyD

have not been characterised. HyD 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

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Vitamin D or 25-OH-D3. The use of protective clothing should be sufficient to avoid adverseeffects in users.

SAFETY FOR THE ENVIRONMENTThere is no necessity to perform an environmental risk assessment for this type of naturally

existing compounds, under the conditions of the proposed use.

MONITORING

Validated methods were described allowing monitoring of the components in the initial product(25-OH-D3), the premixes and feedingstuffs.

RECOMMENDATIONS

Since 25-OH-D3 is more potent in its vitamin D activity than vitamin D3, but higher potencydepends on and varies with the criterion assessed and the dosage applied, reliable information to

the user of the product HyD can scientifically not be given in terms of IU of vitamin D. Thereforethe FEEDAP Panel strongly recommends labelling of 25-OH- D3 in g. If for practical reasons thisis not immediately possible then the label of the product should include (i) the potency (IU vitaminD, 1 g of 25-OH-D3should be considered as 80 IU Vitamin D3) and (ii) the source of the vitamin(from vitamin D3, Vitamin D2or Calcifediol).

As long as no more specific data on target animal safety are available and considering the aboverecommendation for labelling 25-OH-D3in g kg

-1complete feed, the highest 25-OH-D3level forchickens for fattening and laying hens should be set with 100 g 25-OH-D3kg

-1complete feed (alevel proven as safe). The same level could also be applied for turkey feed regarding the higherpotency of 25-OH-D3 compared to vitamin D3 and the existing regulations for vitamin D3(maximum content: 5000 IU vitamin D3kg

-1turkey feed).

The FEEDAP Panel recommends that only 25-hydroxylcholecalciferol will be specified in theannex entry including the minimum content requested (>94%).

The addition of both vitamin D sources, vitamin D3 and 25-OH-D3, should not be permitted andthis information should be included in the annex entry.

DOCUMENTATION PROVIDED TO EFSA

1. Letter, dated 23-04-2004 with ref. D(2004)410196, from Ms. Paola TESTORI COGGIfrom the Health & Consumer Protection Directorate-General requesting a consultation ofthe scientific Panel on the evaluation of the safety and efficacy of HyD (calcifediol).

2. Submission of the dossier on 25-Hydroxyvitamin D3 as a vitamin additive for poultry

feeds to be marketed as HyD. ROCHE Vitamins Ltd.

3. Additional dossier on: Answers to questions and comments from EU Member StatesVolume I (Nov/Dec 2002 and April 2003). ROCHE Vitamins Ltd.

4. Additional dossier on: Answers to questions and comments from EU Member StatesVolume II (December 2003 to April 2004). DSM Nutritional Products Ltd.

5. Additional dossier on: Answers to questions and comments from EU Member StatesVolume III (January 2005). DSM Nutritional Products Ltd.

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REFERENCESBaker, D.H., Biehl, R.R. and Emmert, J.L. 1998. Vitamin D3 requirement of young chicks

receiving diets varying in calcium and available phosphorus. British Poult. Sci. 39 (3),413-417.

Bar, A., Sharvit, M., Noff, D., Edelstein, S. and Hurwitz, S. 1980. Absorption and excretion ofcholecalciferol and of 25-hydroxycholecalciferol and metabolites in birds. J. Nutr. 110,1930-1934.

Bar, A., Razaphkovsky, V., Vax, E. and Planvnik, L. 2003. Performance and bone developmenton broiler chickens given 25-hydroxycholecalciferol. Brit. Poult. Sci. 44(2), 224-233.

Blomstrand, R. and Forsgren, L. 1967. Intestinal absorption and esterification of vitamin D31,2-3H in man. Acta Chem. Scand. 21, 1662-1663.

Blunt, J.W., Tanaka, Y. and DeLuca, H.F. 1968. The biological activity of 25-hydroxycholecalciferol, a metabolites of vitamin D3. Proc. Natl. Acad. Sci USA 61, 1503-1516.

Chan, W., Brown, J., Lee, S.M. and Buss, D.H. 1995. Meat, poultry and game. Fifthsupplement to the Fifth Edition of McCance and Widdowsons the Composition of foods.Cambridge/London, Royal Society of Chemistry/Ministery of Agriculture, Fisheries andFood.

De Luca, H.F. 2004. Overview of general physiologic features and functions of vitamin D. Am.J. Clin. Nutr. 80 (suppl.), 1689S-1696S

EC (European Commission). 2002. Scientific Committee on Food. Opinion on the TolerableUpper intake level of vitamin D. http://europa.eu.int/comm/food/fs/sc/scf/index_en.html

EC (European Commission) 2004. Reports on Tasks for Scientific Cooperation (SCOOP) Task3.2.11. Assessment of the dietary exposures to arsenic, cadmium, lead and mercury ofthe population of the EU Member States Directorate General Health and Consumer

Protection.

Feldmann, D., Pike, J.W. and Glorieux, F. 2005. Vitamin D. Volume 1 and 2. Elsevier-

Fritts, C.A. and Waldroup, P.W. 2003. Effect of Source and Level of Vitamin D on LivePerformance and Bone Development in Growing Broilers. J. Appl. Poult. Res. 12:45-52.

Gesellschaft fr Ernhrungsphysiologie -GfE-. 1999. Empfehlungen zur Energie- undNhrstoffversorgung der legehenne

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