EFSA Journal - 2011 - Scientific Opinion On Safety and Efficacy of Choline Chloride As A Feed Additive For All Animal

EFSA Journal 2011;9(9):2353
SCIENTIFIC OPINION
Scientific Opinion on safety and efficacy of choline chloride as a feed

additive for all animal species1
EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP)2,3
European Food Safety Authority (EFSA), Parma, Italy
ABSTRACT
Choline contributes to vital biological functions, including synthesis of phospholipids. It is a precursor of
intracellular messenger molecules and of acetylcholine and a methyl group donor. The use levels of choline
chloride supplementation to commercial feeds are considered safe for the target animals. However, the margin of
safety compared to the use level in poultry and pigs is small. A maximum safe content in feed could not be
established because of the limited and inconsistent dataset available and uncertainties arising from differences in
diet composition. The simultaneous use of supplemental choline in feed and in water for drinking should be
avoided. The use of choline chloride as a nutritional additive does not give rise to concern for consumers. The
risk of choline chloride preparations causing inhalation toxicity of exposed workers is regarded as low, as the
particles from the formulations applied are unlikely to reach the alveoli, and the exposure of the upper
respiratory tract to choline chloride caused no adverse effect. Aqueous solutions of up to 70 % choline chloride
are non-irritant to skin and eyes. There is no evidence to indicate that more concentrated and solid choline
chloride formulations would be irritant, although in the absence of studies this possibility cannot be entirely
excluded. The possibility that occupational exposure to choline chloride might cause skin sensitisation cannot be
discounted. The use of choline chloride in animal nutrition is not expected to substantially increase the
concentration in the environment; consequently, no risk for the environment is foreseen. Due to its established
nutritional role in domestic animals, choline chloride is regarded as an effective source of choline.
© European Food Safety Authority, 2011
KEY WORDS
Nutritional additive, vitamin, provitamin, choline, safety, efficacy
1
On request from the European Commission, Question No EFSA-Q-2010-00872, adopted on 6 September 2011.
2
Panel members: Gabriele Aquilina, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël
Dierick, Mikolaj Antoni Gralak, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Reinhard Kroker, Lubomir Leng, Anne-
Katrine Lundebye Haldorsen, Secundino Lopez Puente, Alberto Mantovani, Giovanna Martelli, Miklós Mézes, Derek
Renshaw, Maria Saarela, Kristen Sejrsen and Johannes Westendorf. Correspondence: FEEDAP@efsa.europa.eu
3
Acknowledgement: The Panel wishes to thank the members of the Working Group on Vitamins, including Annette
Schuhmacher, for the preparatory work on this scientific opinion.
Suggested citation: EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP); Scientific
Opinion on safety and efficacy of choline chloride as a feed additive for all animal species. EFSA Journal 2011;9(9):2353.
[15 pp.] doi:10.2903/j.efsa.2011.2353. Available online: www.efsa.europa.eu/efsajournal
© European Food Safety Authority, 2011

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Choline chloride for all species
SUMMARY
Following a request from the European Commission, the Panel on Additives and Products or
Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and
efficacy of choline chloride, supplied via feed or water, as feed additive for all animal species.
Choline chloride is applied for use in all animal species and categories without a maximum limit. The
additive (in a liquid or different solid forms) can be incorporated in feed directly or via premixtures.
The only form which is applied for use in water for drinking is the liquid formulation.
Choline contributes to vital biological functions, as for example in the synthesis of the phospholipids,
which are structural cell components, as precursor for the intracellular messenger molecules, and for
acetylcholine. Choline is the major source of methyl groups.
Oral administration routes of choline chloride via feed or water are considered as bioequivalent.
The FEEDAP Panel concludes that recommended and use levels of choline chloride supplementation
to feed are safe for the target animals. However, studies indicate that the margin of safety compared to
the use level in poultry and pigs is small. The limited and inconsistent dataset available together with
the uncertainties arising from differences in diet composition does not allow the FEEDAP Panel to
conclude on the maximum safe content in feed. The simultaneous use of supplemental choline in feed
and water for drinking should therefore be avoided.
The FEEDAP Panel concludes that the use of choline chloride as a nutritional additive does not give
rise to concern for consumers.
The risk of choline chloride preparations causing inhalation toxicity to the lungs of exposed workers is
regarded as low, as the particles from the formulations applied are unlikely to reach the alveoli, and
the exposure of the upper respiratory tract to choline chloride caused no adverse effect. Aqueous
solutions of up to 70 % choline chloride are non-irritant to skin and eyes. There is no evidence to
indicate that more concentrated and solid choline chloride formulations would be irritant, although in
the absence of studies this possibility cannot be entirely excluded. The possibility that occupational
exposure to choline chloride might cause skin sensitisation cannot be discounted.
Choline occurs widely in nature and is readily biodegradable. The use of choline chloride in animal
nutrition is not expected to substantially increase the concentration in the environment. Therefore, a
risk for the environment resulting from the use of choline chloride in animal nutrition is not foreseen.
Due to its established nutritional role in domestic animals, choline chloride is regarded as an effective
source of choline.
EFSA Journal 2011;9(9):2353 2

TABLE OF CONTENTS
Abstract ....................................................................................................................................................1
Table of contents ......................................................................................................................................3
Background ..............................................................................................................................................4
Terms of reference ...................................................................................................................................4
Assessment ...............................................................................................................................................6
1. Introduction ......................................................................................................................................6
2. Characterisation ................................................................................................................................6
2.1. Characterisation of the active substance .................................................................................6
2.2. Manufacturing process ............................................................................................................7
2.3. Stability and homogeneity ......................................................................................................7
2.4. Conditions of use ....................................................................................................................8
2.5. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL)8
3. Safety ................................................................................................................................................8
3.1. Safety for the target species ....................................................................................................9
3.2. Safety for the consumer ........................................................................................................10
3.3. Safety for the user .................................................................................................................11
3.4. Safety for the environment....................................................................................................12
4. Efficacy ..........................................................................................................................................12
5. Post-market monitoring ..................................................................................................................12
Conclusions and recommendations ........................................................................................................13
Documentation provided to EFSA .........................................................................................................13
References ..............................................................................................................................................13
Appendix ................................................................................................................................................15
EFSA Journal 2011;9(9):2353 3

BACKGROUND
Regulation (EC) No 1831/20034 establishes the rules governing the Community authorisation of
additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any
person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an
application in accordance with Article 7. In particular Article 10(2) of that Regulation also specifies
that for existing products within the meaning of Article 10(1), an application shall be submitted in
accordance with Article 7, at the latest one year before the expiry date of the authorisation given
pursuant to Directive 70/524/EEC for additives with a limited authorisation period, and within a
maximum of seven years after the entry into force of this Regulation for additives authorised without
time limit or pursuant to Directive 82/471/EEC.
The European Commission received a request from the VITAC EEIG Vitamins Authorisation
Consortium5 for (i) authorisation of a new use (i.e. use in water for drinking) and (ii) re-evaluation of
the product choline chloride to be used as a feed additive for all animal species (category: nutritional
additive; functional group: vitamins, provitamins and chemically well-defined substances having
similar effect) under the conditions mentioned in Table 1.
According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the
application to the European Food Safety Authority (EFSA) as an application under Article 4(1)
(authorisation of a feed additive or new use of a feed additive) and under Article 10(2) (re-evaluation
of an authorised feed additive. EFSA received directly from the applicant the technical dossier in
support of this application.6 According to Article 8 of that Regulation, EFSA, after verifying the
particulars and documents submitted by the applicant, shall undertake an assessment in order to
determine whether the feed additive complies with the conditions laid down in Article 5. The
particulars and documents in support of the application were considered valid by EFSA as of 30 June
2010.
Choline chloride has been authorised without time limit under Council Directive 70/524/EEC7 for its
use in all animal species as a nutritional additive
The Panel on Food additives and Nutrient Sources added to Food issued an opinion on choline
chloride orthosilicic acid (H4SiO4) added for nutritional purposes to food supplements (EFSA, 2009a).
The Scientific Committee on Consumer Products (SCCP) issued an opinion on choline chloride (EC,
2008).
TERMS OF REFERENCE
According to Article 8 of Regulation (EC) No 1831/2003, EFSA shall determine whether the feed
additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the
safety for the target animal(s), consumer, user and the environment and the efficacy of choline
chloride, when used under the conditions described in Table 1.
4
OJ L 268, 18.10.2003, p.29
5
VITAC EEIG Vitamins Authorisation Consortium, 130A Avenue Louise, B-1050 Brussel, Belgium. Companies: Balchem
Italia s.r.l.; BASF SE; Chr. Olesen Group, representing Be-Long Enterprise Inc. – Nanjing, China; Andres Pintaluba S.A.
representing Shandong Aocter Chemical Co. LTD Gaotang, Shandong, China; Taminco N.V.
6
EFSA Dossier reference: FAD-2010-0024
7
OJ C 50, 25.2.2004, p.1
EFSA Journal 2011;9(9):2353 4

Table 1: Description and conditions of use of the additive as proposed by the applicant
Additive Choline chloride

Registration number/EC
Not appropriate
No/No (if appropriate)
Category(-ies) of additive Nutritional additives (3)
Vitamins, pro-vitamins and chemically well defined substances having a
Functional group(s) of additive
similar effect
Description
Chemical Purity criteria Method of analysis
Composition, description
formula (if appropriate) (if appropriate)
Choline chloride min
Choline chloride C5H14ClNO Ion chromatography
99 % pure
Trade name (if appropriate) Not appropriate - non-holder specific authorisation

Name of the holder of
Not appropriate - non-holder specific authorisation
authorisation (if appropriate)
Conditions of use
Species or Minimum content Maximum content Withdrawal
Maximum
category of period
Age mg/kg of complete feedingstuffs
animal (if appropriate)
All animal species No

No restriction
and categories restriction - -
Other provisions and additional requirements for the labelling
Specific conditions or restrictions for

Applicable in premix, feed and water
use (if appropriate)
Specific conditions or restrictions for
-
handling (if appropriate)
Post-market monitoring
-
(if appropriate)
Specific conditions for use in
complementary feedingstuffs -
(if appropriate)
Maximum Residue Limit (MRL) (if appropriate)

Species or category of Target tissue(s) or Maximum content in
Marker residue
animal food products tissues
- - - -
EFSA Journal 2011;9(9):2353 5

ASSESSMENT
This opinion is based in part on data provided by a consortium of companies involved in the
production/distribution of choline chloride. It should be recognised that this data covers a fraction of
existing additives containing choline chloride. The application is for the active substance and the
composition of the additive formulations is not the subject of the application. The Panel has sought to
use the data provided together with data from other sources to deliver an opinion and to produce
recommendations for the authorisation, which would secure the safety of future uses of choline
chloride as feed additive.
1. Introduction
Choline contributes to vital biological functions of body, as for example for the synthesis of the
phospholipids, which are structural cell components, as precursors for the intracellular messenger
molecules, and for acetylcholine. Choline is the major source of methyl groups.
Choline chloride is authorised without a time limit under Council directive 70/524/EEC8 for it use in
all species as a nutritional additive; no maximum levels of choline chloride in feeds are established in
the EU. The applicant, a consortium of five companies, asks for the re-evaluation of the use of choline
chloride as additive to feed and for new use of this additive (use in water for drinking) for all animal
species and categories without restrictions for age, content in feedingstsuff and without withdrawal
time.
Choline chloride is authorised for addition for specific nutritional purposes in foods for particular
nutritional uses (Regulation (EC) No 953/2009),9 to processed cereal based foods and baby foods for
infants and young children (Directive 2006/125/EC, Annex IV)10 and to infant formulae and follow-on
formulae when reconstituted as instructed by the manufacturer (Directive 2006/141/EC, Annex III).11
Choline-stabilised orthosilicic acid is authorised as a mineral substance which may be used in the
manufacture of food supplements (Regulation (EC) No 1170/2009, Annex II).12
2. Characterisation
The dossier contains data from five sources of choline chloride all obtained by chemical synthesis.
Several additives containing choline chloride are commercially available in Europe. They may differ
in the physical properties (liquid, powder) and choline chloride concentration.
2.1. Characterisation of the active substance

Choline chloride [IUPAC name: ethanaminium 2-hydroxy-N,N,N-trimethyl-, chloride; synonyms: (2-
hydroxyethyl)trimethylammonium chloride; (β-hydroxyethyl) trimethylammonium chloride;
ammomium, (2-hydroxyethyl)trimethyl- chloride; choline hydrochloride; cholinium chloride;
trimethyl(2-hydroxyethyl) ammonium chloride)], is identified with CAS number 67-48-1 and EINECS
number 200-655-4. The structural formula is presented in Figure 1.
Figure 1. Structural formula of choline chloride

8
OJ C 50, 25.2.2004, p.1
9
OJ L 269, 14.10.2009, p.9
10
OJ L 339 6.12.2006, p. 16
11
OJ L 401 30.12.2006, p. 1
12
OJ L 314 1.12.2009, p. 36
EFSA Journal 2011;9(9):2353 6

The molecular formula of choline chloride is C5H14NO.Cl and its molecular weight is 139.63. It has a
melting point of 247 °C, shows a bulk density of 1.1 g/cm3 and is soluble in water (800 g/L at 25 °C)
and ethanol, insoluble in ether and benzene.
Choline chloride is the active substance under application and is a white crystalline powder.
Specifications set by producers allow a minimum of 99 % purity (anhydrous basis). Analysis of
twenty-five batches of the 75 % choline chloride liquid from five sources (five batches each) showed
high content uniformity and negligibly small differences between the products (producer 1: > 99.90 %;
producers 2, 3 and 4: > 99.85 %; producer 5, 99.96 %, mean values calculated on anhydrous basis).13
Main organic impurities resulting from the synthesis, i.e. unreacted trimethylamine (ranging from <
100 to < 500 mg/kg), ethylene glycol (from 64 to < 500 mg/kg), and chlorethanol (10–55 mg/kg)
formed by reaction of hydrochloric acid with ethylene oxide, were below 500 mg/kg each. Their sum
was below 1500 mg/kg (< 250 to 622 mg/kg), heavy metals (expressed as lead) < 20 mg/kg (from < 2
to < 20 mg/kg),13 all parameters complying with OECD SIDS (2004). Dioxins and dioxin-like PCBs
were also determined in five batches with upper limits of 0.5 ng dioxins WHO-PCDD/F-TEQ/kg and
0.35 ng WHO-PCDD/F-PCB-TEQ dioxin-like PCB´s /kg.14
2.1.1. Formulated products

Choline chloride obtained from chemical synthesis is hygroscopic and is further processed to obtain
the formulation that is placed on the market. Four main formulations are described: choline chloride
liquid, which contains 70 to 80 % active substance in water; choline chloride on plant based carrier
which contains active substance between 50 and 70 % and is obtained absorbing choline chloride
solution on a plant based carrier and drying this mixture to a moisture below 3 %; choline chloride on
silica carrier, which is produced by mixing choline chloride solution with silica to a concentration of
typically 50 % with a water content of 15–20 %; and choline chloride spray dried, obtained by spray
drying of choline chloride solution to a concentration of 96 % and a moisture below 0.5 %.
The applicant provided some selected data on particle size distribution of the different products. The
critical fraction below 50 µm is < 1 % for all solid products.15 No data on dusting potential were
provided.
2.2. Manufacturing process

The main synthetic route starts from trimethylamine, ethylene oxide and hydrochloric acid in aqueous
solution. The selectivity of the reaction is determined by the molar ratio of three reagents, together
with pressure, temperature and pH. The concentration of the final product choline chloride (ethylene
oxide free) depends on the raw materials used. Sodium hydroxide (NaOH) is added as catalyst in some
processes.
2.3. Stability and homogeneity
2.3.1. Shelf-life
The applicant provided information on the recovery of one choline chloride formulation (60 % on
plant based carrier, three batches) when kept at 25 °C (60 % relative humidity, RH) up to 24 months.
The recovery values showed no reduction of the content of choline chloride over time.16
The stability of three batches of the liquid product (75 %) was tested for two years at room
temperature. At the end of the test period, no choline chloride loss was observed.17
13
Technical dossier/Section II/Annex 2.1.3 and supplementary information November 2010
14
Technical dossier/Supplementary information November 2010/Annexes 2.5-2.9
15
Technical dossier/Section II/Annex 2.1.5
16
Technical dossier/Section II/Annex 2.4.1a
17
Technical dossier/Section II/Annex 2.4.1.b
EFSA Journal 2011;9(9):2353 7

2.3.2. Stability of the additive when added to premixtures, feed and water for drinking
According to Whitehead (2002), the average choline chloride loss per month in premixtures is 2 %.
Stability during feed preparation depends on the processing conditions and may vary from 100 %
retention (at 93 °C x 30 s) to 96 % retention (at 149 °C x 30 s).
The applicant provided information on the stability of choline chloride (60 % on plant based carrier,
three batches) when incorporated in a premixture containing trace elements at 10 % wt and kept in
sealed aluminum bags at 35 °C up to six months. Choline chloride recovery was 90.5 % after six
months.18
The applicant provided information on the stability of the additive choline chloride (75 % solution in
water, three batches) in mash feed (choline chloride supplementation 2 g/kg) when kept at 20 °C and
at 35 °C for three months. The initial concentration was 2.5 g choline chloride/kg feed. After a storage
period of three months the mean recovery was 97.6 % of the initial concentration.18
Since the 75 % choline chloride liquid is the only formulation for use in water for drinking, the shelf-
life of this product (25 % water) could be used as an indicator of the choline chloride stability in water
for drinking.
2.3.3. Homogeneity
For a solid choline chloride formulation (60 % choline chloride) the applicant provided a statistical
method (Jansen, 1992) to calculate homogeneity in feed. The calculations resulted in a coefficient of
variation (CV %) of about 6 %.
The 75 % choline chloride liquid was used for studies on homogeneous distribution in 18 feed
samples. The CV was 9.4 %.
Choline chloride is highly soluble in water and therefore homogeneity in water for drinking needs not
to be demonstrated.
2.4. Conditions of use

Choline chloride is to be used in all animal species and categories without a maximum limit. The
additive (in a liquid or solid form) can be incorporated in feed directly or via premixtures. The only
form which is applied for use in water for drinking is the liquid formulation.
2.5. Evaluation of the analytical methods by the European Union Reference Laboratory
(EURL)
EFSA has verified the EURL report as it relates to the methods used for the control of choline chloride
in animal feed. The Executive Summary of the EURL report can be found in the Appendix.
3. Safety
According to Regulation (EC) No 429/2008, tolerance, metabolism and residue, and toxicological
(with concern to consumer safety) studies are not required for vitamins, pro-vitamins and chemically
defined substances having similar effects which are already authorised as feed additives under
Directive 70/524/EEC and which do not have the potential to accumulate, which FEEDAP considers is
the case for choline chloride. However, these exemptions are generally set without prejudice to any
request of supplementary information made by EFSA according to Article 8(2) of Regulation (EC) No
1831/2003.
18
Technical dossier/Supplementary information November 2010/Annex 2.11
EFSA Journal 2011;9(9):2353 8

3.1. Safety for the target species

Requirements for choline (NRC, see McDowell, 2000) are in the range of 300–2000 mg/kg feed for
pigs and poultry, 400–1500 mg/kg feed for fish and 1250–2400 mg/kg feed for pets. The requirement
for adult ruminants is not known. Typical poultry diets provide already 70–80 % of the requirements
for choline from feed materials. Different factors make it difficult to characterise the requirements for
supplemental choline in farm animals: (i) the range of choline content in plant feed materials (e.g.,
maize ~ 500 mg/kg, rapeseed ~ 6800 mg/kg), (ii) the differences in bioavailability of choline from
feed materials (25–70 %), (iii) the dietary supply with other substances involved in transmethylation
reactions (methionine, betaine, homocysteine, cobalamine and folic acid), (iv) the absence of data on
the amount of endogenously synthesised choline and of the supply with other transmethylating
substances, and (v) the remarkable increase in performance of poultry and pigs and nutritional quality
of complete feed in the period from the time when the studies cited were conducted and today.
Vitamin supplementation of commercial compound feed is mostly oriented towards recommendations

for supplemental choline, which are in the range of 200–800 mg/kg feed for pigs and poultry (1200
mg/kg for turkeys), 600–1000 mg/kg feed for fish and 1200–1400 mg/kg feed for pets (AWT, 2001).
A survey on vitamin supplementation of commercial feeds for pigs and poultry in Europe (Belgium,
Denmark, Germany, Italy, Netherlands, Portugal, Spain, United Kingdom) identified a range of
supplemental 0–1000 mg choline chloride/kg pig and poultry diet as commercial use levels (Gropp,
1994). A more recent publication (Whittemore et al., 2002) gives the adjusted range of dietary
additions of choline in pig diets found in Denmark, Germany, Netherlands, United Kingdom, United
States, Spain and Australia for weaned piglets up to 15 kg bw with 200–500, for growing pigs of 15–
50 kg bw with 100–300, for growing pigs of 50–150 kg bw with 0–200, for gestating sows with 300–
950, and for lactating sows with 300–950 mg/kg feed.
In male leghorns, 2500 mg supplemented choline chloride/kg diet had no adverse effect on growth
(Ketola and Nesheim 1974). Deeb and Thornton (1959) found supplementation of a semipurified diet
with 880–1760 mg choline chloride/kg diet was optimal for growth, but levels exceeding 2200 mg/kg
diet depressed growth. Saville et al. (1967) reported signs of pyridoxine deficiency (hyperexcitability,
muscular incoordination) in chickens for fattening after supplementing a diet with 2200 mg choline
chloride/kg and growth depression after even lower doses (1320 mg/kg diet).
In laying hens no adverse effects were observed with the supplement of 7330 mg choline chloride/kg
diet (Crawford et al., 1969). Choline chloride is in the wide range (90 mg/kg diet for three weeks up to
1800 mg/kg) without any influence on the performance of laying hens and on hatchability of eggs
(Jeroch et al., 1991). In another study (March and MacMillan 1979) the adverse effect of
supplementing 3834–5228 mg choline chloride/kg diet affected product quality (fishy taint of some
eggs).
Fishy taint of eggs is a well known problem, not only related to several known dietary factors.
According to a review of Zentek (2003), fishy taint has been demonstrated to be related to increased
levels of trimethylamine (TMA) mainly in the egg yolk. Diet can affect egg taint either by increased
dietary TMA levels (i.e. by fish meal, precursors of TMA like choline and betaine), by providing
precursors for the TMA formation to the intestinal microflora (i.e. sinapine from rapeseed) or by
provision of inhibitors of the endogenous TMA oxidase (i.e. rapeseed). Moreover, egg taint may occur
even if no known disposing factor is contained and when dietary levels of choline are normal.
Adverse effects in dogs (reduced erythrocyte numbers) were reported at levels of choline chloride
equivalent to three times the apparent choline requirements (NRC, 1987). Southern et al. (1986) found
that 2000 mg supplemental choline per kg of a maize-soybean diet, administered over the whole
growing period, depressed growth of pigs.
EFSA Journal 2011;9(9):2353 9

3.1.1. Conclusions
to feed are safe for the target animals. However, studies indicate that the margin of safety compared to
the use level in poultry and pigs is small. The limited and inconsistent dataset available together with
the uncertainties arising from differences in diet composition do not allow the FEEDAP Panel to
conclude on the maximum safe content in feed. The simultaneous use of supplemental choline in feed
and water for drinking should therefore be avoided.
3.2. Safety for the consumer
3.2.1. Metabolic and residue studies

Choline and compounds derived from choline serve a number of vital biological functions, (i) choline
is used in the synthesis of the phospholipids, phosphatidylcholine and sphingomyelin, which are
structural cell components essential for the structural integrity of cell membranes, (ii) the choline-
containing phospholipids, phosphatidylcholine and sphingomyelin, are precursors for the intracellular
messenger molecules, diacylglycerol and ceramide. Two other choline metabolites, platelet activating
factor (PAF) and sphingophosphorylcholine, are also known to be cell signaling molecules, (iii)
choline is a precursor for acetylcholine, an important neurotransmitter involved in muscle control,
memory, and many other functions, (iv) choline is involved in the transport and metabolism of lipids
(fat) and (v) choline is the major source of methyl groups. It can be oxidized in the body to form
betaine, which serves as methyl donor in methylation reactions. Methyl groups from betaine may be
used to convert homocysteine (methyl acceptor) to methionine, a cobalamin dependent process. The
enzyme hepatic betaine-homocysteine methyltransferase (BHMT) is stimulated by choline (or betaine)
when methionine deficient diets arte given to chicks (Emmert et al., 1996). Elevated levels of
homocysteine in the blood of humans have been associated with increased risk of cardiovascular
diseases (EFSA, 2009b). The demethylated betaine (dimethyl-glycine) is further metabolised to
glycine/serine. Glycine is also a methyl donor in the one carbon pool, the methyl being transferred to
tetrahydrofolic acid.
Animals can synthesize choline in small amounts by converting phosphatidylethanolamine to

phosphatidylcholine (de novo synthesis of choline). Three methylation reactions are required, each
using the S-adenosyl methionine as a methyl group donor. Because phosphatidylcholine can be
synthesized and metabolized to provide choline, choline was not previously considered an essential
micronutrient. However, the de novo synthesis does not provide enough choline if the requirement is
high (juvenile animals, dietary factors).
As the body is not known to store choline, in situations where excess choline is fed, it is catabolised
via the choline oxidation pathway.
3.2.2. Toxicological studies
3.2.2.1. Acute toxicity

Animal studies with choline chloride show a low acute toxicity after oral uptake (with a range of
LD50s of 3150 – ≥ 6000 mg/kg bw determined in different studies with rats, and 3900-6000 mg/kg bw
for mice, OECD SIDS, 2004).
3.2.2.2. Genotoxicity studies including mutagenicity

Choline chloride does not show a mutagenic potential when tested in vitro (Ames test, yeast gene
conversion, clastogenicity, and sister chromatid exchange). It has no structural alerts (OECD SIDS,
2004).
3.2.2.3. Sub-chronic repeated dose oral toxicity studies

No data available.
EFSA Journal 2011;9(9):2353 10

3.2.2.4. Chronic oral toxicity studies

No data available.
3.2.2.5. Reproduction and developmental toxicicity studies

No developmental toxic effects were observed in mice after oral doses of 1250 mg/kg bw/day on
gestation days 1 to 18. Doses above the levels recommended currently (4160 mg/kg bw/day), and
associated with maternal toxicity, did produce developmental toxic effects, but these were secondary
to the maternal toxicity at the excessive doses used (OECD SIDS, 2004).
3.2.3. Assessment of consumer safety

No UL has been established in Europe to date. The OECD reported an UL of 3500 mg/person/day for
adults defined by US Standing Committee on the Scientific Evaluation of Dietary Reference Intakes
(OECD SIDS, 2004). Hypotension was considered as the critical effect for choline: an uncertainty
factor of 2 was applied to the human NOAEL because of the limited database and the large inter-
individual variation.
Since choline is not known to show any significant deposition in farm animals, the use of choline
chloride as feed additive under good animal nutrition practice is unlikely to elicit any appreciable
increase of consumer’s exposure. Thus, the use of choline chloride in animal husbandry is considered
safe for consumers.
3.3. Safety for the user
3.4.1. Exposure by inhalation

As less than 1 % of the weight of three solid preparations consist of particles of less than 50 µm
diameter, the amount of dust any that can be inhaled into the alveoli of the lungs is considered to be
minimal. Mehta, et al. (2009) showed that choline is well tolerated in mice upon intranasal
administration (200 mg/kg bw on alternate days for 28 days). As the particles are unlikely to reach the
alveoli and the exposure to the upper respiratory tract caused no adverse effect, the risk of choline
chloride preparations causing inhalation toxicity to the lungs of exposed workers is regarded as low.
3.4.2. Irritancy
Full reports of studies of irritancy were not available, but a review publication (OECD SIDS, 2004)
summarised the results of some studies, including skin irritancy studies in rabbits and humans and an
eye irritancy study in rabbits.
In a study that was not in line with current methodological guidelines, a 70 % aqueous solution of
choline was applied to the shaved backs of two female White Viennese rabbits under an occlusive
dressing for 20 hours. Assessment for skin reactions was performed at 24 hrs and 2, 5 and 8 days after
application. Reddening that was described as “questionable” was seen on one rabbit.
In a 21-day skin irritation study of 25 humans with self-perceived sensitive skin, a 0.5 % aqueous
solution of choline chloride, a soap bar containing 5 % choline chloride and a liquid body soap
containing 5 % choline chloride were evaluated and compared to their respective controls. There were
no significant differences between the response to samples containing choline chloride and their
respective choline chloride free controls.
In a non-GLP eye irritancy study that was broadly in line with OECD test guideline 405, a 70 %
aqueous solution of choline was applied to one female and one male rabbit. Reddening of the eye and
slight secretion of tears was observed after application. These effects were no longer seen three hours
after the administration.
EFSA Journal 2011;9(9):2353 11

The available data suggest that aqueous solutions of up to 70 % choline are non-irritant. There is no
evidence to indicate that undiluted choline chloride would be irritant, although in the absence of
studies this possibility cannot be entirely excluded.
3.4.3. Sensitisation
No data were available on skin sensitisation potential in laboratory animals. However, a review
publication (OECD SIDS, 2004) summarised a test for skin sensitisation in humans and one case
report of sensitisation in a human.
In a human repeated-insult patch test on 202 subjects, an aqueous solution of 0.5 % (w/v) choline
chloride was applied during the induction phase and a 0.2 % (w/v) aqueous solution during the
challenge phase was tested and compared to controls. The results of the study showed no evidence of
dermal sensitisation reactions elicited by choline chloride.
One case of acute contact dermatitis was reported in a woman who worked in a garden centre. Patch
testing was positive for a 1 % aqueous solution of choline chloride.
The human data on sensitisation do not allow an unequivocal conclusion to be made about the
potential to cause skin sensitisation of exposure to choline chloride in the workplace as a result of its
use in animal feeds. The concentrations of choline chloride used in the repeated-insult patch test were
too low for this purpose. The case report of contact dermatitis indicates that some people might
develop skin sensitisation to choline chloride, but gives no indication about how common such a
reaction may be. The possibility that occupational exposure to choline chloride might cause skin
sensitisation cannot be discounted.
3.4. Safety for the environment

Choline occurs widely in nature and is readily biodegradable (OECD SIDS, 2004). The use of choline
chloride in animal nutrition is not expected to substantially increase the concentration in the
environment. Therefore, a risk for the environment resulting from the use of choline chloride in animal
nutrition is not foreseen.
4. Efficacy
According to Regulation (EC) No 429/2008, efficacy studies are not required for vitamins, pro-
vitamins and chemically defined substances having similar effects which are already authorised as
feed additives under Directive 70/524/EEC and which do not have the potential to accumulate, which
FEEDAP considers is the case for choline chloride.
Due to its established nutritional role in domestic animals, choline chloride is regarded to be an
effective source of choline.
Choline has been globally used in animal nutrition for decades. Data on requirement, allowances and
recommendations for feed supplementation are easily accessible as standard literature for animal
nutrition experts.
5. Post-market monitoring
The FEEDAP Panel considers that there is no need for specific requirements for a post-market
monitoring plan other than those established in the Feed Hygiene Regulation19 and Good
Manufacturing Practice.
19
OJ L 35, 8.2.2005, p. 1.
EFSA Journal 2011;9(9):2353 12

CONCLUSIONS AND RECOMMENDATIONS

CONCLUSIONS
Oral administration routes of choline chloride via feed or water are considered as bioequivalent.
to feed are safe for the target animals.
Studies indicate that the margin of safety compared to the use level in poultry and pigs is small.
However, the FEEDAP Panel cannot conclude on the maximum safe content in feed because of the
limited and inconsistent dataset available and the uncertainties arising from differences in diet
composition.
The FEEDAP Panel concludes that the use of choline chloride as a nutritional additive does not give
rise to concern for consumers.
Aqueous solutions of up to 70 % choline chloride are non-irritant to skin and eyes. There is no
evidence to indicate that more concentrated and solid choline chloride formulations would be irritant,
however in the absence of studies this possibility cannot be entirely excluded. The possibility that
occupational exposure to choline chloride might cause skin sensitisation cannot be discounted. The
risk of choline chloride preparations causing inhalation toxicity to exposed workers is regarded as low.
Choline occurs widely in nature and is readily biodegradable. The use of choline chloride in animal
nutrition is not expected to substantially increase the concentration in the environment. Therefore, a
risk for the environment resulting from the use of choline chloride in animal nutrition is not foreseen.
Due to its established nutritional role in domestic animals, choline chloride is regarded as an effective
source of choline.
RECOMMENDATIONS
Given the potential to exceed a safe dose, the simultaneous use of supplemental choline in feed and
water for drinking should be avoided.
DOCUMENTATION PROVIDED TO EFSA

1. Choline chloride as a feed additive for all animal species. May 2010. Submitted by VITAC
EEIG Vitamins Authorisation Consortium.
2. Choline chloride as a feed additive for all animal species. Supplementary information.
November 2010 and June 2011. Submitted by VITAC EEIG Vitamins Authorisation
Consortium.
3. Evaluation report of the European Union Reference Laboratory for Feed Additives on the
methods(s) of analysis for choline chloride.
4. Comments from Member States received through the ScienceNet.
REFERENCES
AWT (Arbeitsgemeinschaft Wirkstoffe in der Tierernährung) 2001. Vitamins in Animal Nutrition,
Bonn. ISBN 3-86037-155-X. Agrimedia GmbH in Bergen.
Crawford JS, Griffith M, Teekell RA, Watts AB, 1969. Poult. Sci. 38, 620-626.
Deeb SS, Thornton PA, 1959. The choline requirement of the chick. Poult. Sci. 38, 1198.
EFSA Journal 2011;9(9):2353 13

EC (European Commission), 2008, online. Opinion of the Scientific Committee on Consumer

Products (SCCP) on Choline chloride. Available from:
http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_132.pdf
EFSA (European Food Safety Authority), 2009a. Opinion of the Scientific Panel on Food additives
and Nutrient Sources added to Food on choline chloride orthosilicic acid added for nutritional
purposes to food supplements. The EFSA Journal, 948, 1-23.
EFSA (European Food Safety Authority), 2009b. Opinion of the Scientific Panel on Additives,
Products or Substances used in Animal Feed on the safety and efficacy of guanidinoacetic acid as
feed additive for chicken for fattening. The EFSA Journal, 988, 1-30.
Emmert JL, Garrow TA, Baker DH, 1996. Hepatic betaine-homocysteine methyltransferase activity in
the chicken is influenced by dietary intake of sulphur amino acids, choline and betaine. J. Nutr.
126, 2050-2058.
Gropp JM. 1994. Vitamin Fortification Levels in European Commercial Poultry and Swine Diets.
Proceedings of the Arkansas Nutrition Conference, Fayetteville, Arkansas, 105-134.
Jansen HD, 1992. Mischtechnik im Futtermittelbetrieb. Anforderungen an Mischenlage, Arbeits- und
Mischgenauigkeit. Die Mühle+ Mischfuttertechnik, 129, 265-270.
Jeroch H, Ludke C, Richter G, Hennig A, 1991. Influence of Choline Supply on the Reproductive
Performance of Breeding Hens. 3 Symposium, Vitamine und weitere Zusatzstoffe bei Mensch und
Tier. Friedrich-Schiller-Universitat, Jena, pp. 270-272.
Ketola HG and Nesheim MC, 1974. Influence of dietary choline and methionine levels on the
requirements of choline by chickens. J. Nutr. 104, 1484-1489.
March BE and MacMillan C, 1979. Trimethylamine production in the caeca and small intestine as a
cause of fishy taints in egs. Poult. Sci. 58, 93-98.
McDowell LR. 2000. Vitamins in animal and human nutrition, Second Edition, Iowa State University
Press. ISBN 0-8138-2630-6, p. 577.
Mehta AK, Arora N, Gaur SN, Singh BP, 2009 Acute toxicity assessment of choline by inhalation,
intraperitoneal and oral routes in Balb/c mice. Reg. Toxicol. Pharmacol. 54, 82-86.
NRC (National Research Council), 1987. Vitamin tolerance of animals. National Academic Press,
Washington DC, 77-83.
OECD SIDS, 2004. OECD SIDS choline chloride UNEP Publications SIDS. Initial Assessment
Report for SIAM 19 Berlin, Germany, 19-22 October 2004.
Saville DG, Solvyns A, Humphries C, 1967. Choline induced pyridoxine deficiency in broiler
chickens. Austr. Vet. J. 43, 346-348.
Southern LL, Brown DR, Werner DD, Fox MC, 1986. J. Anim. Sci. 62, 992-996.
Whitehead CC, 2002. Vitamins in feedstuffs. In: Poultry Feedstuffs. Supply, Composition and
Nutritive Values. Poultry Science Symposium Series. Volume Twenty-Six. Eds: McNab JM and
Boorman KN. CABI Publishing. ISBN 0 85199 464 4, 181-190.
Whittemore CT, Close WH, Hazzledine MJ, 2002. The need for nutrient requirement standards for
pigs. A report of the British Society of Animal Science nutritional standards working group: pigs.
Pig News and Information, 23, 67N-74N.
Zentek J, 2003. Egg taint. A problem of practical importance. Lohmann information, No. 28, 1-4.
EFSA Journal 2011;9(9):2353 14

APPENDIX
Executive Summary of the Evaluation Report of the European Union Reference Laboratory for
Feed Additives on the Method(s) of Analysis for choline chloride
In the current application authorisation is sought for choline chloride under the category nutritional
additives, functional group '3a' vitamins, pro-vitamins and chemically well-defined substances having
similar effect, according to the classification system of Annex I of Regulation (EC) No 1831/2003.
Specifically, authorisation is sought for the use of choline chloride for all animal species and
categories. The active substance is a white crystalline powder containing at least 99 % total (on
anhydrous basis) expressed as choline chloride. It is intended to be used in premixtures, feedingstuffs
as a formulated product and in water as pure substance. According to the applicant, the typical
formulation is in the range of 70 to 80 wt % if liquid, of 50 to 70 wt. % if on vegetable carrier and
around 50 wt. % on silica carrier. The applicant does not propose any minimum or maximum choline
chloride concentration in feedingstuffs or water.
For the determination of choline chloride in the feed additive, premixtures, feedingstuffs and water the
applicant proposed a single laboratory validated and further verified ion chromatographic method with
conductivity detection (IC-CD). The method is based on the determination of the choline chloride
content in an aqueous solution. The following performance characteristics derived from the validation
and verification studies - performed at choline chloride concentrations ranging from 100 to 25000
mg/kg - were reported by the applicant:
- a relative standard deviation for repeatability (RSDr) ranging from 0.34 to 4.1 %,
- a relative standard deviation for intermediate precision (RSDip) ranging from 0.31 to 3.3 %,
and
- a recovery rate (RRec) ranging from 91.5 to 99.5 %.
Based on the above mentioned performance characteristics the CRL recommends for official control
this single laboratory validated and further verified IC-CD method for the determination of choline
chloride in feed additive, premixtures, feedingstuffs and water within the concentration range covered
by the validation study.
Further testing or validation of the methods to be performed through the consortium of National
Reference Laboratories as specified by article 10 (Commission Regulation (EC) No 378/2005) is not
considered necessary.
EFSA Journal 2011;9(9):2353 15

EFSA Journal - 2011 - Scientific Opinion On Safety and Efficacy of Choline Chloride As A Feed Additive For All Animal

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EFSA Journal 2011;9(9):2353

Scientific Opinion on safety and efficacy of choline chloride as a feed

European Food Safety Authority (EFSA), Parma, Italy

© European Food Safety Authority, 2011

© European Food Safety Authority, 2011

EFSA Journal 2011;9(9):2353 2

EFSA Journal 2011;9(9):2353 3

EFSA Journal 2011;9(9):2353 4

Additive Choline chloride

Trade name (if appropriate) Not appropriate - non-holder specific authorisation

All animal species No

Other provisions and additional requirements for the labelling

Specific conditions or restrictions for

Maximum Residue Limit (MRL) (if appropriate)

EFSA Journal 2011;9(9):2353 5

2.1. Characterisation of the active substance

Figure 1. Structural formula of choline chloride

EFSA Journal 2011;9(9):2353 6

2.1.1. Formulated products

2.2. Manufacturing process

2.3. Stability and homogeneity

EFSA Journal 2011;9(9):2353 7

2.4. Conditions of use

EFSA Journal 2011;9(9):2353 8

3.1. Safety for the target species

Vitamin supplementation of commercial compound feed is mostly oriented towards recommendations

EFSA Journal 2011;9(9):2353 9

3.2. Safety for the consumer

3.2.1. Metabolic and residue studies

Animals can synthesize choline in small amounts by converting phosphatidylethanolamine to

3.2.2. Toxicological studies

3.2.2.1. Acute toxicity

3.2.2.2. Genotoxicity studies including mutagenicity

3.2.2.3. Sub-chronic repeated dose oral toxicity studies

EFSA Journal 2011;9(9):2353 10

3.2.2.4. Chronic oral toxicity studies

3.2.2.5. Reproduction and developmental toxicicity studies

3.2.3. Assessment of consumer safety

3.3. Safety for the user

3.4.1. Exposure by inhalation

EFSA Journal 2011;9(9):2353 11

3.4. Safety for the environment

EFSA Journal 2011;9(9):2353 12

CONCLUSIONS AND RECOMMENDATIONS

DOCUMENTATION PROVIDED TO EFSA

4. Comments from Member States received through the ScienceNet.

EFSA Journal 2011;9(9):2353 13

EC (European Commission), 2008, online. Opinion of the Scientific Committee on Consumer

EFSA Journal 2011;9(9):2353 14

EFSA Journal 2011;9(9):2353 15

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