HyD EFSA

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 HyD (calcifediol), based
on 25-hydroxylcholecalciferol/25-hydroxy-pre-cholecalciferol, as feed
additive in accordance with Council Directive 70/524/EEC.

(Question NEFSA-Q-2004-065)

Adopted on 26 May 2005
SUMMARY

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. HyD 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 HyD (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 (1g 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 HyD 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 g. If for practical reasons this
is not immediately possible then the label of the product should include (i) the potency (IU vitamin
D, 1 g 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 g 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 g kg
-1
complete feed, the
maximum 25-OH-D
3
content for chickens for fattening should be set with 100 g 25-OH-D
3
kg
-1

complete feed (a level proven as safe). A similar deduction leads to a proposal of 80 g 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 g 25-OH-D
3
kg
-1
. 100
g 25-OH-D
3
kg
-1
feed level could be applied for turkeys for fattening as maximum content.
Opinion on the additive HyD (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 retro-
conversion 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, HyD 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 g 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 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-D
3
from HyD be used as a substitute of vitamin D
3
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 D
3
by 25-OH-D
3
from HyD 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 HyD 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, sub-
chronic 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 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 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.

TABLE OF CONTENTS
1. Introduction .................................................................................................................... 5
1.1. Composition and characteristics of HyD............................................................ 6
1.2. Chemical and physical characteristics of 25-OH-D
3
............................................ 6
1.3. Stability............................................................................................................... 8
1.3.1. Shelf life of HyD................................................................................................ 8
1.3.2. Premixes ............................................................................................................ 9
1.3.3. Complete feeds and treatments.......................................................................... 9
1.4. Control methods ................................................................................................. 9
2. Efficacy of the product in target species....................................................................... 10
2.1. Recommendations for vitamin D
3
in poultry ...................................................... 10
2.2. Efficacy trials .................................................................................................... 10
2.2.1. Chickens for fattening....................................................................................... 11
2.2.1.1.Growth performance and feed conversion ratio................................................ 11
2.2.1.2.Bone mineralization.......................................................................................... 12
2.2.2. Laying hens...................................................................................................... 13
2.2.3. Turkeys............................................................................................................. 13
2.3. Studies on the quality of animal produce .......................................................... 14
2.4. Bioequivalence and labelling. ........................................................................... 15
2.5. Conclusion........................................................................................................ 16
3. Safety studies on target species .................................................................... 16
3.1. Tolerance studies ............................................................................................. 16
3.1.1. Chickens for fattening....................................................................................... 17
3.1.2. Laying hens...................................................................................................... 19
3.1.3. Turkeys............................................................................................................. 20
3.1.4. Conclusions on the safety for the target animals............................................... 20
3.2. Fate of 25-OH-D
3
and body deposition ............................................................. 21
3.2.1. Fate of 25-OH-D
3
.............................................................................................. 21
3.2.2. Deposition ........................................................................................................ 21
3.2.2.1.Chickens for fattening....................................................................................... 21
3.2.2.2.Laying hen eggs............................................................................................... 22
3.2.2.3.Turkeys ............................................................................................................ 23
3.2.3. Conclusions...................................................................................................... 24
4. Studies on laboratory animals. ..................................................................................... 24
4.1. Conclusions...................................................................................................... 25
5. Safety evaluation for the human consumer .................................................................. 25
5.1. Human use of 25-OH-D
3
................................................................................... 25
5.2. Status of 25-OH-D
3
in humans.......................................................................... 25
5.3. Biological activity of 25-OH-D
3
.......................................................................... 25
5.4. The Tolerable Upper Limit (UL) for humans...................................................... 26
5.5. Consumer exposure to 25-OH-D
3
..................................................................... 26
5.6. Conclusion........................................................................................................ 28
6. User safety assessment ............................................................................................... 28
Skin irritation ................................................................................................................ 28
Eye irritation................................................................................................................. 28
6.1. Pure 25-OH-D
3
................................................................................................. 28
6.2. Formulated product in beadlets (1.25% 25-OH-D
3
)........................................... 29
6.3. HyD supplemented feed.................................................................................. 29
6.4. Conclusions...................................................................................................... 29
7. Safety for the Environment ........................................................................................... 29
Conclusions ..................................................................................................................... 29
Documentation provided to EFSA.................................................................................... 32
References ...................................................................................................................... 33
Scientific Panel Members................................................................................................. 35

Acknowledgement............................................................................................................ 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 HyD (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 HyD (calcifediol), based on 25-hydroxylcholecalciferol/25-
hydroxy-pre-cholecalciferol)
Minimum
content
Maximu
m
content
No.
(or
EC
No.)
Additive
Chemical
formula,
description
Species
or
category
of animal M
a
x
i
m
u
m

a
g
e

IU
2
kg
-1
of complete
feedingstuff
Other provisions

Calcifediol
25
hydroxyl-
chole
calciferol/2
5-hydroxy-
pre-chole
calciferol

Chickens
for
fattening

Turkeys

Laying
Hens
- -
5000

5000

3000
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 HyD (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 nL 270 of 14.12.1970, p.1
2
Reference standard: cholecalciferol (IU)
1 g 25 hydroxycholecalciferol is considered by the notifier as equivalent to 40 IU

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).

1.1. Composition and characteristics of Hy D
HyD 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 anti-
caking agents respectively, and butylated hydroxy toluene (BHT) (20 g kg
-1
) and citric acid (0.5 g
kg
-1
) as anti-oxidants.
3

4

5
The 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.
6
Analysis of production batches demonstrated that HyD averaged a
content of 25-OH-D
3
of 13.5 g kg
-1
(variation of 106-110% of the claim on the labels).
7
HyD 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 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 <10 m in diameter.
8

1.2. 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.

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 HyD 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 (3,5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-3,25-
diol monohydrate
CAS No. 63283-36-3
Empirical formula C
27
H
44
O
2
.H
2
O
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, 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 1512.
12

9
Volume 1-6. Section II. 1 and 2.
10
11
Volume 6. Annex 8.
12
Answers to the Questions from the EU Member States. Volume I. Annex 8. November, 2003.
13
14 9
8 10
17
12
11
15
16
7 5
6
20
CH
3
18
CH
2
19
1
23
22
4
C H
3
21
24
2
3
O H
CH
3
OH
C H
3
13
14 9
8 10
17
12
11
15
16
7 5
6
20
CH
3
18
CH
2
19
1
23
22
4
C H
3
21
24
2
3
O H
CH
3
C H
3

The preparation process including extraction, chemical then photochemical transformation and
purification (crystallisation) steps ensure the complete elimination of the yeast cells.
13

14

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
3
.
15
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 HyD 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-OH-
previtamin 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
3
.
16
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
3
.
17

1.3. 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 HyD 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).
18

19

1.3.1. Shelf life of Hy D
The stability of HyD was determined by maintaining HyD in commercial polyethylene
containers at 25C and 40C for 52 weeks.
20

The applicant reported that the loss of 25-OH-D
3
from HyD beadlets was in the range 0-8% at
25C and 11-17% at 40C 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 <25C) 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, 2003.
15
Volume 6. Annex 7.
16
Volume 6. Annex 10 and 11.
17
Volume 6. Annex 14.
18
% recovery = {[concn](t=n)/[concn](t=0)}*100
19
Volume 6. Annex 19.
20
Volume 6. Annex 10, 11 and 12.
21

1.3.2. Premixes
Recent studies
22

23
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 25C and 35C for 3 and 1
month respectively. A 70% recovery was obtained when the premix was stored at 35C 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 25C only 65% of 25-OH-D
3
could be recovered. When stored for 2 months at 35C in
the presence of trace minerals recovery averaged about 23% where the starting concentrations
were 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 3
months 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-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 HyD 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 75C and 85C.
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 25C
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 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 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
3
.
27

28

22
Answers to the Questions from the EU Member States. Volume I. Annex A-9. November, 2003.
23
Answers to the Questions from the EU Member States. Volume II. Annex A-2. July, 2004.
24
25
Volume 6. Annex 10 and 18.
26
Answers to the Questions from the EU Member States. Volume I. Annex 12 and 13. November, 2003.
27
28
Volume 6. Annex 23, 24 and 25.

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.
Recommendation
Species/category
Age
(weeks)
IU kg
-1
diet g kg
-1
diet
Remark Reference
0-6 450 11.25 On DM base GfE 1999
0-8 200 5
Requirement
Diet at 90% DM
NRC 1994
Chickens for
fattening
0-6 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 27.5
Requirement
Male and female
NRC 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 at
90% DM
NRC 1994
Laying hens
>18 1000 25 INRA 1989
DM =Dry matter
NRC: 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 D
3
added to the diets of domestic birds (37.5-
75 g for chickens for fattening and for layers, 100 g kg
-1
for turkeys) are 2-3 fold higher than
the requirement. No recommendation is presently available for 25-OH-D
3
.

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 in
the dossier.
29
The trials have been carried out mainly in the United States in 1990-1995 for
chickens,
30
1995-1997 for laying hens,
31
and 1993-1998 for turkeys.
32

29
Volume 7A - 7E.
30
Volume 7A, 7B and 7C.
31
Volume 7D.
32
Volume 7E.

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
3
.

2.2.1. Chickens for fattening
2.2.1.1. Growth performance and feed conversion ratio
One trial has been carried out (Table 4)
33
which evaluates in the range of 0 to 20 g kg
-1
diet, the
levels of 25-OH-D
3
or D
3
showing that 20 g (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. Dose response of vitamin D
3
and its metabolite 25-OH-D
3
(in the form of HyD)
at
low dietary supply on body weight and feed conversion of chickens for
fattening.
80 birds x 10 replicates/group; duration 46 days

Body weight Feed conversion
g
vitamin
kg
-1
diet
Vitamin D
3

(kg)
25-OH-D
3

(% of vit. D
3
)

Vitamin D
3
(kg feed kg
-1

gain)
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
1.61 2.28
a

2.5 0.32
b
0.64
b
105* 116* 1.53 2.17
b
96 97*
5.0 0.34
c
0.79
c
104* 131* 1.45 2.07
c
98 96*
10 0.35
d
1.09
d
104* 115* 1.41 1.93
d
99 98 *
20 0.37
e
1.37
e
104* 110* 1.40 1.88
e
96 96*
*Difference statistically significant at 5 % level to the corresponding vitamin D groups
a, b, cMeans with different letters in the same column differ significantly at p 0.05.

33
Volume 7A. Annex 12.

This trial shows that the requirement for vitamin D
3
is 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 for
Vit D
3
or 25-OH-D
3
, Bar et al., 2003; 5 g vit D
3
, Baker et al., 1998). However, Fritts and
Waldroup (2003) observed optimal body weight when supplying 20 g kg
-1
of vitamin D
3
or only
5 g kg
-1
25-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 g 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 g 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.
34

35
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 g 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 g, the body weight can be improved, suggesting that 25-OH-D
3
has a higher efficacy
than vitamin D
3
.
36

37

Three field studies
38
confirm, in large numbers of birds, that the performance of chickens for
fattening are similar when fed 35 to 103 g kg
-1
vitamin D
3
of 25-OH-D
3
.

2.2.1.2. 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 (g kg
-1
diet) Vitamin D
3
25-OH-D
3
1
39

80 x 10

0
2.5
5
10
20
33.7
g

36.4
f

38.6
de

39.8
c

41.6
b

33.7
g

38.0
e

39.5
cd

41.1
b

43.4
a

2
40

74 x 10

1.56
6.25
27.5
f

-
30.9
e

37.3
c

34
Volume 7A. Annex 10, 13 and 17.
35
Volume 7C. Annex 1, 3 and 4.
36
Volume 7A - 7E.
37
38
Volume 7A. Annex 4, 5 and 6.
39
Volume 7A.Annex 12.
40
Volume 7A. Annex 17.

31.2
62.5
38.8
b

39.7
ab

39.2
b

40.4
a

3
41

74 x 10

1.56
6.25
31
62
28.6
f

-
36.2
c

39.8
a

30
e

37
bc

37
b

40
a

4
42

74 x 10

31
62
78
94
-
39.5
bc

40.0
bc

39.4
bc

33.4
d

40.6
ab

41.8
a

39.7
bc

Means with different letter are significantly different (comparison between all treatments)
1 g 25-OH-D
3
is nearly equivalent to 2 g vit. D.

Bone ash increased with the dietary levels of vitamin D
3
and 25-OH-D
3
from 0 to 62 g 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 g 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 g kg
-1
diet.

2.2.2. 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 g kg
-1
of 25-OH-D
3
which were compared to a single level
of vitamin D
3
(69 g kg
-1
). The feed conversion ratio was decreased in hens fed the hydroxylated
form of Vitamin D
3
when hens were supplied at 82 g 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 g 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 g).
Recently, Keshavarz (2003) compared vitamin D
3
and HyD supplied at 69 g 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.

2.2.3. 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 7.
42
Volume 7B. Annex 8-10.
43
Volume 7D. Annex 1 and 2.
44
Volume 7D. Annex 17.
45
Volume 7E. Annex 8 and 9.
46
Volume 7E. Annex 2.

Table 6. Summary the three acceptable turkey experiments conducted in the USA
Final body
weight (kg)
Feed conversion
(g feed g-
1
gain)
No. of
animals x
replicates
Duration
(days)
Dose level
(1)

(g kg
-1
feed)
D
3

25-OH-
D
3

D
3

25-OH-
D
3

Ref
38x12
16x12
84 (phase
1)
42 (phase
2)
40/35 (phase 1/2)
138/94 (phase 1/2)
-
14.6
15.1
14.8
-
3.02
2.96
2.97
47

25 x 5 (f)
21 x 5 (m)
112
49.5
69
99
-
11.0
11.2
-
11.3*
-
2.46
2.43

2.41*
48

31 x 13
101 (f)
133 (m)
49.5
96-69
(2)

99
-
8.6
-
8.7

8.9*
-
2.45
-
2.35

2.26
49

(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 96-69.
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 g kg
-1
25-
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 g 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,
50
25-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 g 25-OH-D
3
and 62.5 g 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
3
.
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-D
3
and Vitamin D
3
. It was improved in two comparisons amongst 22 comparisons. The

47
Volume 7E. Annex 1.
48
Volume 7E. Annex 3-6.
49
Volume 7E. Annex 7.
50
Volume 7E. Annex 8 and 9.
51
Volume 7E. Annex 10-14.

breast meat yield was also measured in 22 comparisons at similar levels of both compounds.
52

53

54
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.

2.4. 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 0.025 g 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 g ergocalciferol or cholecalciferol as a mass standard.
The applicant is aware of this system and ask consequently for labelling purposes 25-hydroxy-
cholecalciferol (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 g 25-OH-D
3
is equal to 40 IU vitamin D
following the international standard for (unhydroxylated) vitamin D compounds (1 IU = 0.025 g
vitamin D). The companys 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 g 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
2+
1.5 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 16.
53
Volume 7B. Annex 1-6, 8 and 10.
54
Volume 7C. Annex 1-3, 6, 7 and 9.

Bone ash low P 2 - 2.5 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 g, 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 g 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 g 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).

2.5. 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 g kg
-1
. At lower doses (2.5 and 25 g 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
g 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 g 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 g 25-OH-D
3
is equal to
1 g vitamin D
3
or 40 IU vitamin D. The companys deduction is based on a considerable
number of experiments on poultry with mostly higher dosages (>30g) 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 g 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 g, which is scientifically correct.
3
3
. To avoid
3
, vitamin D
2
or Calcifediol).

3. Safety studies on target species
3.1. Tolerance studies


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.
3.1.1. 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 g 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 g vitamin D
3
, 69,
207 and 690 g 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 g 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 g
vitamin D
3
and the 69 g 25-OH-D
3
kg
-1
group, it was significantly lower in the 690 g 25-OH-D
3

kg
-1
group, with the 207 g 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 13800 g kg
-1
, respectively. Because of high toxicity
and morbidity, the treatments with 6900 and 13800 g 25-OH-D
3
kg
-1
had to be terminated after
22 days, treatment with 3450 g 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 g 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 6.
56
Volume 8A. Annex 7-9 and 10-11. Volume 8B. Annex 1-6.
57
Volume 8C. Annex 3-12. Volume 8D-8E.
58
Volume 8F-8G.

Dietary level (g kg
-1
) 69 690 3450 6900 13800
Mortality (%)
a
, vitamin D
3
5.43 4.29 4.84 9.14 13.14
Mortality (%)
a
, 25-OH-D
3
4.57 6.29 (16.9)
b
(8.88)
c
(17.5)
c

Body weight (kg), vitamin D
3
2.49 2.50 2.19 1.23 0.87
Body weight (kg), 25-OH-D
3
2.51 2.22*
g feed g
-1
gain, vitamin D
3
1.87 1.89 1.92 2.30 2.72
g feed g
-1
gain, 25-OH-D
3
1.86 1.87
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 (g kg
-1
) 69 690 3450 6900 13800
No. of birds, examined, vitamin D
3
28 28 28 28 59
No. of birds, , examined, 25-OH-D
3
28 28 (66)
b
(41)
c
(59)
c

Renal calcification (%)
a
, vitamin D
3
17.9 7.1 55.2 89.3 100
Renal calcification (%)
a
, 25-OH-D
3
7.1 96.4 (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 g kg
-1
, such
in an increase occurred for 25-OH- D
3
already at 690 g 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 10000 g vitamin D
3
, but for 25-OH-D
3
already at 100 g 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 g 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 g,
significant difference to 35 g), but 560 g 25-OH-D
3
kg
-1
feed was lower and numerically equal
to 35 g 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 g 25-OH-D
3
kg
-1
feed showed lowest values (significant for Plasma Ca). Apparent
Ca- and P- retention was increased up to 280 g 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.

Two more recent studies (Bar et al., 2003; Fritts and Waldroup 2003) showed that 100 g 25-OH-
D
3
kg
-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 fed
diets containing 69 g vitamin D
3,
41.24, 82.5, 412.5, and 825.0 g 25-OH-D
3
kg
-1
feed,
respectively. The duration of experimental phase I was 16 weeks, 270 layers remaining (9
replicates 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 D
3
25-OH-D
3

Dietary level (g kg
-1
)
and source
69 41.25 82.5 412.5 825.0
Egg 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
-1
eggs 2.29 2.25 2.24 2.35* 2.48*
* figures significantly different (p <0.05) from the vitamin D
3
group
All results listed in Table 10 were less favourable in the group with 825 g 25-OH-D
3
kg
-1
diet
compared to vitamin D
3
. But also 412.5 g 25-OH-D
3
resulted in lower egg weight. The two lower
25-OH-D
3
dosages improved egg weight and egg shell thickness.
Hematology and organ weights (heart, liver, spleen, bursa of Fabricius, brain, thymus, ovaries,
and kidneys) did not show any difference in the 25-OH-D
3
or vitamin D
3
groups. The egg quality
criteria, not dependent on egg weight, were not significantly influenced by the treatment.
Table 11. Tolerance study on laying hens, week 42 to week 58 of age, phase II
Vitamin D
3
25-OH-D
3
Dietary level (g kg
-1
)
and source
69 41.25 82.5 412.5 825.0
Egg production (%) 66.7 64.5 68.3* 65.6 63.8*
Egg weight (g) 61.2 61,4 61.9* 60.4* 58.3*
Egg shell thickness (mm) 0.16 0.16 0.17* 0.16 0.14*
Body weight change (g) 73 66 70 72 75
Feed consumption (g d
-1
) 124.2 123.9 122.9 126.5 127.9
kg feed kg
-1
eggs 3.14 3.07 2.98* 3.27* 3.52*
3
group

60
Volume 8C. Annex 1.

In phase II, 825 g 25-OH-D
3
kg
-1
negatively influenced egg production, egg weight and feed
efficiency data (feed kg
-1
eggs) compared to the vitamin D
3
group (Table 11). Also egg weight
and feed consumption kg
-1
eggs of the 412.5 g 25-OH-D
3
group differed significantly from the
vitamin D
3
group. 82.5 g 25-OH-D
3
kg
-1
feed improved significantly egg production, egg weight,
egg shell thickness and the feed efficiency parameters. Egg quality criteria, not dependent on
egg weight, were not significantly influenced by the treatment.
No increase in mortality was observed in both phases. The pathology report indicates the
presence of granular, basophilic material in the kidneys of birds sacrificed at days 7 and 112, but
does 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) were
fed diets containing 68.9 g vitamin D
3
and 44.5, 99, 495, and 990 g 25-OH-D
3
kg
-1
for 16
weeks.
Final body weight and feed efficiency was not significantly affected by the 25-OH-D
3
treatment.
However, 99 g 25-OH-D
3
showed a significantly higher body weight and a better feed
conversion rate than the vitamin D
3
control.
All mortality and observations made during the course of the trial were found to be consistent with
commercial practices. Total mortality was significantly higher for the groups with 495 (13.1%),
and 990 g 25-OH-D
3
kg
-1
(14.8%) compared to 6.0% in the control vitamin D
3
group. However
the increase in mortality was particularly seen in males and occurred mainly in the initial test
phase due to an increased incidence of air sacculitis, omphalitis, and colibacillosis. No mortality
was observed between day 84 and day 112. Five cases of soft bones were recorded in the 990
g 25-OH-D
3
group, one in the 495 g 25-OH-D
3
group, and zero in the other groups.
Total disorders observed (dehydration, reduced pigmentation score -legs- and feathering
condition) finally increased with 990 g 25-OH-D
3
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 per
replicate on day 112 from the 99, 495, and 990 g 25-OH-D
3
groups were sacrificed for
hematology, 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 attributable
to the highest 25-OH-D
3
treatment. 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 of
the females. Tubular mineralisation was present in the kidneys in four males and two females in
the groups receiving vitamin D
3
, in one male receiving 25-OH-D
3
at 99.0 g, two males and two
females receiving 495 g 25-OH-D
3
, and in five males and one female receiving 990 g 25-OH-
D
3
kg
-1
feed.
3.1.4. Conclusions on the safety for the target animals
The studies on chickens for fattening clearly show that 25-OH-D
3
has a higher toxic potential than
vitamin D
3
. Because of large steps between the dosages in the crucial experiment II, the
tolerance 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-D
3
may have a 5-10 higher toxic
potential for chickens for fattening than vitamin D
3
. Although a margin of safety for the upper level
recommended by the notifier (70 g 25-OH-D
3
kg
-1
) can not be given, it is certainly less than 10,

because 690 g 25-OH-D
3
kg
-1
caused weight gain depression and lead to a higher occurrence
of renal calcifications.
For laying hens a comparison concerning a potentially different tolerance of vitamin D
3
and 25-
OH-D
3
(recommended level by the notifier: 75 g 25-OH-D
3
kg
-1
) can not be made due to the
study design. The study on laying hens, based on production parameters, showed that 825 g
25-OH-D
3
kg
-1
diet are not tolerated well by layers. Even 412.5 g 25-OH-D
3
kg
-1
feed resulted for
two 16 week periods in smaller egg weight and increased feed consumption kg
-1
eggs. It is
concluded that the margin of safety is smaller than 5.5 (412.5/75). A field study allows
establishing 82.5 g 25-OH-D
3
kg
-1
diet as safe.
As long as no more detailed data is available it would be prudent to accept 100 g 25-OH-D
3
kg
-1

complete feed as the upper tolerated limit for chickens for fattening and 80 g 25-OH-D
3
kg
-1
for
laying hens.
In contrast to chickens for fattening and laying hens, turkeys seem to tolerate higher doses of 25-
OH-D
3
well (recommended level by the notifier: 100 g 25-OH-D
3
kg
-1
). The significantly higher
mortality observed for 495 and 990 mg 25-OH-D
3
kg
-1
feed compared to 69 g vitamin D
3
may be
regarded as incidental, because the figures are not clearly attributable to 25-OH-D
3
treatment.
However, for 990 g 25-OH-D
3
kg
-1
feed there is a weak evidence for mild intolerance. Therefore
the margin of safety of the upper recommended level could be given as about 5 (495/100).

3.2. Fate of 25-OH-D
3
and body deposition

3.2.1. Fate of 25-OH-D
3

The comparative absorption and excretion of cholecalciferol and 25-OH-D
3
in birds (Bar et al.,
1980) has shown that: i) the relative net absorption of 25-OH-D
3
is higher than that of
cholecalciferol in chickens (84 and 67% respectively) while cholecalciferol absorption was
significantly 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 as
polar (chloroform-methanol extractable) metabolites was 20% and 14% in the chicken and turkey
respectively, that of 25-OH- D
3
representing only 7% for both species.
In mammals and birds, 25-OH-D
3
is the initial metabolite of cholecalciferol resulting from
hydroxylation in the liver (Haussler and Rasmussen, 1972). In birds, it represents the most
significant metabolite in the plasma, kidney and bone. Subsequent metabolism at the kidney level
gives rise to the biologically active metabolite 1,25-(OH)
2
-D
3
(Holick et al., 1971) which is
markedly present in the same tissues. 25-OH-D
3
is metabolized also to 25,26-(OH)
2
-D
3,
21,25--
(OH)
2
-D
3
and several other more polar and excretable compounds that are detectable in the
plasma.
No retroconversion of 25-OH-D
3
to Vitamin D
3
occurs. It is very likely that the metabolic fate of
orally administered 25-OH-D
3
would be qualitatively the same as that of the endogenously
produced 25-OH-D
3
.

3.2.2. Deposition
3.2.2.1. Chickens for fattening
One-day old (male and female) chickens for fattening were given commercial-type feeds
supplemented with the same quantity of vitamin D
3
or 25-OH-D
3
(69 g kg-
1
feed) but also higher
doses (x3 and x10) of 25-OH-D
3
(207 and 690 g kg-
1
feed).
61

62
After 49 days administration, a

61
Volume 8A. Annex 10-15.

longer period than usual in Europe, the birds were slaughtered and serum and tissues sampled
for 25-OH-D
3
but also 1,25-(OH)
2
-D
3
determination. No gender difference was observed. Serum
levels of 25-OH-D
3
were 3 times higher in birds fed 25-OH-D
3
when compared to a similar dose of
vitamin D
3
(43 and 13 ng L
-1
respectively). As dietary 25-OH-D
3
increased from 207 to 690 g kg-
1
feed, serum 25-OH-D
3
increased from 113 to 246 ng L
-1
. However, serum 1,25-(OH)
2
-D
3

concentration fell from 68 to 54 pg mL
-1
despite an 18-fold increase in serum 25-OH-D
3

concentrations from the birds fed vitamin D
3
to those fed the highest level of 25-OH-D
3
which
indicates a tight regulation of this key metabolite. The results of the analysis of 25-OH-D
3
in
tissues (Table 12) indicate that its concentration is about 3 times higher in the animals
administered directly this compound in comparison to a similar amount of vitamin D
3
. A significant
positive correlation is observed between the serum and tissue 25-OH-D
3
concentrations. The
slope for skin/fat concentration is 3 times that for breast muscle.

Table 12. 25-OH-D
3
concentration in tissues of chicken for fattening fed vitamin D
3
or
25-OH-D
3
for 49 days (g kg-
1
wet tissue)
Vitamin D source and inclusion rate (g kg
-1
feed)
25-OH- D
3

Tissues
Vitamin D
3

69 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 D
3
group
3.2.2.2. Laying hen eggs
The incidence of vitamin D
3
supplementation of feed on the concentration of vitamin D
3
and 25-
OH-D
3
in egg yolk has been studied in laying hens
63
(Mattila et al., 1999). A metabolic equilibrium
was reached after the first time point retained, i.e. 4 weeks exposure. At a dose currently used in
laying hen feed, i.e. 62 g vitamin D
3
kg
-1
, 25-OH-D
3
concentration in egg yolk was about 30 % of
that of vitamin D
3
(Table 13). The increase of the concentration of vitamin D
3
in feed (x 3.5)
resulted in a considerable increase (x 7) of vitamin D
3
deposition in egg yolk whereas 25-OH-D
3

concentration was multiplied by a factor of 1.5 only.

Table 13. Vitamin D
3
and 25-OH-D
3
concentration in eggs of laying hens fed vitamin D
3

for 4 to 6 weeks (g per 100 g egg yolk) (Mattila et al., 1999)

Vitamin D
3
inclusion (g kg
-1
feed)
26.6 62.4 216
Vitamin D
3
1.4 3.4 23
25-OH-D
3
0.5 1.0 1.5

Laying hens 20 weeks of age were pre-conditioned for 28 days on a standard diet then assigned
to 5 groups of 9 animals that received either vitamin D
3
at 69 g kg-
1
feed (control group) or 25-
OH-D
3
at 41, 82, 413 and 825 g kg-
1
feed (x0.5, x1, x5 and x10 the dose proposed for use

62
Volume 8B. Annex 1-6.
63
Volume 8C. Annex 3-12. Volume 8D 8E.

respectively) for 28 days measured values were close to the target dosage at the beginning of
the study but decline by 20% at the end of the study. Eggs were collected the 56
th
day. The
animals were slaughtered and tissues sampled. 25-OH-D
3
concentration was determined in the
eggs and tissues, using an HPLC/IRC method (see 1.4). The results (Table 14) indicate that 25-
OH-D
3
concentrations were not significantly different in the eggs laid by either the vitamin D3 or
25-OH-D
3
supplemented 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-D
3
levels were
not 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-D
3
dosage increased the residual levels by 2.5, 4, 2.5
and 3 in the liver, skin/fat, breast muscle and thigh muscle respectively.

Table 14. 25-OH-D
3
concentration in eggs and tissues of laying hens fed vitamin D
3
or
25-OH-D
3
for 28 days (g kg-
1
wet tissue)
Vitamin D source and inclusion rate (g kg
-1
feed)
25-OH-D
3

Tissues
Vitamin D
3

69 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 - -
3
group

3.2.2.3. Turkeys
One day-old turkeys were assigned to 5 groups of 12 animals (6 males and 6 females) that
received for 112 days a feed containing either vitamin D
3
at 69 g kg-
1
(control group) or 25-OH-
D
3
at 50, 99, 495 and 990 g kg-
1
feed (x0.5, x1, x5 and x10 the dose proposed for use
respectively).
64
Major values were 39, 83, 315 and 732, respectively. The animals were
slaughtered and tissues sampled for 25-OH-D
3
determination, using an HPLC/IRC method. The
results given on Table 15 indicate that 25-OH-D
3
concentrations were not significantly different in
the liver, skin/fat, breast muscle and thigh muscle of animals that received 25-OH-D
3
at 99 g kg-
1
feed when compared to those of the vitamin D3. For the higher 25-OH-D
3
dosage (x5) residual
levels were not increased in the liver but in the skin/fat (x6), breast muscle (x4) and thigh muscle
(x5).

Table 15. 25-OH-D
3
concentration in tissues of turkeys fed vitamin D
3
or 25-OH-D
3
for
112 days (g kg-
1
wet tissue)
Vitamin D source and inclusion rate
(g kg
-1
feed)
Vitamin D
3
25-OH-D
3

Tissues
69 99 495
Breast muscle 2.3 2.3 8.6*
Thigh muscle 2.8 2.0 10.7*
Skin plus fat 6.5 8.2 52.9*

64
Volume 8F. Annex 6-12. Voume 8G.

Liver 3.1 6.4 6.5
Kidney - 6.4 -
3
group

3.2.3. Conclusions
In birds the ingested 25-OH-D
3
behaves qualitatively in the same way as the endogenous
metabolite derived from vitamin D
3
metabolism. No retro-conversion of 25-OH-D
3
to Vitamin D
3

occurs.
Deposition of 25-OH-D
3
in the laying hen egg or turkey tissues is not significantly different in the
birds that received either vitamin D
3
or a similar quantity of 25-OH-D
3
corresponding to the dose
proposed for use. However, deposition of 25-OH-D
3
in chicken tissues resulting from 25-OH-D
3

supplementation of the diet at the dose proposed for use is about three times higher than that
found following the use of the same quantity of vitamin D
3
(69 g kg-
1
).
4. Studies on laboratory animals.

As a general principle the FEEDAP Panel considers conventional toxicology studies to be
inappropriate for testing pure substances which are dietary nutrients (essential dietary nutrients).
Such substances have a physiological concentration which is optimum for health and
performance. Dietary intakes of such substances which lead to amounts which are significantly
below or above that which is optimum for health and performance will inevitably cause a
physiological imbalance and consequent adverse effects. This principle applies to substances
where the purity is well established, with the source and method of production sufficiently well
characterised for reassurance that no toxic contaminants will be present in the product. The
testing appropriate to such substances will need to be judged on a case-by-case basis but in
circumstances where the use of the substance leads to no increase in human intake there is no
requirement for further toxicity data. Where there are major impurities, or the source is poorly
characterised some evidence of safety from toxicological studies will be required. The testing
programme would need to be designed to cover all likely areas of potential consumer risk. Where
toxicological studies are already available due account will be taken of the data, but conduct of
new studies for evaluation of the safety of such products should form part of a clear defined
strategy relevant to assessing consumer safety. Justification for omitting studies of key effects
from any submission should be made by the applicant as part of this defined strategy.

In the particular case of HyD some studies are available and were performed in the 1970s and
1980s as part of the original regulatory submission on the human drug Calcifediol (Calderol)
which is the same as 25-OH-D
3
.

The studies considered by the FEEDAP Panel were, acute toxicity in mice and rats, repeat dose
sub-chronic toxicity in rats, two mutagenicity studies and reproduction studies in both rat and
rabbit.
65

66
From the studies conducted there is no reason to suspect 25-OH-D
3
of having a
genotoxic effect. Effects seen in the toxicity studies which have been conducted are entirely
consistent with a physiological overload of Vitamin D or its metabolites and are not indicative of
any unexpected toxicity arising from the source or production method of the substance.

65
Volume 8H.
66
Answers to the Questions from the EU Member States. Volume III. Annex 1 and 4. January, 2005.

4.1. Conclusions
inappropriate for testing chemically defined pure substances which are dietary nutrients, which is
3
from HyD 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, sub-chronic and reproductive toxicological effects observed are entirely consistent with a
3
or its metabolites.

5. Safety evaluation for the human consumer
5.1. Human use of 25-OH-D
3

25-OH-D
3
is used in human medicine to prevent rickets, to manage metabolic bone disease and
for the treatment of hypocalcemia.
67
No incompatibilities are known, and any-side effects are
described related to overdosing in susceptible individuals.
68

5.2. Status of 25-OH-D
3
in humans
As 25-OH-D
3
is a normal metabolite in vitamin D metabolism in man, it is very likely that
exogenous sources of 25-OH-D
3
in the diet will be metabolised in the same way as the
endogenously produced 25-OH-D
3
. Several studies in humans and animals have demonstrated
that while vitamin D is absorbed mainly into the lymph, the more polar metabolite 25-OH-D
3
, at
physiological concentrations, is also absorbed more rapidly and efficiently from the proximal
jejunum 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 D
increase serum 25-OH-D
3
concentrations in a dose dependent manner, both doses leading to
plateau concentrations after 2-3 months (Vieth et al., 2001). The concentration of 25-OH-D
3
in
the 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
-1
across a wide
range of daily supplies of vitamin D (20 to 250 g), suggesting that an homeostatic control
system exists and regulates serum 25-OH-D
3
. This metabolite is further metabolized in the
kidney to the main active metabolite 1,25-(OH)
2
D which is strictly regulated by parathyroid
hormone and calcium. The blood level of 1,25-(OH)
2
D is maintained within a narrow range
independant of normal variations of vitamin D supply and in circulating vitamin D or 25-OH-D
3
.
Pharmacologic doses of 25-OH-D
3
do not change or may even decrease plasma levels of 1,25-
(OH)
2
D unless vitamin D deficiency is present (review from Ovesen et al., 2003).
Vitamin D
3
in high doses is very toxic to man and during the toxic episode the serum
concentration of 25-OH-D
3
reaches very high levels. There are no systematic studies on the
toxicity of 25-OH-D
3
in the human.

5.3. Biological activity of 25-OH-D
3

Vitamin D status in humans is determined by measuring serum 25-OH-D
3
concentration. The
biological activity of 25-OH-D
3
is greater than that of vitamin D. However, there is not yet
consensus on the conversion factor that should be used for 25-OH-D
3
to calculate vitamin D
equivalence. Depending on the testing system used but always referring to the rat, the factor
varies from 1.5 to 5 (Blunt et al., 1968; Reeve et al., 1982). The activity factor of 1.5 is used in the
American Dietary Allowances (Institute of Medicine, 1997) whereas a factor of 5 is used in the
British Food composition tables (Chan et al., 1995). It must be noted that the half-life of 25-OH-D
3

(20-28 days) is much shorter than that of vitamin D
3
(Haddad and Rojanasathit 1976).

67
Volume 6. Annex 21.
68
Volume 8H. Annex 10.

5.4. The Tolerable Upper Limit (UL) for humans
The UL for vitamin D intake in humans, based on symptoms of hypercalcaemia that appear as
the first toxicity sign, has been set to 50 g day
-1
in adults and 25 g day
-1
in children up to the
age of 11 (EC, 2002; Institute of Medicine, 1997). The FEEDAP Panel suggests an UL for 25-
OH-D
3
estimated using the relative biological activity factor of 5 which represents the most
conservative approach considering either the results obtained in the rat (see 5.3.) or the chicken
(see Table 7). It would represent 10 g day
-1
in the adult and 5 g day
-1
in children.

5.5. Consumer exposure to 25-OH-D
3
Vitamin D supply to humans includes vitamin D
3
produced in the skin under the influence of UV-
light and dietary vitamin D
3
and vitamin D
2
. Studies have shown that dietary vitamin D
3
activity is
mainly composed of native vitamin D
3
. However, in some foodstuffs 25-OH-D
3
contributes to
vitamin D activity (Mattila et al., 1993; Mattila, 1995; review from Ovesen et al., 2003). 25-OH-D
3

contents are typically low (<1 g kg
-1
) in milk and similar to vitamin D. 25-OH-D
3
contents in fish
are generally low <1 g kg
-1
, whereas vitamin D is highly variable between and within species (2-
477 g kg
-1
), the highest levels being found in fatty fish (salmon, sardines, mackerel). 25-OH-D
3

concentrations range from 2 to 5 g kg
-1
in meat (e.g. 2.5 g kg
-1
in chicken meat) and offal and
up to 10 g kg
-1
in egg yolk. Mushrooms are the main source of vitamin D
2
in the human diet.
Spring butter may contain small but detectable amounts of vitamin D
2
. There does not seem to
be any 25-OH-D
2
in mushrooms, but in beef liver small amounts of 25-OH-D
2
have been detected
(Mattila et al., 1995).
The eventual use of HyD

as feed additive would be substitutive to vitamin D
3
. Considering the
dose of 25-OH-D
3
from HyD proposed for use for laying hens, the resulting concentrations of 25-
OH-D
3
in the eggs and edible tissues are not significantly different from those found when using
vitamin D
3
and therefore the consumer exposure to 25-OH-D
3
would remain unchanged. A similar
situation is observed with the consumption of turkey tissues. When chicken tissues are
concerned the substitution of vitamin D
3
with 25-OH-D
3
increases by a factor of 3 the consumer
exposure to 25-OH-D
3
. It must be noted that these data are based on single studies.
The daily exposure of the consumer to 25-OH-D
3
has been calculated by the FEEDAP Panel
considering:

i) the 25-OH-D
3
contents in tissues and eggs corresponding to the highest 25-OH-D
3
dosage recommended for use in poultry feed by the FEEDAP Panel, i.e. 100 g kg
-1

feed. Experimental values were available for turkeys (see Table 15). For chickens and
laying hens (eggs) the figures were calculated by interpolation of experimental data
(see Table 12 and 14) from animals receiving higher and lower dosages in feed,
assuming the linearity of the deposition vs. administered dose ratio. Another
approximation resulted from the fact that no data were available for the 25-OH-D
3
contents of chicken liver and kidney and values from laying hens were taken instead,

ii) the theoretical worst case consumption figures as laid down under Directive
2001/79/EC
69
fixing guidelines for assessment of additives in animal nutrition.

The higher values found (Table 16), i.e. 6.4 g day
-1
when considering chicken meat plus egg or
3.5 g day
-1
when considering turkey meat plus egg comply with the suggested provisional UL for
adults but exceed the suggested provisional UL for children, in the case of chicken meat plus
egg.

69
Directive 2001/79/EC fixing guidelines for the assessment of additives in animal nutrition. OJ. L 267,
6.10.2001, p. 1.

Table 16. 25-OH-D
3
human intake based on consumption model from Directive
2001/79/EC.
Amount
consumed
Content of 25-OH-D
3
( g kg
-
1
)
in chicken in turkey
Intake of 25-OH-D
3
( g day
-1
)
From chicken From
turkey
and egg and egg
300 g Muscle 7.7
a
2.3
d
2.31 0.69
100 g Liver 10.0
b
6.4
d
1.00 0.64
10 g Kidney 9.2
c
6.4
d
0.09 0.06
90 g Skin/fat 18.1
a
8.2
d
1.63 0.74
100 g Egg 13.8
b
1.38 1.38
Total 6.41 3.51
a)
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 estimation
has been performed taking into consideration more realistic data, based on intake data for the
mean 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 the
provisional UL for the adult and children respectively, considering that the childrens consumption
of 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-D
3
to vitamin D
3,
the
vitamin D
3
contents of tissues or products (eggs) from animals fed 25-OH-D
3
supplemented diets
should be very low when compared to those from animals that receive vitamin D
3
in their diet.

Table 17. 25-OH-D
3
human intake based on SCOOP data (EC, 2004).
(Maximum meat
a
intake: 175 g day
-
1, maximum egg intake 36 g day
-1
)
Amount consumed Content of 25-OH-D
3
( g
kg
-1
) in chicken tissue and
egg
a

25-OH-D
3
intake

( g day
-
1
)

from chicken and egg
105 g Muscle
b
7.7 0.81
35 g Liver
b
10.0 0.35
3.5 g Kidney
b
9.2 0.03
31.5 g Skin/fat
b
18.1 0.57
36 g Egg 13.8 0.50
Total 2.26
a)
See Table 16
b)
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.


5.6. Conclusion
3
3

D
3
(3.5 g day
-1
FEEDAP Panel, represents 35% and 70% of the provisional UL for the adult and children
respectively.
When chicken for fattening is concerned, a similar calculation leads to a consumer exposure
-1
exposure appears to be below the provisional UL for both the adult (23%) and children (46%).
3
3
for chickens for
-1
feed, and for
-1
3
by 25-OH-D
3
3


6. User safety assessment
Skin irritation
A GLP compliant study
70
of skin irritation of Hy-D beadlets was conducted in 3 rabbits according
to OECD guideline 404. The HyD

beadlets were non-irritant to rabbit skin.

Eye irritation
A GLP compliant study
71
of eye irritation of HyD

beadlets was conducted in three rabbits
according to OECD guideline 405. The HyD

beadlets caused some irritation to the conjunctiva
which 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 occupational
exposure assessment has been carried out by the applicant relevant to the production and
handling of the product in its various forms.

6.1. Pure 25-OH-D
3

The advised protective measures include use of externally supplied air when handling this
product, thus inhalation exposure is not considered further. Since bioavailability via the dermal
route is unknown, exposure by this route is calculated, based on an assumption of 10% of
exposed dose. Since the maximum exposed dose used in this analysis is more than 100 times
higher than a measured atmospheric level during normal handling this approximation is
considered by the FEEDAP Panel to be acceptable. Based on this assessment it is concluded
that exposure in the absence of protective clothing could result in a dose equivalent to 5 times
the therapeutic dose of 25-OH-D
3
or 125 g.
Use of externally supplied air and full protective clothing while handling the product is thus
concluded to be necessary but sufficient to avoid exposures which may result in chronic effects.

70
Answers to the Questions from the EU Member States. Volume III. Annex 2. January, 2005.
71
Answers to the Questions from the EU Member States. Volume III. Annex 3. January, 2005.

Since the acute toxicity is relatively low the protective measures applied are also considered to
be sufficient to prevent any acute effects.
The notifier reports monitoring of workers
72
on one occasion for blood levels of 25-OH-D
3
and
1,25-diOH-D
3
after handling product during normal operations showed no pattern of change in
blood values. Atmospheric levels achieved during handling pure 25-OH-D
3
were 0.33 mg 25-OH-
D
3
m
-3
.

6.2. Formulated product in beadlets (1.25% 25-OH-D
3
)
Since HyD

is a product formulated into beadlets to minimise respirable dusts this reduces the
potential exposure of users to 25-OH-D
3
.
73

74
About 95% of particles have diameters in the 20-
80m while 3-4% have diameters of <10 m. The main exposure that may occur is at the point of
manual addition of this product to the feed production process. Ventilation at such points is
generally specifically designed to minimise exposure to any dusts. The applicant recommends
the use of protective clothing for handling this product and use of this should be sufficient to
minimise risk of adverse effects. Despite the use of ventilation at the point of maximum potential
exposure the use of a protective dust mask is considered necessary since the risk to the operator
from respiratory exposure has not been characterised.

6.3. HyD supplemented feed
Following FEEDAP Panels recommendations the maximum concentration likely to occur in feed
is 100 g 25-OH-D3 kg
-1
feed. Since the upper tolerable limit of 10 g day
-1
in adults would
therefore be contained in 100 g of feed the risk of adverse effects from exposure is considered to
be negligible.

6.4. Conclusions
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
Vitamin D or 25-OH-D
3
. The use of protective clothing should be sufficient to avoid adverse
effects in users.

7. Safety for the Environment
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, as it has been stated in the Directive 2001/79/EC.

CONCLUSIONS
From the assessment of the data submitted for the additive HyD (Calcifediol), the FEEDAP
Panel draws the following main conclusions:

CHARACTERISATION OF THE ADDITIVE

72
73
74

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 D
3
(25-
OH-D
3
). No DNA from the production process is expected to be present in the final product.

EFFICACY
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 g kg
-1
. At lower doses (2.5 and 25 g 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
g 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 g 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 g 25-OH-D
3
is equal to
1 g vitamin D
3
or 40 IU vitamin D. The companys deduction is based on a considerable
number of experiments on poultry with mostly higher dosages (>30g) 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 g 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 g, which is scientifically correct.
3
3
. To avoid
3
, vitamin D
2
or Calcifediol).

SAFETY FOR THE TARGET SPECIES
The studies on chickens for fattening clearly show that 25-OH-D
3
has a higher toxic potential than
vitamin D
3
. Because of large steps between the dosages in the crucial experiment II, the
tolerance 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-D
3
may have a 5-10 higher toxic
potential for chickens for fattening than vitamin D
3
. Although a margin of safety for the upper level
recommended by the notifier (70 g 25-OH-D
3
kg
-1
) can not be given, it is certainly less than 10,
because 690 g 25-OH-D
3
kg
-1
caused weight gain depression and lead to a higher occurrence
of renal calcifications.
For laying hens a comparison concerning a potentially different tolerance of vitamin D
3
and 25-
OH-D
3
(recommended level by the notifier: 75 g 25-OH-D
3
kg
-1
) can not be made due to the
study design. The study on laying hens, based on production parameters, showed that 825 g
25-OH-D
3
kg
-1
diet are not tolerated well by layers. Even 412.5 g 25-OH-D
3
kg
-1
feed resulted for
two 16 week periods in smaller egg weight and increased feed consumption kg
-1
eggs. It is
concluded that the margin of safety is smaller than 5.5 (412.5/75). A field study allows
establishing 82.5 g 25-OH-D
3
kg
-1
diet as safe.

As long as no more detailed data is available it would be prudent to accept 100 g 25-OH-D
3
kg
-1

complete feed as the upper tolerated limit for chickens for fattening and 80 g 25-OH-D
3
kg
-1
for
laying hens.
In contrast to chickens for fattening and laying hens, turkeys seem to tolerate higher doses of 25-
OH-D
3
well (recommended level by the notifier: 100 g 25-OH-D
3
kg
-1
). The significantly higher
mortality observed for 495 and 990 mg 25-OH-D
3
kg
-1
feed compared to 69 g vitamin D
3
may be
regarded as incidental, because the figures are not clearly attributable to 25-OH-D
3
treatment.
However, for 990 g 25-OH-D
3
kg
-1
feed there is a weak evidence for mild intolerance. Therefore
the 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-D
3
behaves qualitatively in the same way as the endogenous
metabolite derived from vitamin D
3
metabolism. No retro-conversion of 25-OH-D
3
to Vitamin D
3

occurs.
Deposition of 25-OH-D
3
in the laying hen egg or turkey tissues is not significantly different in the
birds that received either vitamin D
3
or a similar quantity of 25-OH-D
3
corresponding to the dose
proposed for use. However, deposition of 25-OH-D
3
in chicken tissues resulting from 25-OH-D
3

supplementation of the diet at the dose proposed for use is about three times higher than that
found following the use of the same quantity of vitamin D
3
(69 g kg-
1
).
inappropriate for testing chemically defined pure substances which are dietary nutrients, which is
3
from HyD 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, sub-chronic and reproductive toxicological effects observed are entirely consistent with a
3
or its metabolites.
3
3

D
3
(3.5 g day
-1
FEEDAP Panel, represents 35% and 70% of the provisional UL for the adult and children
respectively.
When chicken for fattening is concerned, a similar calculation leads to a consumer exposure
-1
exposure appears to be below the provisional UL for both the adult (23%) and children (46%).
3
3
for chickens for
-1
feed, and for
-1
3
by 25-OH-D
3
3


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

Vitamin D or 25-OH-D
3
. The use of protective clothing should be sufficient to avoid adverse
effects in users.

SAFETY FOR THE ENVIRONMENT
There 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-D
3
), the premixes and feedingstuffs.

RECOMMENDATIONS
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 HyD 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 g. If for practical reasons this
is not immediately possible then the label of the product should include (i) the potency (IU vitamin
D, 1 g of 25-OH-D
3
3
) and (ii) the source of the vitamin
(from vitamin D
3
, Vitamin D
2
or Calcifediol).
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 g kg
-1
complete feed, the highest 25-OH-D
3
level for
chickens for fattening and laying hens should be set with 100 g 25-OH-D
3
kg
-1
complete feed (a
level proven as safe). The same level could also be applied for turkey feed regarding the higher
potency of 25-OH-D
3
compared to vitamin D
3
and the existing regulations for vitamin D
3

(maximum content: 5000 IU vitamin D
3
kg
-1
turkey feed).
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.
DOCUMENTATION PROVIDED TO EFSA

1. Letter, dated 23-04-2004 with ref. D(2004)410196, from Ms. Paola TESTORI COGGI
from the Health & Consumer Protection Directorate-General requesting a consultation of
the scientific Panel on the evaluation of the safety and efficacy of HyD (calcifediol).
2. Submission of the dossier on 25-Hydroxyvitamin D
3
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 States
Volume I (Nov/Dec 2002 and April 2003). ROCHE Vitamins Ltd.
Volume II (December 2003 to April 2004). DSM Nutritional Products Ltd.
Volume III (January 2005). DSM Nutritional Products Ltd.


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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 of
cholecalciferol 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 development
on broiler chickens given 25-hydroxycholecalciferol. Brit. Poult. Sci. 44(2), 224-233.
Blomstrand, R. and Forsgren, L. 1967. Intestinal absorption and esterification of vitamin D
3

1,2-
3
H 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 D
3
. Proc. Natl. Acad. Sci USA 61, 1503-
1516.
Chan, W., Brown, J., Lee, S.M. and Buss, D.H. 1995. Meat, poultry and game. Fifth
supplement to the Fifth Edition of McCance and Widdowsons the Composition of foods.
Cambridge/London, Royal Society of Chemistry/Ministery of Agriculture, Fisheries and
Food.
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 Tolerable
Upper 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) Task
3.2.11. Assessment of the dietary exposures to arsenic, cadmium, lead and mercury of
the population of the EU Member States Directorate General Health and Consumer
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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 Live
Performance and Bone Development in Growing Broilers. J. Appl. Poult. Res. 12:45-52.
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Nhrstoffversorgung der legehennen und Masthhner (Broiler). DLG-Verlag, Frankfurt. 7,
185.
Gesellschaft fr Ernhrungsphysiologie -GfE-. 2004. Empfehlungen zur Energie- und
Nhrstoffversorgung von Mastputen. Proc. Soc. Nutr. Physiol. 13, 199-233
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3
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Holick, M.F., Schnoes, H.K., DeLuca, H.F., Suda, T. and Cousins, R.J. 1971. Isolation and
identification of 1-25-dihydroxycholecalciferol. A metabolite of vitamin D active in intestine.
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INRA. 1989. Lalimentation des animaux domestiques: porc, lapin, volaille. INRA ed. Paris
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Keshavarz, K. 2003. A comparison between cholecalciferol and 25-OH-cholecalciferol on
performance and eggshell quality of hens fed different levels of calcium and phosphorus.
Poult Sci. 82, 1415-1422.
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metabolites: in vivo study in the rat. Isr J Med Sci. 23 (11), 1114-7.
Mattila, P., Piironen, V., Uusi-Rauva, E. and Koivistoinen, P. 1993. Determination of 25-
hydroxycholecalciferol content in egg yolk by HPLC. J. Food Compos. Anal. 6, 250-255.
Mattila, P. 1995. Analysis of cholecalciferol, ergocholecalciferol and their 25-hydroxylated
metabolites in foods by HPLC (dissertation). EKT-series 995. University of Helsinki,
Department of applied chemistry and microbiology.
Mattila, P., Piironen, V., Uusi-Rauva, E. and Koivistoinen, P. 1995. Contents of cholecalciferol,
ergocholecalciferol and their 25-hydroxylated metabolites in milk products and raw meat
and liver as determined by HPLC. J. Agric. Food Chem. 43, 2394-2399.
Mattila, P., Lehikoinen, K., Kiiskinen, T. and Piironen, V. 1999. Cholecalciferol and 25-
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1977. Relative Toxicity and Metabolic Effects of Cholecalciferol and 25-
Hydroxycholecalciferol in Chicks. J. Nutr. 107, 1027-1034.
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Washington
Ovesen, L, Brot, C. and Jakobsen, J. 2003. Food contents and biological activity of 25-
hydroxyvitamin D: a vitamin d metabolite to be reckoned with? Ann. Nutr. Metab. 2003.
47, 107-113.
Raiten, D.J. and Picciano, M.F. 2004. Vitamin D and health in the 21
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Reeve, L.E., Jorgensen, N.A. and DeLuca, H.F. 1982. Vitamin D compounds in cows milk.
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Sitrin, M.D., Pollack, K.L., Bolt, M.J.G. and Rosenberg, I.H. 1982. Comparison of

vitamin D
3
and 25-hydroxy-vitamin D
3
absorption in the rat. Am. J. Physiol. 242, G326-G332.
Soares, J.H., Kerr, J.M. and Gray, R.W. 1995. 25-Hydroxycholecalciferol in poultry nutrition.
Poultry Sci. 74; 1919-1934.
Sutton, A.L. and Mac Donald, P.N. 2003. Vitamin D: More than a bone a-fide hormone. Mol.
Endocrinol. 17; 777-791.
Thompson, G.R., Lewis, B. and Booth, C.C. 1966. Absorption of vitamin D
3
-
3
H in control
subjects and patients with intestinal malabsorption. J. Clin. Invest. 21, 1662-1663.
Vieth, R., Chan, P.C.R. and McFarlane, G.D. 2001. Effiicacy and safety of vitamin D
3
intake
exceeding the lowest observed adverse effect level. Am. J. Clin. Nutr. 73, 288-294.
Yarger, J.G., Quarles, C.L., Hollis, B.W. and Gray, R.W. 1995. Safety of 25-
Hydroxycholecalciferol as a Source of Cholecalciferol in Poultry Rations. Poult. Science.
74:1437-1446.

SCIENTIFIC PANEL MEMBERS
Arturo Anadn, Margarita Arboix Arzo, Georges Bories, Paul Brantom, Joaquim Brufau de
Barber, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Nol Dierick, Gerhard
Flachowsky, Anders Franklin, Jrgen Gropp, Anne-Katrine Lundebye Haldorsen, Ingrid Halle,
Alberto Mantovani, Kimmo Peltonen, Guido Rychen, Pascal Sanders, Amadeu Soares, Pieter
Wester and Wilhelm Windisch.

ACKNOWLEDGEMENT
The Scientific Panel on Additives and Products or Substances used in Animal Feed wishes to
thank Professor Thomas Acamovic, Professor Christel Lamberg-Allardt and Professor Yves Nys
for their contributions on the preparation of the document on the efficacy and safety assessment
of HyD (calcifediol), based on 25-hydroxylcholecalciferol/25-hydroxy-pre-cholecalciferol.

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The EFSA Journal (2005) 224, 1-35

Opinion of the Scientific Panel on Additives and Products or

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