Upload
dindarachma
View
32
Download
3
Embed Size (px)
DESCRIPTION
food safety
Citation preview
EFSA Journal 2012;10(6):2737
Suggested citation: EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP); Scientific
Opinion on the safety and efficacy of beta-carotene as a feed additive for all animal species and categories. EFSA Journal
2012;10(6):2737. [33 pp.] doi:10.2903/j.efsa.2012.2737. Available online: www.efsa.europa.eu/efsajournal
© European Food Safety Authority, 2012
SCIENTIFIC OPINION
Scientific Opinion on the safety and efficacy of beta-carotene as a feed
additive for all animal species and categories1, 2
EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP)3,4
European Food Safety Authority (EFSA), Parma, Italy
ABSTRACT
The use of beta-carotene is safe for the target animals. Setting a maximum content in feed legislation is not
considered necessary. However, this conclusion assumes that triphenylphosphine oxide does not exceed
100 mg/kg additive. In all food-producing animals (except veal calves) and laboratory rodents, beta-carotene is
almost fully metabolised. In contrast, humans, non-human primates and ferrets absorb relatively high quantities
of beta-carotene unchanged. Investigations with ferrets and hamsters as well as intervention studies in humans
may indicate a certain dose-dependent potential of beta-carotene to promote lung carcinoma, particularly in
smokers. A systematic literature review and meta-analysis of nine randomised controlled trials demonstrated an
increased risk of lung and stomach cancers in smokers and asbestos workers at dose levels ≥ 20 mg/day.
However, increased risk of lung cancer at such doses could be observed only if plasma levels of beta-carotene
exceeded 3 µmol/L. The FEEDAP Panel considers it prudent, in the absence of an acceptable daily intake, that
supplemental beta-carotene in animal feed should not significantly add to consumer exposure from other
sources. The use of supplemental beta-carotene in feeds of food-producing animals, except veal calves, would
not result in a significant additional exposure of consumers to beta-carotene; however, consumption of liver
from veal calves could lead to additional exposure. Beta-carotene is not an irritant to eyes or skin and is not a
skin sensitiser. Respiratory exposure from handling beta-carotene-containing additives is considered potentially
hazardous. Taking the widespread occurrence of beta-carotene in nature and its oxidative susceptibility into
account, the FEEDAP Panel considered it unlikely that the use of beta-carotene in animal nutrition at the
recommended feed concentrations would pose a risk to the environment. Beta-carotene is utilised for the
synthesis of retinol in almost all animal species except the cat. Effects on reproduction and immunity were not
sufficiently demonstrated.
© European Food Safety Authority, 2012
KEY WORDS
Nutritional additive, vitamins and provitamins, beta-carotene, safety, efficacy
1 On request from the European Commission, Question No EFSA-Q-2009-00884 adopted by the FEEDAP Panel on 23 May
2012. 2 This scientific opinion has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No
1831/2003. The modified sections are indicated in the text. 3 Panel members: Gabriele Aquilina, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël
Albert Dierick, Mikolaj Antoni Gralak, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Lubomir Leng, Secundino López
Puente, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Giovanna Martelli, Miklós Mézes, Derek Renshaw,
Maria Saarela, Kristen Sejrsen and Johannes Westendorf. Correspondence: [email protected] 4 Acknowledgement: The Panel wishes to thank the members of the Working Group on Fat-soluble Vitamins, including
Reinhard Kroker and Annette Schuhmacher, for the preparatory work on this scientific opinion.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 2
SUMMARY
Following a request from the European Commission, the Panel on Additives and Products or
Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and
efficacy of beta-carotene as an additive to feed and water for drinking for all animal species.
Beta-carotene, an isoprenoid compound, is synthesised by plants and microorganisms. It is used in
animal nutrition mainly as provitamin A.
The FEEDAP Panel concludes that the use of beta-carotene is safe for the target animals. Setting a
maximum content in feed legislation is not considered necessary. However, this conclusion assumes
that triphenylphosphine oxide does not exceed 100 mg/kg additive.
In all food-producing animals (except the calves) and laboratory rodents, beta-carotene is almost fully
metabolised in the enterocytes. In contrast, humans, non-human primates and ferrets absorb relatively
high quantities of beta-carotene unchanged. Consequently, toxicological data resulting from studies
with laboratory rodents cannot be used for conclusions on consumer safety. Investigations with ferrets
and hamsters as well as intervention studies in humans indicate a dose-dependent potential of beta-
carotene to promote lung carcinoma, particularly in smokers (and asbestos workers). A systematic
literature review and meta-analysis of nine randomised controlled trials demonstrated an increased
risk of lung and stomach cancers in smokers and asbestos workers supplemented with beta-carotene at
doses equal to or greater than 20 mg/day. However, increased risk of lung cancer at such doses could
be observed only if plasma levels of beta-carotene exceeded 3 µmol/L, which suggests that internal
exposure indicators, e.g. plasma levels, may be more appropriate than oral intake to characterise a
risk. The same review did not record an increased cancer incidence at supplemental dose levels
varying from 6 to 15 mg/day for about 5–7 years.
The FEEDAP considers it prudent, in the absence of an acceptable daily intake, that supplemental
beta-carotene in animal feed should not significantly add to consumer exposure from other sources.
The FEEDAP Panel considers that the use of supplemental beta-carotene in feeds of food-producing
animals, except veal calves, would not result in a significant additional exposure of consumers to
beta-carotene. However, consumption of liver from preruminant calves treated with beta-carotene
could lead to a significant additional exposure of the consumer. Although frequent calf liver
consumption is limited, the FEEDAP Panel concludes that unlimited use of beta-carotene as an
additive to milk replacers may be of concern as regards consumer safety.
Beta-carotene from chemical synthesis and from fermentation is not an irritant to eyes or skin and is
not a skin sensitiser. For one additive, respiratory exposure of users was calculated to be considerably
above the guidance value for oral intake (15 mg/person/day). In the absence of any information on
inhalation toxicity, such exposure is considered potentially hazardous.
Taking the widespread occurrence of beta-carotene in nature and its oxidative susceptibility into
account, the FEEDAP Panel considers it unlikely that the use of beta-carotene in animal nutrition at
the recommended feed concentrations would pose a risk to the environment.
The FEEDAP Panel concludes that beta-carotene is utilised for the synthesis of retinol in almost all
animal species except the cat. Effects on reproduction and immunity are not sufficiently
demonstrated.
The FEEDAP Panel formulated some recommendations, particularly concerning the specifications of
the additive, including triphenylphosphine oxide, a maximum content in milk replacers, the restriction
of its use to premixtures and the avoidance of any use in water for drinking.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 3
TABLE OF CONTENTS
Abstract .................................................................................................................................................... 1 Summary .................................................................................................................................................. 2 Table of contents ...................................................................................................................................... 3 Background .............................................................................................................................................. 4 Terms of reference ................................................................................................................................... 4 Assessment ............................................................................................................................................... 6 1. Introduction ..................................................................................................................................... 6 2. Characterisation ............................................................................................................................... 7
2.1. Characterisation of the active substance ................................................................................... 7 2.1.1. Beta-carotene produced by chemical synthesis .................................................................. 7 2.1.2. Beta-carotene obtained by fermentation ............................................................................. 8
2.2. Formulated products ................................................................................................................. 9 2.3. Stability and homogeneity ........................................................................................................ 9
2.3.1. Shelf life ............................................................................................................................. 9 2.3.2. Stability in premixtures and feed ...................................................................................... 10 2.3.3. Homogeneity in feed ........................................................................................................ 10 2.3.4. Stability and homogeneity in water for drinking .............................................................. 10
2.4. Physico-chemical incompatibilities in feed ............................................................................ 10 2.5. Conditions of use .................................................................................................................... 11 2.6. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) 11
3. Safety ............................................................................................................................................. 11 3.1. Metabolic pathways of beta-carotene ..................................................................................... 11
3.1.1. Tissue concentrations ....................................................................................................... 12 3.2. Safety for the target species .................................................................................................... 14
3.2.1. Conclusions on target animal safety ................................................................................. 15 3.3. Safety for the consumer .......................................................................................................... 15
3.3.1. Toxicological studies ........................................................................................................ 15 3.3.2. Assessment of consumer safety ........................................................................................ 17
3.4. Safety for the user ................................................................................................................... 19 3.4.1. Effects on skin and eyes ................................................................................................... 19 3.4.2. Exposure by inhalation and effects on the respiratory system .......................................... 19 3.4.3. Conclusions on safety for the user .................................................................................... 20
3.5. Safety for the environment ...................................................................................................... 20 4. Efficacy .......................................................................................................................................... 20
4.1. Reproduction and immunology ............................................................................................... 20 4.2. Conclusions on efficacy .......................................................................................................... 21
5. Post-market monitoring ................................................................................................................. 21 Conclusions and recommendations ........................................................................................................ 21 Remark ................................................................................................................................................... 22 Documentation provided to EFSA ......................................................................................................... 23 References .............................................................................................................................................. 23 Appendices ............................................................................................................................................. 31 Appendix A ............................................................................................................................................ 31 Appendix B ............................................................................................................................................ 32 Appendix C ............................................................................................................................................ 33 Potential exposure of users handling beta-carotene ............................................................................... 33 Estimate of risk mitigation ..................................................................................................................... 33 Calculation of exposure by inhalation during a working day ................................................................. 33
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 4
BACKGROUND
Regulation (EC) No 1831/20035 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 addition, 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 a
time limit or pursuant to Directive 82/471/EEC.
The European Commission received a request from the VITAC EEIG Vitamins Authorisation
Consortium6 for (i) authorisation of a new use (i.e. use in water for drinking) and (ii) re-evaluation of
the product beta-carotene when 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.7 According to Article 8 of that Regulation, EFSA, after verifying the
particulars and documents submitted by the applicant, shall undertake an assessment in order to
determine whether the feed additive complies with the conditions laid down in Article 5. The
particulars and documents in support of the application were considered valid by EFSA as of 2 March
2010.
Beta-carotene (E160 a) has been authorised without time limit under Council Directive 70/524/EEC8
for its use for all animal species as a nutritional additive and for canaries as a sensory additive.
The Scientific Committee on Food expressed an opinion on the tolerable upper intake level of beta-
carotene (EC, 2000). The Panel on Dietetic Products, Nutrition and Allergies (NDA) issued four
opinions on substantiation of several health claims related to beta-carotene pursuant to Article 13(1) of
Regulation (EC) No 1924/2006 (EFSA, 2009a, 2010a, 2011a, 2011b). The Panel on Food Additives
and Nutrient Sources added to Food (ANS) issued an opinion on the re-evaluation of mixed carotenes
(E 160a (i)) and β-carotene (E 160a (ii)) as a food additive (EFSA, 2012a).
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 animals, consumer, user and the environment and the efficacy of the product beta-
carotene, when used under the conditions described in Table 1.
5 Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use
in animal nutrition. OJ L 268, 18.10.2003, p. 29. 6 VITAC EEIG Vitamin Authorisation Consortium, Avenue Louise 130A, B-1050 Brussels, Belgium; Companies: BASF
SE, Ludwigshafen, Germany; DSM Nutritional Products Ltd., Delft, The Netherlands; Europe-Asia Import-Export GmbH,
Hamburg, Germany; Feed Additive Technologies SARL, Etrembières, France; Lohmann Animal Health GmbH &Co. KG,
Cuxhaven, Germany; Sunvit GmbH, Bardowick, Germany. 7 EFSA Dossier reference: FAD-2009-0046. 8 Commission List of the authorised additives in feedingstuffs published in application of Article 9t (b) of Council Directive
70/524/EEC concerning additives in feedingstuffs (2004/C 50/01). OJ C 50, 25.2.2004, p. 1.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 5
Table 1: Description and conditions of use of the additive as proposed by the applicant
Additive Beta Carotene
Registration number/EC
No/No (if appropriate) 3a 160 a
Category(-ies) of additive 3. Nutritional additives
Functional group(s) of additive a. Vitamins, provitamins and chemically well defined substances
having a similar effect
Description
Composition, description Chemical
formula
Purity criteria
(if appropriate)
Method of analysis
(if appropriate)
Beta-carotene C40H56
Min. 96 %
(max. 3 % other
colouring matters)
JECFA
PhEur
Trade name (if appropriate) Not appropriate
Name of the holder of
authorisation (if appropriate) Not appropriate
Conditions of use
Species or
category of
animal
Maximum
Age
Minimum content Maximum content Withdrawal
period
(if appropriate)
mg or Units of activity or CFU kg-1 of complete
feedingstuffs, supplementary feed (based on end
feed) and in water*
All animal species
All categories - - - -
Other provisions and additional requirements for the labelling
Specific conditions or restrictions for
use (if appropriate)
Only for manufacture of animal feeds. Declaration to be made on
the label: mg Beta-Carotene.
Beta-Carotene shall be placed on the market in the form of a
formulation containing min. 10 % Beta-Carotene.
If used in water a formulation has to be made in such a way that it
is soluble or dispersible in water.
Specific conditions or restrictions for
handling (if appropriate) None.
Post-market monitoring
(if appropriate)
No specific requirement other than the traceability and complaint
system implemented in compliance with the requirement of
Regulation No 183/2005
Specific conditions for use in
complementary feedingstuffs
(if appropriate)
Maximum Residue Limit (MRL) (if appropriate)
Marker residue Species or category of
animal
Target tissue(s) or
food products
Maximum content in
tissues
- - - -
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 6
ASSESSMENT
This opinion is based in part on data provided by a consortium of companies involved in the
production/distribution of beta-carotene. It should be recognised that these data cover only a fraction
of existing additives containing beta-carotene. The composition of the additives is not the subject of
the application. The FEEDAP Panel has sought to use the data provided together with data from other
sources to deliver an opinion.
The application contains data from five sources of beta-carotene obtained by chemical synthesis and
from one source obtained by fermentation.
1. Introduction
Beta-carotene and carotenoids in general are isoprenoid compounds synthesised by plants and
microorganisms. About 700 naturally occurring carotenoids have been identified so far. About 10 % of
these carotenoids can be found in the human diet, and about 20 of these compounds have been found
in plasma and tissues of mammals. Some dietary carotenoids, such as beta-carotene, serve as
provitamin A.
Beta-carotene (E-160a) is included in the European Union Register of Feed Additives pursuant to
Regulation (EC) No 1831/2003. It is authorised without a time limit in application of Article 9t (b) of
Council Directive 70/524/EEC9 concerning additives in feedingstuffs (2004/C 50/01) for its use in all
animal species as a nutritional additive and for canaries as a sensory additive.
The applicant, a consortium of six companies, asks for the re-evaluation of the use of beta-carotene as
an additive to feed and for a new use of beta-carotene (use in water for drinking). The substance is
intended as a nutritional additive under the functional group vitamins, provitamins and chemically
well-defined substances having similar effects, for all animal species and categories.
Beta-carotene is authorised for use in food (Regulation (EC) No 1925/2006,10 amended by Regulation
(EC) No 1170/2009)11 and in food supplements (Directive 2002/46/EC, Annex II),12 for addition for
specific nutritional purposes in foods for particular nutritional uses (Regulation (EC) No 953/2009)13
and for addition to processed cereal-based foods and baby foods for infants and young children
(Directive 2006/125/EC, Annex IV).14 Beta-carotene can be used as a colourant in foodstuffs
(Directive 2001/50/EC).15 It is authorised in cosmetics as a skin conditioner (Commission Decision
2006/257/EEC).16
Beta-carotene is described in the European Pharmacopoeia (PhEur) in Monograph (MG) 1069.
9 Commission List of the authorised additives in feedingstuffs published in application of Article 9t (b) of Council Directive
70/524/EEC concerning additives in feedingstuffs (2004/C 50/01). OJ C 50, 25.2.2004, p. 1. 10 Regulation (EC) No 1925/2006 of the European Parliament and of the Council of 20 December 2006 on the addition of
vitamins and minerals and of certain other substances to foods. OJ L 404 30.12.2006, p. 26. 11 Commission Regulation (EC) No 1170/2009 of 30 November 2009 amending Directive 2002/46/EC of the European
Parliament and of the Council and Regulation (EC) No 1925/2006 of the European Parliament and of the Council as
regards the lists of vitamin and minerals and their forms that can be added to foods, including food supplements. OJ L 314
1.12.2009, p. 36. 12 Directive 2002/46/EC of the European Parliament and of the Council of 10 June 2002 on the approximation of the laws of
the Member States relating to food supplements. OJ L 183 12.7.2002, p. 51. 13 Commission Regulation (EC) No 953/2009 of 13 October 2009 on substances that may be added for specific nutritional
purposes in foods for particular nutritional uses. OJ L 269, 14.10.2009, p. 9. 14 Commission Directive 2006/125/EC of 5 December 2006 on processed cereal-based foods and baby-foods for infants and
young children. OJ L 339 6.12.2006, p. 16. 15 Commission Directive 2001/50/EC of 3 July 2001 amending Directive 95/45/EC laying down specific purity criteria
concerning colours for use in foodstuffs. OJ L 190, 12.7.2001, p. 14. 16 Commission Decision 2006/257/EC of 9 February 2009 amending Decision 96/335/EC establishing an inventory and a
common nomenclature of ingredients employed in cosmetic products. OJ L 97 5.04.2006, p. 1.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 7
The Joint FAO/WHO Expert Committee on Food Additives (JECFA) assessed beta-carotene,
chemically synthesised and produced from fermentation by Blakeslea trispora, several times, most
recently in 2011 (JECFA, 2011).
2. Characterisation17
Beta-carotene occurs naturally in feedingstuffs. De novo synthesis of beta-carotene in animals does not
occur.
2.1. Characterisation of the active substance
Beta-carotene (IUPAC name: (all-E)-3,7,12,16-tetramethyl-1,18-bis(2,6,6-trimethylcyclohex-1-enyl)-
octadeca-1,3,5,7,9,11,13,15,17-nonaene; synonyms: , -carotene, all-trans- -carotene, provitamin A,
CI Food Orange 5) is identified by the INS number 160a, CAS (Chemical Abstracts Service) number
7235-40-7 and EINECS (European Inventory of Existing Chemical Substances) number 230-636-6.
The structural formula of beta-carotene is shown in Figure 1.
Figure 1: Structural formula of beta-carotene
The molecular formula of beta-carotene is C40H56 and its molecular weight is 536.88. It has a melting
point of 176–183 C, a density of 0.6–1 g/cm3 (20 °C), an octanol–water coefficient of 17.6 and a very
high pKa value (> 14). It is insoluble in water and ethanol, and soluble in chloroform. It has limited
solubility in vegetable oils.
Beta-carotene occurs in the form of red to brownish-red to violet crystals or crystalline powder and
contains predominantly the all-trans (Z) isomer of beta-carotene with varying amount of the cis isomer
depending on the different formulations and other carotenoids (< 3 %). Beta-carotene can be produced
by chemical synthesis or from fermentation by Blakeslea trispora. It contains, by specifications in
compliance with the PhEur (MG 1069), at least 96 % beta-carotene (in the dried substance) in the total
colouring matter.
2.1.1. Beta-carotene produced by chemical synthesis
Two synthetic processes are described in the dossier submitted by the applicant.
Analysis of five datasets (five batches each)18 showed an average beta-carotene content (expressed as
% of dried substance or of total colouring matter) of 98.2 ± 0.96 % (mean value source 1, 98.9 %;
source 2, 98.5 %; source 3, 98.6 %; source 4, 96.6 %; and source 5, 98.8 %), the lowest value out of
the 25 samples being 96.2 %. The mean concentration of carotenoids other than beta-carotene was
0.85 ± 0.95 %. The highest value out of 25 determinations was 2.7 %.19 These data confirm that the
active substance beta-carotene complies with the specifications and corresponding data of food
legislation (Regulation (EC) No 231/2012).20
The loss on drying (threshold of the PhEur 0.2 %) was submitted for three products with values
varying between 0 and 0.1 %. The thresholds in food legislation for sulphated ash and lead in beta-
17 This section has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/2003. 18 Technical dossier/Confidential information C1 to C5. 19 Technical dossier/Confidential information C1 to C5 and Supplementary information January 2012 and April 2012. 20 Commission Regulation (EC) No 231/2012 of 9 March 2012 laying down specifications for food additives listed in
Annexes II and III to Regulation (EC) No 1333/2008 of the European Parliament and of the Council. OJ L 83 22.03.2012,
p. 1.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 8
carotene are ≤ 0.1 % and ≤ 2 mg/kg respectively. The data submitted by the applicant (for two sources
heavy metals instead of lead)17 show that all comply with the criteria for food additives (Regulation
(EC) No 231/2012).18
The applicant provided a representative survey of the residual solvents, which vary according to the
different production procedures. Analytical data provided for acetone, ethanol, methanol, methylene
chloride, trichloromethane, benzene, diethylketone, ethyl acetate, isobutyl alcohol and 2-propanol
show that the active substances under application can comply with the thresholds proposed by VICH
(International Cooperation on Harmonisation of Technical Requirements for Registration of
Veterinary Medicinal Products).17
Triphenylphosphine oxide (TPPO), a reaction by-product, occurs in all beta-carotene sources
submitted. Levels of TPPO range from 18 to 205 mg/kg in the active substance and depend on the
source. However, the highest value found in an additive containing 10 % beta-carotene was 60 mg/kg,
likely corresponding to 600 mg of TPPO/kg in the active substance.21
2.1.2. Beta-carotene obtained by fermentation
Blakeslea trispora Thaxter slant strains XCPA 07-05-1 and XCPA 07-05-2 are active producers of
beta-carotene. This fungus exists in (+) and (–) forms, of which the (+) form synthesises trisporic acid,
a precursor of beta-carotene. On mating the two types in a specific ratio, the (–) form then synthesises
large amounts of beta-carotene. The (+) and (–) forms differ slightly from each one from another
according to their cultural and morphological properties. The (+) form has aerial mycelia that are
yellow-orange to brownish in colour whereas the (–) form has aerial mycelia that are grey-whitish to
brownish in colour.
The strains are deposited at the China General Microbiological Culture Collection Center with the
deposition numbers CGMCC 7.44 for XCPA 07-05-1 and CGMCC 7.45 for XCPA 07-05-2.22 Genetic
stability of the strains has been shown for six generations.23
The microorganisms are not known to produce toxins, virulence factors or antibiotics. In this respect,
the Scientific Committee on Food (SCF) (EC, 2000) and JECFA (2001) did not raise safety concerns
for beta-carotene produced by Blakeslea trispora. Data on antibiotic resistance of the production
strains were not provided as the applicant argued that the product does not contain the production
microorganism.
The product obtained from fermentation is filtered, extracted and then subjected to crystallisation.
Analysis of five batches24 showed an average beta-carotene content (expressed as % of total colouring
matter) of 99.5 ± 0.29 % and of carotenoids other than beta-carotene of 0.99 ± 0.21 %.
The applicant provided data on impurities in three batches of beta-carotene obtained by fermentation.25
Data on sulphated ash (0.01–0.02 %), lead (≤ 0.001 %), microbiological parameters (moulds, yeasts
and Salmonella) show that all parameters comply with the criteria for food additives (Regulation (EC)
No 231/2012). Analytical data for residual solvents (dichloromethane < 0.003 %, ethanol < 0.025 %,
ethyl acetate < 0.12 % and acetone < 0.23 %) show that the active substances under application
comply with the thresholds proposed by Regulation (EC) No 231/2012 (ethyl acetate and ethanol) and
by VICH. Mycotoxins were not detected in beta-carotene obtained by fermentation (thresholds in
Commission Directive 2008/128/EC but no longer present in Regulation (EC) No 231/2012).
21 Technical dossier/Supplementary information January 2011 and January 2012. 22 Technical dossier/Supplementary Information January 2012 and Confidential information C3. 23 Technical dossier/Section II/Annex 2202. 24 Technical dossier/Confidential Information C3. 25 Technical dossier/Supplementary information April 2012.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 9
2.2. Formulated products
Beta-carotene is sensitive to oxidation, light and heat; therefore, the final formulations require
stabilised forms, which can be achieved by specific product formulation. Additives available in the
market may differ in the origin of beta-carotene and in their physico-chemical properties. The most
common beta-carotene content of the additives present on the European market is 10 %.
The crystalline raw product is subjected to a milling procedure resulting in maximum particle size of
0.5 µm to ensure uniform availability to the target animal. The crystals are then converted by heat
treatment to an amorphous form, which is the basis for water-dispersible forms and the dry powder.
The active substance is embedded in a colloidal matrix of mainly gelatine and also containing
antioxidants (e.g. tocopherol, tocopheryl, ascorbic acid, ethoxyquin), emulsifiers and carbohydrates
(e.g., starch, maltodextrin or sucrose). The dispersion can be dehydrated by spraying (spray drying or
spray cooling). Alternatively, the colloidal suspension is emulsified in paraffin oil (which is removed
later on) and dried in a fluidised bed.
The particles of the active substance included in the matrix can be additionally stabilised by chemical
or thermal cross-linking of the beadlet surface. These dry powders are no longer water dispersible but
show technological advantages for processing in the feed compound industry.
For use in water for drinking the applicant provided as an example data on the composition of a cold
water-dispersible formulation used in the food industry. The product contains 10 % beta-carotene and
(in descending order of weight) modified food starch, glucose syrup, medium-chain triglycerides, DL-
alpha-tocopherol and tricalcium phosphate. The producer recommends storing the product in tightly
sealed, lightproof packaging in a cool place because it is sensitive to oxygen, heat, light and moisture.
Three sets of data were provided for particle size distribution. In the first set, analysis of representative
products showed that 0–1.38 % (w/w) of the particles were smaller than 50 µm;26 in the second set
(three batches of another additive) 0.21–0.59 % (w/w) of the particles were smaller than 50 µm.27 In
contrast, data from two other formulations showed that 99% of particles in one product were smaller
than 15.2 µm whereas no particles smaller than 50 µm were seen in the other product.28 The dusting
potential of these last two additives (one batch each) was determined to be 0.57 g/m3 and 0.21 g/m3
respectively.29 Differences in particle size distribution do not necessarily reflect differences in the
dusting potential.
2.3. Stability and homogeneity
2.3.1. Shelf life
Beta-carotene is sensitive to oxidation, light and heat. Its stability was examined (three batches) when
stored at two different temperatures (room temperature or 5 °C) under artificial conditions (aluminium
foil bag under inert gases) for 18 months. Recovery was higher than 94.5 %.
Additives should contain beta-carotene in a stabilised form. The shelf life of beta-carotene in one
additive (three batches) when stored at 15 °C in an aluminium foil bag was followed for 36 months
under nitrogen and for 24 months without nitrogen. Recoveries after 24 months were higher than
93.5 % for all six measurements. No essential differences were observed for the different storage
conditions. Because recovery of the product stored under nitrogen was still greater than 96 %, the
applicant proposes a shelf-life of 36 months when kept at about 15 °C in the original package.
26 Technical dossier/Section II. 27 Technical dossier/Supplementary Information January 2011. 28 Technical dossier/Supplementary information January 2012 Annexes vii 1 and vii 3. 29 Technical dossier/Supplementary information January 2012 Annexes vii 2 and vii 4.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 10
2.3.2. Stability in premixtures and feed
All data described in this section derived from two formulations from the same producer.
The two additives were incorporated at 1 g/kg into premixtures both containing trace elements and
choline chloride. One additive was tested in a premixture for chickens for fattening, the other in a
premixture for cattle. Samples were stored for one month at 35 °C and for 3 months at 25 °C. After
one month at 35 °C recoveries in the cattle and poultry premixtures were 70 % and 92 %, respectively,
and after 3 months at 25 °C recoveries were 78 % and 80 % respectively.30
Cattle mash feed was supplemented with ~33 mg beta-carotene/kg of feed from two additives and
pelleted. Both products were stable during pelleting (conditioning at 70 °C, pelleting at 80 °C). Losses
after one month at 25 C were about 10 % in one batch; after three months about 29 % and 25 % of the
initial value.24
Pet food was supplemented with ~44 mg beta-carotene/kg feed from two additives and extruded.
There was an unexplained difference between the target value and the concentration in the mash
sample showing recoveries of only 84 % and 80 % in the two products. Subsequent recoveries during
storage were negatively affected by these initial differences. Total losses after three months‘ storage at
25 °C in paper bags were about 60 % and 25 % respectively. Losses after six months were about 75 %
and 40 % respectively. Extrusion did not influence the stability of beta-carotene.24
Shrimp feed was supplemented with ~100 mg beta-carotene/kg feed from two additives followed by
preconditioning (at about 95 °C), pelleting (at 104 °C) or extrusion (95 °C). Pelleting reduced the
initial content by ~5–10 % and extrusion by ~10–15 %. After storage in paper bags at 25 °C for three
months, recovery was ~73–83 % in the pelleted feed and ~78–85 % in the extruded feed; the
differences between the two additives were larger than the difference caused by different physical
treatments.24
2.3.3. Homogeneity in feed
Based on a statistical method (Jansen, 1992), the coefficient of variation (CV) for homogeneity for
two formulated products containing beta-carotene, one with a small particle size (50–550 µm) and
another with a larger particle size (100–850 µm), was calculated to be around 3.23 % and 0.24 %
respectively. However, this method has been developed to test the working accuracy of mixing
equipment.
2.3.4. Stability and homogeneity in water for drinking
Stability of the additives in water for drinking was not investigated under practical conditions with an
additive formulated specifically for use in water. The data provided refer to an additive intended for
feed use and investigated under laboratory conditions at 15 C in flasks closed with a stopper for 4, 8
and 24 hours at a concentration of about 31 mg beta-carotene/100 mL.31 The data are given in terms of
the beta-carotene content of the additive. There was no change in this value (~10 %) after 24 hours.
The data do not allow any conclusion on homogeneity as the sample was kept under continuous
magnetic stirring. The fact that a cold water-dispersible formulation has been used for many years in
beverages cannot be accepted as a replacement for data demonstrating stability and homogeneity in
water for drinking (probably at a higher concentration than is used in beverages).
2.4. Physico-chemical incompatibilities in feed
No physico-chemical incompatibilities or interactions have been reported between beta-carotene and
feed materials, carriers, other approved additives or medicinal products when the additive was added
to premixtures and feed. No such incompatibilities or interactions are expected.
30 Technical dossier/Section II/Annex 2401. 31 Technical dossier/Section II/Annex 2402.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 11
2.5. Conditions of use
Because beta-carotene is not stable to light, oxygen and heat, only formulated products containing
stabilised beta-carotene can be placed on the market. The additive is intended to be incorporated in
feed either directly or using premixtures without minimum or maximum limits.
The applicant recommended a range of 10–70 mg beta-carotene/kg complete feed for dairy cows, 80–
100 mg beta-carotene/kg complete feed for sows, 70–125 mg beta-carotene/kg complete feed for
breeding horses, up to 100 mg beta-carotene/kg milk replacer, up to 20 mg beta-carotene/kg complete
feed for rabbits and up to 30 mg beta-carotene/kg complete feed for other animal species and
categories. The applicant recommends further to use the same doses in water.
2.6. Evaluation of the analytical methods by the European Union Reference Laboratory
(EURL)
EFSA has verified the European Union Reference Laboratory (EURL) report as it relates to the
methods used for the control of beta-carotene in animal feed. The Executive Summary of the EURL
report can be found in Appendix A.
3. Safety
3.1. Metabolic pathways of beta-carotene
Beta-carotene, as a fat-soluble molecule, is absorbed by mucosal cells of the small intestine by passive
diffusion, similar to the absorption pathway of the products of triglyceride digestion. The availability
of beta-carotene varies considerably as the release of (natural) beta-carotene from the feed matrix and
the extent of beta-carotene absorption depend on several factors including animal species, the general
feed matrix, processing of the feed, dietary level and type of fat, presence of other carotenoids, dietary
beta-carotene intake and vitamin A status. The release of beta-carotene from the feed matrix is
followed by solubilisation of beta-carotene into lipid globules and the formation of mixed micelles.
Release and solubilisation of beta-carotene appear to be the most important steps determining beta-
carotene availability. Dietary fat enhances the solubilisation and hence the absorption of beta-carotene;
absorption is higher from monounsaturated than from polyunsaturated fatty acids (Hollander and
Ruble, 1978). Some soluble fibres, particularly citrus pectin, but not oat beta-glucan, reduce intestinal
beta-carotene uptake apparently by disrupting micelle formation (Erdman et al., 1986; Rock and
Swendseid, 1992; Deming et al., 1999, 2000).
The uptake of beta-carotene into the enterocytes of the duodenum occurs by passive diffusion, which
requires a concentration gradient between the micelle and the cell membrane and may become
saturated at high beta-carotene doses (Parker, 1996). In the mucosal cells beta-carotene is converted
mainly to retinal by central cleavage by the enzyme beta-carotene-15,15 -monooxygenase (formerly
dioxygenase), and further to retinol and retinyl esters. The extent of cleavage is highly species
dependent as well as being influenced by the beta-carotene dose and vitamin A status (see Parker,
1996). Estimates of the ‗bioavailability‘ (% absorbed of the ingested dose) of carotenoids range from
1 % to 99 % (van Vliet et al., 1995; Parker et al., 1999) with large treatment, intra-individual, inter-
individual and inter-species variability (e.g. Parker, 1996); also different study designs and methodical
approaches may account for the discrepancies in the published data (Parker et al., 1999). In humans
the utilisation of beta-carotene (absorption as such (Goodman et al., 1966a) and as retinyl esters
(Novotny et al., 1995; van Vliet et al., 1995)) has been estimated in the range of 9–22 %.
Studies in humans also indicate that about 20–75 % of the absorbed beta-carotene is converted in the
intestinal mucosa to retinyl esters and up to 30 % of the beta-carotene is absorbed unchanged
(Goodman et al., 1966a, b; Blomstrand and Werner, 1967; van Vliet et al., 1995; Parker et al., 1997);
however, the proportions of retinyl esters and intact beta-carotene may be reversed at high oral beta-
carotene doses (Parker et al., 1997, 1999). The ferret (Mustela putorius furo), the Mongolian gerbil
(Meriones unguiculatus), preruminant calves and several non-human primates evidently absorb and
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 12
convert beta-carotene in a manner partially similar to that of humans (Ribaya-Mercado et al., 1989,
1992; Krinsky et al., 1990; Gugger et al., 1992; Poor et al., 1992; Wang et al., 1992; White et al.,
1993; Pollack et al., 1994; Bierer et al., 1995; Hoppe et al., 1996; Lee et al., 1998; Slifka et al., 1999).
In contrast, almost all beta-carotene is cleaved in the intestinal mucosal cells of the rat, and only 2 % is
released from the enterocytes as intact beta-carotene (Huang and Goodman, 1965). Also, pigs,
chickens, rabbits and other white-fat animals efficiently convert beta-carotene to retinol, i.e. only
small amounts of intact beta-carotene are available and found in plasma and tissues (Ullrey, 1972;
Chew et al., 1984, 1991; Erdman et al., 1986; Poor et al., 1987; Ben-Amotz et al., 1989; Mokady et al.,
1990; Chew, 1993a; Yap et al., 1997; Slifka et al., 1999; Schweigert et al., 2001). The cat absorbs only
intact beta-carotene and is not able to convert beta-carotene into retinol (Chew et al., 2000a, 2001;
Schweigert et al., 2002).
Beta-carotene is not a prevalent carotenoid in the marine environment. Information on provitamin A
activity of beta-carotene in salmonids is scarce. Poston et al. (1977) claimed that rainbow trout have
the ability to convert beta-carotene to vitamin A only at temperatures above 10 °C. The provitamin A
character of beta-carotene has been established for other fish species such as tilapia (Katsuyama and
Matsuno, 1988), ayu (Matsuno, 1991) and Atlantic halibut (Moren et al., 2004). Conversion of
xanthophylls such as astaxanthin into vitamin A is well established in salmonids (Schiedt et al., 1985;
Al-Khalifa and Simpson, 1988; Christiansen et al., 1994; White et al., 2003).
In the enterocytes the majority of beta-carotene is metabolised mainly by central cleavage yielding two
molecules of retinal and then retinol, which may be esterified with fatty acids forming retinyl esters,
the transport and storage form of vitamin A. Retinal can also be further oxidised to retinoic acid.
Alternately, eccentric cleavage may take place to a minor extent resulting in the formation of beta-apo-
8 -, 10 - or 12 -carotenals and finally retinoic acid. Appendix B illustrates the fate of beta-carotene in
the enterocytes and in tissues.
Some of the long-chain beta-apocarotenals (e.g. 8 , 10 , 12 , 14 ) resulting from the oxidative eccentric
cleavage products of beta-carotene are found in the plasma of humans and experimental animals and
are increased under conditions of oxidative stress and high dietary doses of beta-carotene (see Eroglu
et al., 2012). Eroglu et al. (2012) demonstrated that beta-carotene can generate both nuclear retinoic
acid receptor agonists (all-trans-retinoic acid) and antagonists (e.g. beta-apo-14 -carotenal and beta-
apo-13-carotenone) depending on the extent of cleavage at the central C15–C15' double bond or the
C13–C14 double bond, respectively. The authors suggested that beta-apocarotenoids function as
naturally occurring retinoid antagonists. The antagonism of retinoid signalling by these metabolites
may have implications for the metabolic activities of dietary beta-carotene as a provitamin A (Eroglu
et al., 2012).
After incorporation of intact beta-carotene into nascent chylomicrons within the Golgi apparatus of the
enterocytes, the chylomicrons are secreted by the enterocytes and transported via the lymphatic system
to the bloodstream and further to the liver and/or to other target tissues (see reviews by Parker, 1996;
Deming and Erdman, 1999). The chylomicrons are degraded in the blood by the lipoprotein lipase
(LPL, attached to the endothelium) to chylomicrons remnants, which are taken up by the liver, where
beta-carotene is released and stored or resecreted into the bloodstream in very low-density lipoprotein
(VLDL) (Johnson and Russell, 1992; Deming and Erdman, 1999). VLDL is further converted to low-
density lipoprotein (LDL); beta-carotene released from LDL is taken up by extrahepatic tissues. In the
fasted state about 75 % of the circulating beta-carotene is found in LDL and 25 % in VLDL and HDL
(EC, 2000).
3.1.1. Tissue concentrations
The plasma and tissue concentrations of beta-carotene partially reflect the capacity to absorb intact
beta-carotene. Ruminants other than monogastric calves, rodents, pigs, rabbits, chickens and other
white-fat animals efficiently convert beta-carotene into retinol; thus, the tissue storage of beta-carotene
is low in these species compared with that of humans.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 13
The main sites of beta-carotene deposition and accumulation are the adipose tissue in humans and the
liver in most animal species; however, substantial amounts can also be found in the corpus luteum of
reproducing animals (e.g. Chew et al., 1984; Kirsche et al., 1987; Arikan and Rodway, 2000, 2001;
Weng et al., 2000; Schweigert, 2003). Table 2 summarises the beta-carotene concentrations in
different species in a range of tissues. The data again indicate a large variability in beta-carotene
concentration between different animal species and tissues, which can be partially explained by
differences in beta-carotene supply or application, differences in the efficiency of absorption and
metabolism and/or differences in vitamin A status.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 14
Table 2: Tissue concentration of beta-carotene in different animal species fed graded levels of beta-
carotene (partially recalculated)
Species Beta-carotene concentration
Plasma/serum
(nmol/L)
Liver (ng/g) Adipose
(ng/g)
Corpus
luteum (ng/g)
Lung
(ng/g)
Milk
(nmol/L)
Ref.
Humans 170–5 360
(–)
640–750
(–)
429–10 415
(–)
620
(–)
(–)
(–)
(–)
(–)
(–)
54–859
(–)
(–)
(–)
31–50
1, 2
3
4*
Rat 7.5–11.2
310–340
ND
156 –13 368
n. d.
(–)
(–)
(–)
(–)
(–)
(–)
(–)
5
6
Pig 2–15
55.9
0–487
~100
(–)
(–)
25–172
~100
0 –12
(–)
(–)
(–)
7
8
Cow/cattle 1 751
4 806–10 673
~3 000
(–)
(–)
(–)
~14 200
(–)
(–)
(–)
(–)
147–209
8
9
Pre-
ruminant
calf
2790–17830
63–386
107–40 534
172–338
483–2 684
(–)
(–)
(–)
(–)
(–)
(–)
(–)
10
11
Ferret 11–870
680–1 800
1 –5 750
0–4 700
644
161–42 305
0–300
(~14)
0–752
(–)
(–)
(–)
(–)
23
1–698
(–)
(–)
(–)
5
12
13
Gerbil 0–88
0.1–106
18–497
(–)
–46
(–)
(–)
(–)
11–62
(–)
(–)
(–)
14
15
Cat 93–125
137
(–)
(–)
(–)
(–)
(–)
(–)
(–)
(–)
(–)
(–)
16
17
(–) , not determined or no data available; ND, below detection limit.*From autopsy.
1, Johnson et al. (1995); 2, Parker (1988); 3, Schmitz et al. (1991); 4, Canfield et al. (1997); 5, Ribaya-Mercado et al. (1989);
6, Barua and Olson (2000); 7, Schweigert et al. (2001); 8, Chew et al. (1984); 9, Calderón et al. (2007); 10, Hoppe et al.
(1996); 11, Poor et al. (1993); 12, Gugger et al. (1992); 13, Ribaya-Mercado et al. (1992); 14, Pollack et al. (1994); 15,
Thatcher et al. (1998); 16, Chew et al. (2000a); 17, Schweigert et al. (2002).
3.2. Safety for the target species
Regulation (EC) No 429/2008 states in Annex III 3.3.1.1 that no studies are required for vitamins,
provitamins and chemically well-defined substances having similar effect that do not have a potential
to accumulate already authorised as feed additives under Directive 70/524/EEC. For those additives
that fall within the functional group ‘vitamins, pro-vitamins and chemically well-defined substances
having similar effect‖ and which have a potential to accumulate, tolerance will be required to be
demonstrated only for compounds for which potency is expected or has been demonstrated to be
different from that of the well established vitamin(s). Consequently, tolerance studies with beta-
carotene are not considered necessary.
The FEEDAP Panel notes that beta-carotene leads to the accumulation of vitamin A in target animals,
mainly in the liver. However, the decreasing rate of conversion of beta-carotene to retinol with an
increasing supply of beta-carotene protects the animals from the consequences of an oversupply of
retinol. The beta-carotene absorbed as intact molecules is deposited in tissues in a dose-dependent
manner; however, absorption (by passive diffusion) is also limited because uptake mechanisms
become saturated. Therefore, the FEEDAP Panel considers that beta-carotene as provitamin A is safe
for the target animals.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 15
Chemically synthesised beta-carotene contains TPPO as a reaction by-product. TPPO may be of
concern for target animal safety: the No Observed Effect Level (NOEL, 90-day repeated dose oral
toxicity in rats) was 2 mg/kg body weight (bw) per day based on reduced alkaline phosphatase
activity, changes in organ weight (liver, kidney and adrenals) and vacuolar degeneration of liver cells.
Consequently, the exposure of target animals to TPPO from beta-carotene-containing additives was
estimated. The default values for body weight and feed intake of different animal species and
categories (see guidance on sensory additives; EFSA, 2012b) were used. The following additional
assumptions were made: (i) the safe intake of TPPO is derived from the NOEL applying a safety
factor of 100, (ii) the maximum recommended feed concentrations provided by the applicant were
taken as beta-carotene exposure, (iii) the additive contains 10 % beta-carotene and 100 mg TPPO/kg.
The calculations are summarised in Table 3.
Table 3: TPPO exposure of target animals administered a 10 % beta-carotene-containing additive
with 100 mg TPPO/kg
Animal category Default values Maximum
recommended
beta-carotene
(mg/kg feed)
Intake of
a 10 %
additive
(mg/day)
TPPO intake
Body
weight
(kg)
Feed
intake
(g/day)
Safe
amount*
(µg/day)
Additive with
100 mg
TPPO/kg
(µg/day)
Salmonids 2 40 30 12 40 1.2
Veal calves (milk
replacer) 100 2 000 100 2 000 2 000 200
Cattle for fattening 400 8 000 30 2 400 8 000 240
Pigs for fattening 100 3 000 30 900 2 000 90
Sows 200 6 000 100 6 000 4 000 600
Dairy cows 650 20 000 70 14 000 13 000 1 400
Turkeys for fattening 12 400 30 120 240 12
Piglets 20 1 000 30 300 400 30
Chickens for fattening 2 120 30 36 40 3.6
Laying hens 2 120 30 36 40 3.6
Dogs 15 250 30 75 300 7.5
*Based on a NOEL of 2 mg/kg bw in a 90-day repeated toxicity study with rats applying a safety factor of 100.
The data show that no concern for target animal safety would arise from the use of 10 % beta-
carotene-containing additives at the maximum recommended feed concentrations (see section 2.5)
when the TPPO content is restricted to 100 mg TPPO/kg additive. The margin of safety is between 6
and 40. This estimate shows that the target animals may tolerate additional TPPO exposure from other
additives that may be produced by similar chemical reactions (e.g. astaxanthin).
3.2.1. Conclusions on target animal safety
The use of beta-carotene in animal nutrition at the maximum doses recommended by the applicant is
safe for the target animals and would not need a maximum content to be set to restrict animal supply.
There would be no concern for target animal safety from the use of additives derived from chemical
synthesis and containing about 10 % beta-carotene at the maximum recommended feed concentrations
provided that the TPPO content is restricted to 100 mg TPPO/kg additive.
3.3. Safety for the consumer
3.3.1. Toxicological studies
The toxicity of beta-carotene (and mixed carotenes) has been recently assessed by the Panel on Food
Additives and Nutrient Sources added to Food (ANS) (EFSA, 2012a). The information relevant to the
present assessment is summarised below.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 16
3.3.1.1. Genotoxicity
Beta-carotene derived from Blakeslea trispora did not show any mutagenic effect in a bacterial reverse
mutation assay with Salmonella Typhimurium and Escherichia coli and in chromosomal aberration
assays in Chinese hamster ovary cells. Synthetic beta-carotene was not mutagenic in a bacterial
reverse mutation assay using Salmonella Typhimurium both in the absence and in the presence of the
S9 mix, and did not induce chromosomal aberrations in vitro or chromosomal aberrations or
micronuclei in mouse bone marrow in vivo.
Negative results for the induction of sister chromatid exchanges, chromosomal aberrations or
micronuclei in vitro and in vivo are reported from limited antimutagenicity studies with beta-carotene.
The ANS Panel concluded that beta-carotene is not of concern with respect to genotoxicity.
A metabolite of eccentric cleavage of beta-carotene, beta-apo-8 -carotenal, was reported to induce
increases in micronuclei, chromosomal aberrations and sister chromatid exchange in primary rat
hepatocytes. The ANS Panel did not consider further the data on micronuclei and chromosomal
aberrations for methodological and statistical reasons. It considered the increase in sister chromatid
exchange observed in the presence of beta-apo-8 -carotenal as more credible, but the biological
significance of this indicative assay in relation to genotoxicity is indirect. Another study (comet assay
with human retinal pigment epithelial cells) suggested a genotoxic potential of beta-apo-8 -carotenal.
The FEEDAP Panel recognises that beta-apo-8'-carotenal is produced in small amounts under
physiological conditions (see section 3.2.1) and considers that under these conditions (≤ 10 mg beta-
carotene/person day, see section 3.3.2.1) a genotoxic effect related to beta-apo-8 -carotenal is not
relevant.
3.3.1.2. Toxicological studies
The FEEDAP Panel considers that toxicity data obtained with laboratory rodents are not indicative for
the potential toxicity of beta-carotene in humans because of the differences in beta-carotene
metabolism in these species. The following extract of the summary of the ANS Panel opinion (EFSA,
2012a) considers, therefore, only studies with ferrets and humans.
In a study with ferrets fed synthetic beta-carotene at doses of 0.16 or 2.4 mg/kg bw per day for six
months, increases in the concentration of beta-carotene in both plasma (up to 21-fold) and lung tissue
(up to 300-fold) were found. All animals fed the high dose of beta-carotene showed localised
proliferation of alveolar cells (type II pneumocytes) and alveolar macrophages and keratinised
squamous metaplasia of alveolar wall epithelium.
Three studies in hamsters and two studies in ferrets have been reported in which animals were exposed
to a combination of beta-carotene and cigarette smoke (constituents). One study in hamsters showed
an inhibitory effect of beta-carotene on cigarette smoke-induced respiratory tract tumorigenesis. In the
other two hamster studies, beta-carotene caused increases in overall respiratory tract tumour incidence
and preneoplastic and neoplastic changes in the larynx, trachea and lung induced by cigarette smoke
(constituents). In ferrets fed beta-carotene in the diet, increased cell proliferation and squamous
metaplasia of alveolar epithelium were observed, which was further increased in the animals also
exposed to cigarette smoke.
It was found that a high dose of beta-carotene (equivalent to 30 mg/day in humans), in contrast to a
low dose (equivalent to 6 mg/day in humans), induced alveolar cell proliferation and keratinised
squamous metaplasia in the lung tissue of all ferrets with or without smoke exposure. In ferrets given
the low dose of beta-carotene alone, no pathological changes were observed.
The Alpha Tocopherol Beta Carotene Prevention Study (ATBC) and the Beta CARotene and Retinol
Efficacy (CARET) trial revealed that heavy smokers and asbestos workers (CARET only) receiving
long-time beta-carotene supplementation (ATBC) or beta-carotene + retinol supplementation
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 17
(CARET) at doses well below the previously established group acceptable daily intake (ADI) of
5 mg/kg bw per day had increased rather than decreased incidences of lung cancer. Besides increased
lung cancer incidence, increased stomach cancer mortality was seen in subjects receiving beta-
carotene supplementation in combination with a mixture of vitamins and minerals. It was commented
that, because of the combined exposure, the effects could not be ascribed to beta-carotene only.
The FEEDAP Panel refers to a publication of the German Institute for Risk Assessment
(Bundesinstitut für Risikobewertung) on the use of vitamins in foods (2005).32 The authors compared
the ATBC trial and the CARET trial with the Physician‘s Health Study (USA) and the Heart
Protection Study (UK), in which comparable doses of beta-carotene had no influence on the
occurrence of tumours. The difference in the observations was traced back to differences in plasma
levels. Studies in which plasma levels were in the range of 4.2–5.6 µmol/L showed an increased lung
cancer frequency as a result of beta-carotene supplementation; studies in which plasma levels were in
the range of 1.2–2.2 µmol/L did not.
Druesne-Pecollo et al. (2010) performed a systematic review and meta-analysis of nine randomised
controlled trials investigating beta-carotene supplementation and cancer risk. They found an absence
of any protective effect associated with beta-carotene supplementation with regard to primary cancer
risk. However, their results indicated an increased risk of lung and stomach cancers in smokers and
asbestos workers supplemented with beta-carotene at doses equal to or greater than 20 mg/day. The
authors, however, noted several significant caveats in the interpretation of their findings.
The ANS Panel noted that it was not possible to identify a No Observed Adverse Effect Level
(NOAEL) from the non-rodent data using a margin of safety approach. However, the Panel also noted
that epidemiological studies reported no increased cancer incidence at supplemental dose levels
varying from 6 to 15 mg/day for about 5–7 years (Druesne-Pecollo et al., 2010).
The ANS Panel concluded that the use of beta-carotene as a food colour is not a safety concern,
provided that the estimated combined intake from its use as a food additive and as a food supplement
is not more than the amount likely to be ingested as a result of the regular consumption of foods in
which it occurs naturally (5–10 mg/day). This would ensure that the exposure to beta-carotene from its
use as a food additive and a food supplement would remain below 15 mg/day, the level of
supplemental intake of beta-carotene for which epidemiological studies did not reveal any increased
cancer risk.
3.3.2. Assessment of consumer safety
In 1975 JECFA allocated a group ADI for carotenoids/xanthophylls of 0–5 mg/kg bw (JECFA, 1975).
However, as a consequence of the first observations on lung cancer in intervention studies with beta-
carotene, this ADI has been withdrawn by the SCF (EC, 2000). Following a review of new
information, the ANS Panel concluded that no ADI could be set for beta-carotene (EFSA, 2012a).
3.3.2.1. Consumer exposure
According to the approximate intake estimate by the SCF (EC, 2000), intake of beta-carotene and
related carotenoids as food additives is about 1–2 mg/day in addition to an average 2–5 mg/day
consumed through natural food sources. Consequently, the total intake was considered to be 3–
7 mg/day or up to 10 mg/day depending on seasonal and regional variations.
The ANS Panel (EFSA, 2012a), based on more current data, identified the range of beta-carotene
exposure from the diet as 1.1–3.9 mg/day for children aged 4–6 years and 1.4–5.6 mg/day for adults.
Although not clearly indicated, the data reported are considered to include beta-carotene from natural
sources only and not from its use as a food colour.
32 http://www.bfr.bund.de/cm/350/use_of_vitamins_in_foods.pdf
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 18
Overall, and taking into account important seasonal and regional variations, the combination of dietary
and supplemental sources would amount to an approximate beta-carotene intake of up to 7 mg/day,
and possibly higher. This conclusion is supported by the German National Consumption Study
(2008),33 showing that, whereas the median intake (P50) is slightly higher than 4 mg/day, the P90 is
higher than 8 mg/day in adult men and women and can be around 10.5 mg/day in women aged 35–64
years; the P95 is always above 10 mg/day and above 13 mg/day in women aged 25–64 years. Thus,
the margin between background intake and supplemental intake eliciting adverse effects in smokers
(≥ 20 mg/day) may be as low as three and even lower than two in high (P95) consumers. Accordingly,
the FEEDAP Panel considers that it should be assessed whether the use of supplemental beta-carotene
in animal feed may lead to a significant additional intake of beta-carotene.
The applicant provided a worst case scenario for consumer exposure based on literature data on beta-
carotene content in pig liver (0.487 mg/kg), milk (0.204 mg/L) and eggs (0.132 mg/kg), and applying
the scenario as set in Regulation (EC) No 429/2008. The results of this calculation suggest that the
contribution of beta-carotene from food of animal origin to the total intake is low (0.368 mg/day).
Hoppe et al. (1996) reported a study in which preruminant calves were fed a complete milk replacer
diet low in vitamin A and supplemented with beta-carotene doses of 0, 0.23, 0.46, 0.92, 1.84 or
3.68 µmol/kg bw for 28 days (corresponding to 0, 0.12, 0.25, 0.49, 0.99 and 1.98 mg/kg bw).
Accumulation in fat was low and not clearly related to the dose; accumulation in liver was remarkable
and linearly related to the dose, indicating that ~1.5 % of total intake was recovered in the liver. The
concentrations of beta-carotene in calf liver from the different treatments were < 0.1, 1.8, 3.6, 6.6, 14.3
and 40.5 mg/kg fresh tissue, respectively.
A 100-kg calf consuming 10 L of milk replacer (2.0 kg dry matter) supplemented with 100 mg/kg
beta-carotene will consume 2.0 mg/kg bw beta-carotene, which corresponds to the top
supplementation level of the study. Thus, with the top supplementation level, the consumption of 60 g
of calf liver could lead to an intake of 2.4 mg of beta-carotene, providing a significant addition to the
overall dietary intake. Halving the beta-carotene concentration in milk replacer to 50 mg/kg would
lead to a supplemental intake of 0.9 mg.
Calderón et al. (2007) reported data on the beta-carotene content of milk from cows fed various diets
with increasing beta-carotene content for six weeks (by gradually replacing hay with silage). The beta-
carotene content of feed was approximately 9, 37, 69 and 106 mg/kg dry matter. The corresponding
beta-carotene concentrations in milk were 0.09 mg/L for the first group and 0.13 mg/L for the other
groups. The authors concluded that there is a partial saturation process for the transfer of beta-carotene
from plasma to milk. Other sources (Ollilainen et al., 1989) give mean beta-carotene concentrations in
milk of about 0.2 mg/L. Accordingly, the beta-carotene intake from consumption of 1.5 L/day of
cow‘s milk (see guidance on consumer safety; EFSA, 2012c) would amount to around 0.3 mg/day; the
corresponding intake for toddlers consuming 1.05 L/day would be about 0.2 mg/day. Neither of these
figures indicate a significant contribution to the total beta-carotene intake of consumers from milk.
3.3.2.2. Triphenylphosphine oxide
No data are available on potential residues of TPPO in beta-carotene. The available assessments of
beta-carotene as a food additive by other scientific bodies (e.g. JECFA, 2011; EFSA, 2012a) did not
consider TPPO. Also, Regulation (EC) No 231/201234 does not list TPPO with a threshold value.
Exposure of target animals is low (see Table 3: 200 µg/day for calves, 600 µg/day for sows,
1400 µg/day for dairy cows), assuming 100 mg TPPO/kg additive. However, certain beta-carotene-
containing additives contain only 6–18 mg TPPO/kg, which would reduce the values to 6–18 % of
those given in Table 3. An accumulation in tissues and organs is not expected. The FEEDAP Panel
further considers that exposure of consumers to TPPO from the direct consumption of the same beta-
carotenes as food additives is considerably higher than any potential intake from food from beta-
33 http://www.was-esse-ich.de/uploads/media/NVSII_Abschlussbericht_Teil_2.pdf 34 OJ L 83 22.03.2012, p. 1.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 19
carotene-treated animals. Consequently, TPPO from beta-carotene used as a feed additive is not
considered a concern for consumer safety.
3.3.2.3. Conclusions on safety for the consumer
The FEEDAP Panel considers it prudent, in the absence of an ADI, that supplemental beta-carotene in
animal feed should not significantly add to consumer exposure from other sources.
The FEEDAP Panel considers that the use of supplemental beta-carotene in feeds of food-producing
animals, except veal calves, would not result in a significant additional exposure of consumers to beta-
carotene.
Consumption of liver from preruminant calves treated with beta-carotene could lead to a significant
additional exposure of the consumer. Although frequent calf liver consumption is limited, the
FEEDAP Panel concludes that unlimited use of beta-carotene as an additive to milk replacers may be
of concern to consumer safety.
3.4. Safety for the user
When handling the product the user/worker is exposed to the final form in which the additive is placed
on the market.
3.4.1. Effects on skin and eyes
From studies performed with beta-carotene obtained from Blakeslea trispora the product is to be
considered as not irritant to the eyes or skin of rabbits and not a skin sensitiser in the Buehler test
(JECFA, 2001). It is assumed that beta-carotene from different sources would not behave differently
with respect to direct effects on skin and/or mucosae.
Based on the information submitted on the composition of the additives containing beta-carotene the
FEEDAP Panel considers it unlikely that such formulations could cause skin/eye irritancy. The
FEEDAP Panel notes that a sensitisation risk associated with gelatin is reported in the scientific
literature; however, such findings are restricted to its use in vaccines (Pool et al., 2002; Saito et al.,
2005). Thus, the FEEDAP Panel considers it unlikely that skin sensitisation may arise in workers
exposed to gelatin present in beta-carotene used as a feed additive.
3.4.2. Exposure by inhalation and effects on the respiratory system
Exposure by inhalation was calculated for the two products for which dusting potential was provided
according to the procedure already applied in former opinions (EFSA, 2009b, 2010b) and described in
the guidance on studies concerning the safety of use of the additive for users/workers (EFSA, 2012d).
The details can be found in Appendix C.
For the additive with the smaller particle size (99 % (w/w) of particles < 15 µm) the daily exposure of
a user working in a premixture plant was calculated to be 31 mg of beta-carotene. For the coarser
product, daily exposure was calculated to be about 1 mg of beta-carotene.35 With the use of a P2 filter
mask, inhalatory exposure could be reduced to 2 mg and 0.1 mg of beta-carotene, respectively.
A risk characterisation of respiratory exposure contains some uncertainties as the only guidance value
for beta-carotene exposure refers to oral intake, in which probably not more than 30 % of the ingested
dose is released after absorption. In contrast, the inhaled dose is immediately available in the alveoli of
the lung, where its metabolism is unknown. However, the FEEDAP Panel assumes that the same dose
when inhaled is more toxic than when ingested.
35 Technical dossier/Supplementary information January 2012/Annex vii 4.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 20
3.4.3. Conclusions on safety for the user
Beta-carotene from chemical synthesis and from fermentation is not irritant to eyes or skin and is not a
skin sensitiser.
Only two additives could be assessed concerning the consequences of their dusting potential. For the
additive with the smaller particle size respiratory exposure was calculated to be 31 mg/day of beta-
carotene, which is considerably above the guidance value for oral intake (15 mg/day). In the absence
of any information on inhalation toxicity such exposure is considered potentially hazardous.
3.5. Safety for the environment
According to Regulation (EC) No 429/2008,36 an environmental risk assessment is not considered
necessary if the active ingredient of the feed additive is a natural/physiological substance, the use of
which will not alter the concentration or distribution in the environment. Beta-carotene occurs
abundantly in plants and the terrestrial environment. Taking into account the oxidative susceptibility
of carotenoids, the FEEDAP Panel considers it unlikely that the use of beta-carotene in animal
nutrition at the recommended feed concentrations would pose a risk to the environment.
4. Efficacy
According to Regulation (EC) No 429/2008 efficacy studies are not required for vitamins, provitamins
and chemically well-defined substances having similar effect already authorised as feed additives. It is
not considered necessary to demonstrate efficacy in specific studies when efficacy of the same
substance is generally accepted in the scientific literature, which is the case for beta-carotene.
Beta-carotene, both naturally occurring in feedingstuffs or from supplementation, is utilised by all
animal species except for cats. Its provitamin A function was demonstrated decades ago. The
conversion of beta-carotene to vitamin A under conventional feeding conditions between 8:1 and 12:1
(on a weight basis) depends on many nutritional and species-related factors. Consequently, a
requirement for dietary beta-carotene to replace vitamin A in its classical functions does not exist.
However, its potential role in reproduction and the immune response, which is discussed in more
detail in section 4.1, resulted in the recommendation of daily allowances (e.g. 200 mg beta-
carotene/day/dairy cow).
4.1. Reproduction and immunology
Beta-carotene appears to have positive effects on immune status and on the reproductive performance
of some animal species. Because beta-carotene accumulates in the corpora lutea it is assumed that
beta-carotene or its metabolites exerts a specific effect in the reproductive tissues and/or is required for
reproduction. However, published data are somewhat inconsistent, which is partially the result of
different study designs, experimental conditions, vitamin A supply/depletion, general reproductive
performance of the animals and route of beta-carotene application. It remains unclear whether the
potential positive effect of beta-carotene on reproductive performance can be traced back to the
provitamin A character of beta-carotene or to a specific beta-carotene effect (see Hurley and Doane,
1989).
The positive effects in cows or pigs reported in the literature include decreased service per conception,
increased number of viable embryos/reduced embryonic mortality, improved embryo quality,
improved immunity with reduced incidence of retained placenta and metritis, increased pregnancy
rate, increased percentage of milk fat (with unaffected milk yield), improved protection of the
mammary gland against infection as a result of increased intracellular killing of microbes by
phagocytes, and higher plasma progesterone and oestradiol levels in the cat (Brief and Chew, 1985;
Iwańska et al., 1985; Daniel et al., 1991a; Coffey and Britt, 1993; Preś et al., 1993; Chew et al., 2001;
36 OJ L 133, 22.5.2008, p. 1.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 21
Schweigert et al., 2002; Sales et al., 2008; Spears and Weiss 2008; de Ondarza et al., 2009).
Experimental evidence suggests that beta-carotene can serve as an alternative vitamin A source for the
in situ synthesis of retinoids in the mammalian embryo (Kim et al., 2011). A number of studies,
however, showed no effect of beta-carotene on reproduction (Folman et al., 1979; Bindas et al., 1984
a, b; Damron et al., 1984; Akordor et al., 1986; Wang et al., 1988; Oldham et al., 1991; Peltier et al.,
1997; Gossen and Hoedemaker, 2005) or even adverse effects (Folman et al., 1987). A recent study on
dairy cows supplemented with 1 g/day beta-carotene during the dry period showed no significant
effect on ovarian activity, although higher hydroxyproline and lower neutrophils in blood of
supplemented cows might suggest a positive effect on uterine function and inflammation (Kaewlamun
et al., 2011). Chew and co-workers reported that beta-carotene improves the immune status and
decreases the incidence of reproductive disorders in peripartum cows by increasing lymphocyte and
phagocyte function (Tjoelker et al., 1988a, b; Tjoelker et al., 1990; Daniel et al., 1991a, b; Chew,
1993b; Michal et al., 1994). Beta-carotene appears to improve cellular and humoral immunity in dogs
(Chew et al., 2000b,c; see also reviews by Chew, 1995; Chew and Park, 2004).
4.2. Conclusions on efficacy
Beta-carotene can be utilised for the synthesis of retinol in almost all animal species and is therefore
considered as a provitamin A. It is not a vitamin A source for cats as beta-carotene—although
absorbed—cannot be converted into retinol.
The data on the effects of supplemented beta-carotene on immunity and reproduction remain
inconsistent and no conclusions can be drawn.
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 Regulation37 and Good
Manufacturing Practice.
CONCLUSIONS AND RECOMMENDATIONS
CONCLUSIONS
The FEEDAP Panel concludes that the use of beta-carotene is safe for the target animals. Setting a
maximum content in feed legislation is not considered necessary. However, this conclusion assumes
that TPPO does not exceed 100 mg/kg additive.
In all food-producing animals (except veal calves) and laboratory rodents, beta-carotene is almost fully
metabolised in the enterocytes. In contrast, humans, non-human primates and ferrets absorb relatively
high quantities of beta-carotene unchanged. Consequently, toxicological data resulting from studies
with laboratory rodents cannot be used for conclusions on consumer safety. Investigations with ferrets
and hamsters as well as intervention studies in humans indicate a dose-dependent potential of beta-
carotene to promote lung carcinomas, particularly in smokers (and asbestos workers).
The FEEDAP Panel considers it prudent, in the absence of an ADI, that supplemental beta-carotene in
animal feed should not significantly add to consumer exposure from other sources.
The FEEDAP Panel considers that the use of supplemental beta-carotene in feeds of food-producing
animals, except veal calves, would not result in a significant additional exposure of consumers to beta-
carotene. Consumption of liver from preruminant calves treated with beta-carotene could lead to a
significant additional exposure of the consumer. Although frequent calf liver consumption is limited,
the FEEDAP Panel concludes that unlimited use of beta-carotene as an additive to milk replacers may
be of concern to consumer safety.
37 Regulation (EC) No 183/2005 of the European Parliament and of the Council of 12 January 2005 laying down
requirements for feed hygiene. OJ L 35, 8.2.2005, p. 1.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 22
Beta-carotene from chemical synthesis and from fermentation is not an irritant to eyes or skin and is
not a skin sensitiser. For one additive the respiratory exposure of users was calculated to be
considerably above the guidance value for oral intake (15 mg/person/day). In the absence of any
information on inhalation toxicity such exposure is considered potentially hazardous.
Taking the widespread occurrence of beta-carotene in nature and its oxidative susceptibility into
account, the FEEDAP Panel considers it unlikely that the use of beta-carotene in animal nutrition at
the recommended feed concentrations would pose a risk to the environment.
The FEEDAP Panel concludes that beta-carotene is utilised for the synthesis of retinol in almost all
animal species except the cat. Effects on reproduction and immunity are not sufficiently demonstrated.
RECOMMENDATIONS
Beta-carotene should be authorised only in stabilised forms.
Specifications for beta-carotene should be in accordance with Commission Regulation (EC) No
231/2012.
The maximum content of TPPO should be set at 100 mg/kg additive (as in Commission Regulation
(EC) No 393/200838).
The introduction of a maximum content of 50 mg beta-carotene/kg milk replacer is recommended to
limit liver content.
Because handling of beta-carotene additives is considered a hazard for users, its use should be
restricted to premixtures (premixture operations).
Consequently, any use in water for drinking should be avoided as effective protection for the user
cannot be guaranteed at the farm level. In addition, the FEEDAP Panel notes that no data have been
provided by the applicant to demonstrate stability and homogeneous distribution of an additive under
practical conditions.
Consumers are exposed to multiple sources of TPPO. The FEEDAP Panel considers that a full safety
assessment of this substance is desirable.
REMARK
Beta-carotene is a striking example of the difficulties and contradictions arising from the assessment
of a generic substance based on a dossier that mixes data on the active substance with data on the
additive. In this case, the applicant has stressed the fact that additives are not the subject of the
authorisation and consequently declined to supply data on the additive. The FEEDAP Panel notes that
some subjects of the safety assessment (safety for the target animal, as influenced by stability and
homogeneous distribution, and safety for the user) can be assessed only on the basis of the additive.
As a consequence, the assessment is restricted to some examples and does not cover all products
entering the market. On the other hand, concerns over user safety for a single additive might not
prevent the additive entering the market as the authorisation is for the active substance. A
generalisation of the conclusions of the assessment is more valid the broader the basis for the
assessment. This means that the larger the number and variety of examples of additives provided, the
higher the probability that the assessment is valid for the generic compound.
38 Commission Regulation (EC) No 393/2008 of 30 April 2008 concerning the authorisation of astaxanthin dimethylsuccinate
as a feed additive. OJ L 117, 1.4.2008, p. 20.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 23
DOCUMENTATION PROVIDED TO EFSA
1. Beta-carotene for all animal species and categories. October 2009. Submitted by Vitamin
Authorisation Consortium European Economic Interest Grouping (VITAC EEIG).
2. Beta-carotene for all animal species and categories. Supplementary information. January 2011.
Submitted by Vitamin Authorisation Consortium European Economic Interest Grouping (VITAC
EEIG).
3. Beta-carotene for all animal species and categories. Supplementary information. January 2012.
Submitted by Vitamin Authorisation Consortium European Economic Interest Grouping (VITAC
EEIG).
4. Beta-carotene for all animal species and categories. Supplementary information. April 2012.
Submitted by Vitamin Authorisation Consortium European Economic Interest Grouping (VITAC
EEIG).
5. Evaluation report of the European Union Reference Laboratory for Feed Additives on the
methods(s) of analysis for beta-carotene.
6. Comments from Member States received through ScienceNet.
REFERENCES
Akordor FY, Stone JB, Walton JS, Leslie KE and Buchanan-Smith JG, 1986. Reproductive
performance of lactating Holstein cows fed supplemental beta-carotene. Journal of Dairy Science,
69, 2173–2178.
Al-Khalifa AS and Simpson KL, 1988. Metabolism of astaxanthin in the rainbow trout (Salmo
gairdneri). Comparative Biochemistry and Physiology, 91B, 563–568.
Arikan S and Rodway RG, 2000. Effects of high density lipoprotein containing high or low beta-
carotene concentrations on progesterone production and beta-carotene uptake and depletion by
bovine luteal cells. Animal Reproduction Science, 62, 253–263.
Arikan S and Rodway RG, 2001. Seasonal variation in bovine luteal concentrations of beta-carotene.
Turkish Journal of Veterinary and Animal Sciences, 25, 165–168.
Bachmann H, Desbarats A, Pattison P, Sedgewick M, Riss G, Wyss A, Cardinault N, Duszka C,
Goralczyk R and Grolier P, 2002. Feedback regulation of beta, beta-carotene 15,15'-
monooxygenase by retinoic acid in rats and chickens. Journal of Nutrition, 132, 3616–3622.
Barua AB and Olson JA, 2000. Beta-carotene is converted primarily to retinoids in rats in vivo.
Journal of Nutrition, 130, 1996–2001.
Ben-Amotz A, Mokady S, Edelstein S and Avron M, 1989. Bioavailability of a natural isomer mixture
as compared with synthetic all-trans-beta-carotene in rats and chicks. Journal of Nutrition, 119,
1013–1019.
Bierer TL, Merchen NR and Erdman JW Jr, 1995. Comparative absorption and transport of five
common carotenoids in preruminant calves. Journal of Nutrition, 125, 1569–1577.
Bindas EM, Gwazdauskas FC, Aiello RJ, Herbein JH, McGilliard ML and Polan CE, 1984a.
Reproductive and metabolic characteristics of dairy cattle supplemented with beta-carotene.
Journal of Dairy Science, 67, 1249–1255.
Bindas EM, Gwazdauskas FC, McGilliard ML and Polan CE, 1984b. Progesterone responses to
human chorionic gonadotropin in dairy cattle supplemented with beta-carotene. Journal of Dairy
Science, 67, 2978–2985.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 24
Blomstrand R and Werner B, 1967. Studies on the intestinal absorption of radioactive -carotene and
vitamin A in man. Conversion of -carotene into vitamin A. Scandinavian Journal of Clinical and
Laboratory Investigation, 19, 339–45.
Brief S and Chew BP, 1985. Effects of vitamin A and beta-carotene on reproductive performance in
gilts. Journal of Animal Science, 60, 998–1004.
Calderón F, Chauveau-Duriot B, Pradel P, Martin B, Graulet B, Doreau M and Nozière P, 2007.
Variations in carotenoids, vitamins A and E, and color in cow‘s plasma and milk following a shift
from hay diet to diets containing increasing levels of carotenoids and vitamin E. Journal of Dairy
Science, 90, 5651–5664.
Canfield LM, Giuliano AR, Neilson EM, Yap HH, Graver EJ, Cui HA and Blashill BM, 1997. Beta-
carotene in breast milk and serum is increased after a single beta-carotene dose. American Journal
of Clinical Nutrition, 66, 52–61.
Chew BP, Holpuch DM and O‘Fallon JV, 1984. Vitamin A and beta-carotene in bovine and porcine
plasma, liver, corpora lutea, and follicular fluid. Journal of Dairy Science, 67, 1316–1322.
Chew BP, Wong TS, Michal JJ, Standaert FE and Heirman LR, 1991. Kinetic characteristics of beta-
carotene uptake after an injection of beta-carotene in pigs. Journal of Animal Science, 69, 4883–
4891.
Chew BP, 1993a. Effects of supplemental beta-carotene and vitamin A on reproduction in swine.
Journal of Animal Science, 71, 247–252.
Chew BP, 1993b. Role of carotenoids in the immune response. Journal of Dairy Science, 76, 2804–
2811.
Chew BP, 1995. Antioxidant vitamins affect food animal immunity and health. Journal of Nutrition,
125(6 Suppl), 1804S–1808S.
Chew BP, Park JS, Weng BC, Wong TS, Hayek MG and Reinhart GA, 2000a. Dietary beta-carotene
absorption by blood plasma and leukocytes in domestic cats. Journal of Nutrition, 130, 2322–2325.
Chew BP, Park JS, Weng BC, Wong TS, Hayek MG and Reinhart GA, 2000b. Dietary beta-carotene
is taken up by blood plasma and leukocytes in dogs. Journal of Nutrition, 130, 1788–1791.
Chew BP, Park JS, Wong TS, Kim HW, Weng BB, Byrne KM, Hayek MG and Reinhart GA, 2000c.
Dietary beta-carotene stimulates cell-mediated and humoral immune response in dogs. Journal of
Nutrition, 130, 1910–1903
Chew BP, Weng BB, Kim HW, Wong TS, Park JS and Lepine AJ, 2001. Uptake of beta-carotene by
ovarian and uterine tissues and effects on steroidogenesis during the estrous cycle in cats.
American Journal of Veterinary Research, 62, 1063–1067.
Chew BP and Park JS, 2004. Carotenoid action on the immune response. Journal of Nutrition, 134,
257S–261S.
Christiansen, R, Lie Ø and Torrisen OJ, 1994. Effect of astaxanthin and vitamin A on growth and
survival during first feeding of Atlantic salmon, Salmo salar L. Aquaculture and Fisheries
Management, 25, 903–914.
Coffey MT and Britt JH, 1993. Enhancement of sow reproductive performance by beta-carotene or
vitamin A. Journal of Animal Science, 71, 1198–1202.
Damron BL, Goodson SR, Harms RH, Janky DM and Wilson HR, 1984. Beta-carotene
supplementation of laying hen diets. British Poultry Science, 25, 349–352.
Daniel LR, Chew BP, Tanaka TS and Tjoelker LW, 1991a. Beta-carotene and vitamin A effects on
bovine phagocyte function in vitro during the peripartum period. Journal of Dairy Science, 74,
124–131.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 25
Daniel LR, Chew BP, Tanaka TS and Tjoelker LW, 1991b. In vitro effects of beta-carotene and
vitamin A on peripartum bovine peripheral blood mononuclear cell proliferation. Journal of Dairy
Science, 74, 911–915.
Deming DM and Erdman JW Jr, 1999. Mammalian carotenoid absorption and metabolism. Pure and
Applied Chemistry, 71, 2213–2223.
Deming DM, Boileau AC, Lee CM and Erdman JW Jr, 1999. Amount of dietary fat and type of
dietary fiber affect beta-carotene bioavailability in the Mongolian gerbil. FASEB Journal, 13,
A553.
Deming DM, Boileau AC, Lee CM and Erdman JW Jr, 2000. Amount of dietary fat and type of
soluble fiber independently modulate postabsorptive conversion of beta-carotene to vitamin A in
Mongolian gerbils. Journal of Nutrition, 130, 2789–2796.
de Ondarza MB, Wilson JW and Engstrom M, 2009. Case study: effect of supplemental -carotene on
yield of milk and milk components and on reproduction of dairy cows. Professional Animal
Scientist, 25, 510–516.
Druesne-Pecollo N, Latino-Martel P, Norat T, Barrandon E, Bertrais S, Galan P and Hercberg S, 2010.
Beta-carotene supplementation and cancer risk: a systematic review and metaanalysis of
randomized controlled trials. International Journal of Cancer, 127, 172–184.
EC (European Commission), 2000, online. Opinion of the Scientific Committee on Food on the
tolerable upper intake level of beta-carotene. Available from
http://ec.europa.eu/food/fs/sc/scf/out80b_en.pdf
EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2009a. Scientific opinion on the
substantiation of health claims related to beta carotene and physiological immune responses of the
skin in relation to UV radiation (sun exposure) (ID 198, 1463) pursuant to Article 13(1) of
Regulation (EC) No 1924/2006. The EFSA Journal, 7(9):1231, 14 pp.
EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2009b.
Scientific opinion on the safety and efficacy of 25-hydroxycholecalciferol as a feed additive for all
animal species. The EFSA Journal, 696, 1–32.
EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2010a. Scientific Opinion on the
substantiation of health claims related to vitamin A (including -carotene) and maintenance of
normal vision (ID 4239, 4701), maintenance of normal skin and mucous membranes (ID 4660,
4702), and maintenance of normal hair (ID 4660) pursuant to Article 13(1) of Regulation (EC) No
1924/2006. EFSA Journal, 8(10):1754, 13 pp.
EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2010b.
Scientific Opinion on the safety and efficacy of Maxiban® G160 (narasin and nicarbazin) for
chickens for fattening. EFSA Journal, 8(4):1574, 45 pp.
EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2011a. Scientific Opinion on the
substantiation of health claims related to beta carotene and protection of DNA, proteins and lipids
from oxidative damage (ID 19, 197, 1262, 1460), protection of the skin from UV-induced
(including photo-oxidative) damage (ID 178, 197, 1263, 1461, 1968, 2320) and maintenance of the
normal function of the immune system (ID 200, 1462) pursuant to Article 13(1) of Regulation (EC)
No 1924/2006. EFSA Journal, 9(4):2021, 22 pp.
EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2011b. Scientific Opinion on the
substantiation of health claims related to a combination of lycopene, proanthocyanidins, vitamin C,
vitamin E, selenium and beta-carotene and contribution to normal collagen formation (ID 1669)
and protection of the skin from UV-induced damage (ID 1669) pursuant to Article 13(1) of
Regulation (EC) No 1924/2006. EFSA Journal, 9(6):2239, 15 pp.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 26
EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), 2012a. Scientific Opinion
on the re-evaluation of mixed carotenes (E 160a (i)) and -carotene (E 160a (ii)) as a food additive.
EFSA Journal, 10(3):2593, 67 pp.
EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2012b
Guidance for the preparation of dossiers for sensory additives. EFSA Journal, 10(1):2534, 26 pp.
EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2012c.
Guidance for establishing the safety of additives for the consumer. EFSA Journal, 10(1):2537, 12
pp.
EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2012d.
Guidance on studies concerning the safety of use of the additive for users/worker. EFSA Journal,
10(1):2539, 5 pp.
Erdman JW Jr, Fahey GC Jr and White CB, 1986. Effects of purified dietary fiber sources on beta-
carotene utilization by the chick. Journal of Nutrition, 116, 2415–2423.
Eroglu A, Hruszkewycz DP, Dela Sena C, Narayanasamy S, Riedl KM, Kopec RE, Schwartz SJ,
Curley RW Jr and Harrison EH, 2012. Naturally-occurring eccentric cleavage products of
provitamin A -carotene function as antagonists of retinoic acid receptors. Journal of Biological
Chemistry, 287, 15886–15895.
European Pharmacopoeia (PhEur), 2010. Beta-carotene, Monograph (MG) 1069, 7th edition. Council
of Europe (COE)—European Directorate for the Quality of Medicines, Strasbourg, France.
Folman Y, Ascarelli I, Herz Z, Rosenberg M, Davidson M and Halevi A, 1979. Fertility of dairy
heifers given a commercial diet free of beta-carotene. British Journal of Nutrition, 41, 353–359.
Folman Y, Ascarelli I, Kraus D and Barash H, 1987. Adverse effects of -carotene in diet on fertility
of dairy cows. Journal of Dairy Science, 70, 357–366.
Goodman DS, Blomstrand R, Werner B, Huang HS and Shiratori T, 1966a. The intestinal absorption
and metabolism of vitamin A and -carotene in man. Journal of Clinical Investigation, 45, 1615–
1623.
Goodman DS, Huang HS and Shiratori T, 1966b. Mechanism of the biosynthesis of vitamin A from
beta-carotene. Journal of Biological Chemistry, 241, 1929–1932.
Gossen N and Hoedemaker M, 2005. Effect of beta-carotin serum concentration on the reproductive
performance in dairy cows. Berliner und Münchener Tierarztliche Wochenschrift, 118, 326–333.
Gugger ET, Bierer TL, Henze TM, White WS and Erdman JW Jr, 1992. Beta-carotene uptake and
tissue distribution in ferrets (Mustela putorius furo). Journal of Nutrition, 122, 115–119.
Hollander D and Ruble PE Jr, 1978. Beta-carotene intestinal absorption: bile, fatty acid, pH, and flow
rate effects on transport. American Journal of Physiology, 235, E686–691.
Hoppe PP, Chew BP, Safer A, Stegemann I and Biesalski HK, 1996. Dietary beta-carotene elevates
plasma steady-state and tissue concentrations of -carotene and enhances vitamin A balance in
preruminant calves. Journal of Nutrition, 126, 202–208.
Huang HS and Goodman DWS, 1965. Vitamin A and carotenoids. I. Intestinal absorption and
metabolism of 14C-labeled vitamin A alcohol and -carotene in the rat. Journal of Biological
Chemistry, 240, 2839–2844.
Hurley WL and Doane RM, 1989. Recent developments in the roles of vitamins and minerals in
reproduction. Journal of Dairy Science, 72, 784–804.
Iwańska S, Lewicki C and Rybicka M, 1985. The effect of beta-carotene supplementation on the beta-
carotene and vitamin A levels of blood plasma and some fertility indices of dairy cows. Archiv für
Tierernhrüng, 35, 563–570.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 27
Jansen HD, 1992. Mischtechnik im Futtermittelbetrieb. Anforderungen an Mischenlage, Arbeits- und
Mischgenauigkeit. Die Mühle+ Mischfuttertechnik, 129, 265–270.
JECFA, 1975. Joint WHO/FAO Expert Committee on Food Additives. Toxicological evaluation of
some food colours, enzymes, flavour enhancers, thickening agents and certain food additives.
WHO Food Additives Series, 6. WHO Press, Geneva.
JECFA, 2001. Joint WHO/FAO Expert Committee on Food Additives. Safety evaluation of certain
food additives and contaminants. WHO Food Additives Series, 48. WHO Press, Geneva.
JECFA, 2011. Evaluation of certain food additives and contaminants. Seventy-third report of the Joint
FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series, no 960. Geneva,
8–17 June 2010. WHO Press, Geneva.
Johnson EJ and Russell RM, 1992. Distribution of orally administered beta-carotene among
lipoproteins in healthy men. American Journal of Clinical Nutrition, 56, 128–135.
Johnson EJ, Suter PM, Sahyoun N, Ribaya-Mercado JD and Russell RM, 1995. Relation between
beta-carotene intake and plasma and adipose tissue concentrations of carotenoids and retinoids.
American Journal of Clinical Nutrition, 62, 598–603.
Kaewlamun W, Okouyi M, Humblot P, Techakumphu M and Ponter AA, 2011. Does supplementing
dairy cows with β-carotene during the dry period affect postpartum ovarian activity, progesterone,
and cervical and uterine involution? Theriogenology, 75, 1029–1038.
Katsuyama M and Matsuno T, 1988. Carotenoid and vitamin A, and metabolism of carotenoids, -
carotene, canthaxanthin, astaxanthin, zeaxanthin, lutein and tunaxanthin in tilapia tilapia nilotica.
Comparative Biochemistry and Physiology, 90B, 131–139.
Kim YK, Wassef L, Chung S, Jiang H, Wyss A, Blaner WS and Quadro L, 2011. Beta-carotene and its
cleavage enzyme beta-carotene-15,15 -oxygenase (CMOI) affect retinoid metabolism in developing
tissues. FASEB Journal, 25, 1641–1652.
Kirsche B, Schlenzig M, Ochrimenko WI and Flachowsky G, 1987. Influence of beta-carotene
supplementation on carotene content of ovaries of heifers. Archiv für Tierernhrüng, 37, 995–9.
Krinsky NL, Mathews-Roth MM, Welankiwar S, Sehgal PK, Lausen NCG and Russett M, 1990. The
metabolism of [14C] -carotene and the presence of other carotenoids in rats and monkeys. Journal
of Nutrition, 120, 81–87.
Lee CM, Lederman JD, Hofmann NE and Erdman JW, 1998. The mongolian gerbil (Meriones
unguiculatus) is an appropriate animal model for evaluation of the conversion of beta-carotene to
vitamin A. Journal of Nutrition, 128, 280–286.
Matsuno T, 1991 Xanthophylls as precursors of retinoids. Pure and Applied Chemistry, 63, 81–88.
Michal JJ, Heirman LR, Wong TS, Chew BP, Frigg M and Volker L, 1994. Modulatory effects of
dietary beta-carotene on blood and mammary leukocyte function in periparturient dairy cows.
Journal of Dairy Science, 77, 1408–1421.
Mokady S, Avron M and Ben-Amotz A, 1990 Accumulation in chick livers of 9-cis versus all-trans
beta-carotene. Journal of Nutrition, 120, 889–892.
Moren M, Naess T and Hamre K, 2004. Conversion of beta-carotene, canthaxanthin and astaxanthin to
vitamin A in Atlantic halibut (Hippoglossus hippoglossus L.) juveniles. Fish Physiology and
Biochemistry, 27, 71–80.
Novotny JA, Dueker SR, Zech LA and Clifford AJ, 1995, Compartmental analysis of the dynamics of
beta-carotene metabolism in an adult volunteer. Journal of Lipid Research, 36, 1825–1838.
Oldham, ER, Eberhart RJ and Muller LD, 1991. Effects of supplemental vitamin A or -carotene
during the dry period and early lactation on udder health. Journal of Dairy Science, 74, 377–381.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 28
Ollilainen V, Heinonen M, Linkola E, Varo P and Koivistoinen P, 1989. Carotenoids and retinoids in
Finnish foods: dairy products and eggs. Journal of Dairy Science, 72, 2257–2265.
Parker RS, 1988. Carotenoid and tocopherol composition of human adipose tissue. American Journal
of Clinical Nutrition, 47, 33–36.
Parker RS, 1996. Absorption, metabolism, and transport of carotenoids. FASEB Journal, 10, 542–551.
Parker RS, Brenna JT, Swanson JE, Goodman KJ and Marmor B, 1997. Assessing metabolism of
beta-[13C]carotene using high-precision isotope ratio mass spectrometry. Methods in Enzymology,
282, 130–140.
Parker RS, Swanson JE, You CS, Edwards AJ and Huang T, 1999. Bioavailability of carotenoids in
human subjects. Proceedings of the Nutrition Society, 58, 155–162.
Peltier MM, Peltier MR, Sharp DC and Ott EA, 1997. Effect of beta-carotene administration on
reproductive function of horse and pony mares. Theriogenology, 48, 893–906.
Pollack J, Campbell JM, Potter SM and Erdman JW Jr, 1994. Mongolian gerbils (Meriones
unguiculatus) absorb beta-carotene intact from a test meal. Journal of Nutrition, 124, 869–873.
Pool V, Braun MM, Kelso JM, Mootrey G, Chen RT, Yunginger JW, Jacobson RM and Gargiullo
PM; VAERS Team. US Vaccine Adverse Event Reporting System, 2002. Prevalence of anti-
gelatin IgE antibodies in people with anaphylaxis after measles-mumps rubella vaccine in the
United States. Pediatrics, 110, e71.
Poor CL, Miller SD, Fahey GC Jr, Easter RA and Erdman JW Jr, 1987. Animal models for carotenoid
utilization studies: evaluation of the chick and the pig. Nutrition Reports International, 36, 229–
234.
Poor CL, Bierer TL, Merchen NR, Fahey CC Jr and Erdman JW Jr, 1992 Evaluation of the pre-
ruminant calf for the study of human carotenoid metabolism. Journal of Nutrition, 122, 262–268.
Poor CL, Bierer TL, Merchen NR, Fahey GC Jr and Erdman JW Jr, 1993. The accumulation of alpha-
and beta-carotene in serum and tissues of preruminant calves fed raw and steamed carrot slurries.
Journal of Nutrition, 123, 1296–1304.
Poston HA, Riis RC, Rumsey GL and Ketola HG, 1977. The effect of supplemental dietary amino
acids, minerals and vitamins on salmonids fed cataractogenic diets. The Cornell Veterinarian, 67,
472–509.
Preś J, Fuchs B and Schleicher A, 1993. The effect of carotene and vitamins A and E supplementation
on reproduction of sows. Archivum Veterinarium Polonicum, 33, 55–64.
Ribaya-Mercado JD, Holmgren SC, Fox JG and Russell RM, 1989. Dietary beta-carotene absorption
and metabolism in ferrets and rats. Journal of Nutrition, 119, 665–668.
Ribaya-Mercado JD, Fox JG, Rosenblad WD, Blanco MC and Russell RM, 1992. Beta-carotene,
retinol and retinyl ester concentrations in serum and selected tissues of ferrets fed beta-carotene.
Journal of Nutrition, 122, 1898–1903.
Rock CL and Swendseid ME, 1992. Plasma beta-carotene response in humans after meals
supplemented with dietary pectin. American Journal of Nutrition, 55, 96–99.
Saito A, Kumagai T, Kojima H, Terai I, Yamanaka T, Wataya Y, Umetsu M, Umetsu A and Yano SA,
2005. Sero-epidemiological survey of gelatin sensitization in young Japanese children during the
1979–1996 period. Scandinavian Journal of Immunology, 61, 376–379.
Sales JN, Dias LM, Viveiros AT, Pereira MN and Souza JC, 2008. Embryo production and quality of
Holstein heifers and cows supplemented with beta-carotene and tocopherol. Animal Reproduction
Science, 106, 77–89.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 29
Schiedt K, Leuenberger FJ, Vecchi M and Glinz E, 1985. Absorption, retention and metabolic
transformation of carotenoids in rainbow trout, salmon and chicken. Pure and Applied Chemistry,
57, 685–692.
Schmitz HH, Poor CL, Wellman RB and Erdman JW Jr, 1991. Concentrations of selected carotenoids
and vitamin A in human liver, kidney and lung tissue. Journal of Nutrition, 121, 1613–1621.
Schweigert FJ, Buchholz I, Schuhmacher A and Gropp J, 2001 Effect of dietary beta-carotene on the
accumulation of beta-carotene and vitamin A in plasma and tissues of gilts. Reproduction Nutrition
Development, 41, 47–55.
Schweigert FJ, Raila J, Wichert B and Kienzle E, 2002. Cats absorb beta-carotene, but it is not
converted to vitamin A. Journal of Nutrition, 132, 1610S–1612S.
Schweigert FJ, 2003. Research note: changes in the concentration of beta-carotene, alpha-tocopherol
and retinol in the bovine corpus luteum during the ovarian cycle. Archiv für Tierernährung, 57,
307–310.
Slifka KA, Bowen PE, Stacewicz-Sapontzakis M and Crissey SD, 1999. A survey of serum and
dietary carotenoids in captive wild animals. Journal of Nutrition, 129, 380–390.
Spears JW and Weiss WP, 2008. Role of antioxidants and trace elements in health and immunity of
transition dairy cows. Veterinary Journal, 176, 70–76.
Thatcher AJ, Lee CM and Erdman JW Jr, 1998. Tissue stores of beta-carotene are not conserved for
later use as a source of vitamin A during compromised vitamin A status in mongolian gerbils
(Meriones unguiculatus). Journal of Nutrition, 128, 1179–1185.
Tjoelker LW, Chew BP, Tanaka TS and Daniel LR, 1988a. Bovine vitamin A and beta-carotene intake
and lactational status. 1. Responsiveness of peripheral blood polymor-phonuclear leukocytes to
vitamin A and beta-carotene challenge in vitro. Journal of Dairy Science, 71, 3112–3119.
Tjoelker LW, Chew BP, Tanaka TS and Daniel LR, 1988b. Bovine vitamin A and beta-carotene intake
and lactational status. 2. Responsiveness of mitogen-stimulated peripheral blood lymphocytes to
vitamin A and beta-carotene challenge in vitro. Journal of Dairy Science, 71, 3120–3127.
Tjoelker LW, Chew BP, Tanaka TS and Daniel LR, 1990. Effect of dietary vitamin A and beta-
carotene on polymorphonuclear leukocyte and lymphocyte function in dairy cows during the early
dry period. Journal of Dairy Science, 73, 1017–1022.
Ullrey DE, 1972. Biological availability of fat-soluble vitamins: vitamin A and carotene. Journal of
Animal Science, 35, 648–657.
Van Vliet T, Schreurs WHP and van den Berg H, 1995. Intestinal β-carotene absorption and cleavage
in men: response of β-carotene and retinyl esters in the triglyceride-rich lipoprotein fraction after a
single oral dose of β-carotene. American Journal of Nutrition, 62, 110–116.
Wang JY, Owen FG and Larson LL, 1988. Effect of beta-carotene supplementation on reproductive
performance of lactating Holstein cows. Journal of Dairy Science, 71, 181–186.
Wang XD, Krinsky NI, Marini RP, Tang G, Yu J, Hurley R, Fox JG and Russell RM, 1992. Intestinal
uptake and lymphatic absorption of β-carotene in ferrets: a model for human β-carotene
metabolism. American Journal of Physiology, 263, G480–G486.
Weng BC, Chew BP, Wong TS, Park JS, Kim HW and Lepine AJ, 2000. Beta-carotene uptake and
changes in ovarian steroids and uterine proteins during the estrous cycle in the canine. Journal of
Animal Science, 78, 1284–1290.
White DA, Ornsrud R and Davies SJ, 2003. Determination of carotenoid and vitamin A concentrations
in everted salmonid intestine following exposure to solutions of carotenoid in vitro. Comparative
Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 136, 683–692.
White WS, Peck KM, Ulman EA, Erdman JW Jr, 1993. The ferret as a model for evaluation of the
bioavailabilities of all-trans-beta-carotene and its isomers. Journal of Nutrition, 123, 1129–1139.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 30
Yap SC, Choo YM, Hew NF and Goh SH, 1997. Distribution of dietary palm carotenes and their
metabolites in the rabbit. Nutrition Research, 17, 1721–1731.
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 31
APPENDICES
APPENDIX A
Executive Summary of the Evaluation Report of the European Union Reference Laboratory for
Feed Additives on the Method(s) of Analysis for beta-carotene39
In the current application authorisation is sought for Beta-Carotene 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 Beta-Carotene for all animal species
and categories. The active substance and the feed additive is Beta-Carotene, which is a crystalline
powder with a purity of at least 96%. It is produced by chemical synthesis and by fermentation from a
strain of Blakeslea trispora. It is intended to be used in premixtures, feedingstuffs and water as a
formulated product. According to the applicant typical formulations contain a minimum of 10% Beta-
Carotene. The applicant does not propose any minimum or maximum concentration of the feed
additive in feedingstuffs or water.
For the determination of the purity of Beta-Carotene (i.e. the percentage mass fraction of Beta-
Carotene in the feed additive), the applicant proposed the European Pharmacopoeia method
(Ph.Eur.3rd, monograph 1069). The EURL-FA considers this method suitable to be used within the
frame of official control.
For the determination of Beta-Carotene in premixtures and feedingstuffs the applicant proposed a High
Performance Liquid Chromatography (HPLC) method, tested for premixtures and feedingstuffs with
concentration ranging from 100 to 2000 mg/kg for premixtures and from 10 to 100 mg/kg in
feedingstuffs. The following performance characteristics derived from a three-laboratories
intercomparison study were reported by the applicant for premixtures and feedingstuffs:
- a recovery rate of circa 100%,
- a repeatability relative standard deviation (RSDr) ranging from 2.9 to 8.9 %,
- a reproducibility relative standard deviation (RSDR) ranging from 3 to 11.5 %, and
- a limit of detection (LOD) of 0.05 mg/kg for feedingstuffs.
Based on these acceptable performance characteristics the CRL considers this method suitable for the
determination of Beta-Carotene in premixtures and feedingstuffs within the concentration range
covered by the collaborative study. Therefore the EURL recommends for
official control the HPLC method submitted by the applicant, for the determination of Beta-Carotene
in premixtures and feedingstuffs.
For the determination of Beta-Carotene in water the applicant did not submit any analytical method
nor experimental data, therefore the EURL cannot evaluate nor recommend any method for the
determination of the active substance in this matrix.
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.
39 The full report is available on the EURL website: http://irmm.jrc.ec.europa.eu/SiteCollectionDocuments/FinRep-FAD-
2009-0046.pdf
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 32
APPENDIX B
Figure: Proposed pathways from beta-carotene to retinoic acid in humans and animals. Central
cleavage (left side) leads mainly to retinol and retinyl esters. Eccentric cleavage (right side) catalysed
by the enzyme , -carotene 9 ,10 -dioxygenase yields apo-10 -carotenal and beta-ionone. The apo-
carotenals are subsequently oxidised to the apo-carotenoic acids and further shortened to retinoic acid
(Bachmann et al., 2002)
Retinal beta-Apo-8 -, 10 - or 12 -carotenals
Retinol Retinyl esters beta-Apo-10 -carotenoic acid
beta-Apo-12 -carotenoic acid
Storage
Retinol
Retinal
Retinoic acid
Retinal oxidase
β,β-Carotene 15,15 -monooxygenase
Beta-carotene for all animal species
EFSA Journal 2012;10(6):2737 33
APPENDIX C
POTENTIAL EXPOSURE OF USERS HANDLING BETA-CAROTENE
There are different operations in a premixture factory during which the worker could be exposed to
dust:
– taking the additive from its bag for weighing in the dispensary
– emptying bags of previously weighed material in the hopper or mixers
– packing the final premixture.
Default values/positions:
– a factory with a large throughput can prepare 40 premixture batches per day (8 hours per shift)
– the maximum time for weighing/emptying is 20 seconds
– total breathed air per worker of 10 m3 per 8 hours = 1.25 m3 per hour
– of premixtures that contain the additive: 100 %
– dusting potential measured: case 1, 0.57 g/m3; case 2, 0.21 g/m3
– concentration of the active substance in dust: case 1: not measured, assumption 10 % as in the
additive because of high amount of fine particles (99 % < 15.2 µm); case 2: 1 %.
ESTIMATE OF RISK MITIGATION
– Estimated reduction (%) of exposure due to the use of personal protection equipment
(coverall, goggles, gloves and masks of the type P2 or P3).
CALCULATION OF EXPOSURE BY INHALATION DURING A WORKING DAY
Batches with potential exposure 40 (batches) × 1 (fraction of batches containing additive) = 40
(batches)
Time of exposure 40 × 20 seconds = 800 seconds
An uncertainty factor of 2 should be introduced
Inhaled air during exposure (Ia), m3 1.25 m3 per hour × 2 × 800/60/60 in hours = 0.55
Active substance in air (Asa), g/m3
Case 1: 0.57 (dust in g/m3) × 0.1 (10 % active substance in
dust) = 0.057
Case 2: 0.21 (dust in g/m3) × 0.01 (1 % active substance in
dust) = 0.002
Active substance inhaled (Asi), mg/day Case 1: 0.057 (Asa) × 0.55 (Ia) × 1 000 = 31
Case 2: 0.002 (Asa) × 0.55 (Ia) × 1 000 = 1
Reduced by filter mask (Asir), mg/day Case 1: 31 (Asi) × 0.1 (by mask type P2) = 3
Case 2: 1 (Asi) × 0.1 (by mask type P2) = 0.1 g/day
The aerosol fraction relevant for occupational health, related to the whole transported aerosol, cannot
be further refined as data on the particle fractions in dust are not available. In case 1 (ultrafine powder)
it should be assumed that all particles of dust are of relevance because virtually all particles of the
product are already smaller than 20 µm.