EFSA Journal 2012;10(6):2737

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 mol/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

On request from the European Commission, Question No EFSA-Q-2009-00884 adopted by the FEEDAP Panel on 23 May 2012. 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. Panel members: Gabriele Aquilina, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Nol Albert Dierick, Mikolaj Antoni Gralak, Jrgen Gropp, Ingrid Halle, Christer Hogstrand, Lubomir Leng, Secundino Lpez Puente, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Giovanna Martelli, Mikls Mzes, Derek Renshaw, Maria Saarela, Kristen Sejrsen and Johannes Westendorf. Correspondence: FEEDAP@efsa.europa.eu 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.

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

Beta-carotene for all animal species

SUMMARY
Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of 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 betacarotene 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 mol/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 57 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.

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Beta-carotene for all animal species

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

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Beta-carotene for all animal species

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 betacarotene (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 -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 betacarotene, when used under the conditions described in Table 1.

7 8

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. 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, Etrembires, France; Lohmann Animal Health GmbH &Co. KG, Cuxhaven, Germany; Sunvit GmbH, Bardowick, Germany. EFSA Dossier reference: FAD-2009-0046. 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.

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Beta-carotene for all animal species

Table 1:
Additive

Description and conditions of use of the additive as proposed by the applicant

Beta Carotene 3a 160 a 3. Nutritional additives a. Vitamins, provitamins and chemically well defined substances having a similar effect Description

Registration number/EC No/No (if appropriate) Category(-ies) of additive Functional group(s) of additive

Composition, description Beta-carotene

Chemical formula C40H56

Purity criteria (if appropriate) Min. 96 % (max. 3 % other colouring matters)

Method of analysis (if appropriate) JECFA PhEur

Trade name (if appropriate) Name of the holder of authorisation (if appropriate)

Not appropriate Not appropriate Conditions of use

Species or category of animal All animal species All categories

Minimum content Maximum Age

Maximum content

mg or Units of activity or CFU kg-1 of complete feedingstuffs, supplementary feed (based on end feed) and in water* -

Withdrawal period (if appropriate) -

Other provisions and additional requirements for the labelling 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. None. No specific requirement other than the traceability and complaint system implemented in compliance with the requirement of Regulation No 183/2005

Specific conditions or restrictions for use (if appropriate)

Specific conditions or restrictions for handling (if appropriate) Post-market monitoring (if appropriate) Specific conditions for use in complementary feedingstuffs (if appropriate)

Maximum Residue Limit (MRL) (if appropriate) Marker residue Species or category of animal Target tissue(s) or food products Maximum content in tissues

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Beta-carotene for all animal species

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.

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.

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Beta-carotene for all animal species

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 176183 C, a density of 0.61 g/cm3 (20 C), an octanolwater 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 beta17 18

This section has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/2003. 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.

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Beta-carotene for all animal species

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 (0.010.02 %), 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 22

Technical dossier/Supplementary information January 2011 and January 2012. 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.

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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 m 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, DLalpha-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 01.38 % (w/w) of the particles were smaller than 50 m;26 in the second set (three batches of another additive) 0.210.59 % (w/w) of the particles were smaller than 50 m.27 In contrast, data from two other formulations showed that 99% of particles in one product were smaller than 15.2 m whereas no particles smaller than 50 m 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. 2.3.1. Stability and homogeneity 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 27

Technical dossier/Section II. 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.

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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 ~510 % and extrusion by ~1015 %. After storage in paper bags at 25 C for three months, recovery was ~7383 % in the pelleted feed and ~7885 % 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 (50550 m) and another with a larger particle size (100850 m), 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 31

Technical dossier/Section II/Annex 2401. Technical dossier/Section II/Annex 2402.

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Beta-carotene for all animal species

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 1070 mg beta-carotene/kg complete feed for dairy cows, 80 100 mg beta-carotene/kg complete feed for sows, 70125 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. 3.1. Safety 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 betacarotene 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, interindividual 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 922 %. Studies in humans also indicate that about 2075 % 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 betacarotene 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

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Beta-carotene for all animal species

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-apo8 -, 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 betaapo-13-carotenone) depending on the extent of cleavage at the central C15C15' double bond or the C13C14 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 lowdensity 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.

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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.

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Table 2:

Tissue concentration of beta-carotene in different animal species fed graded levels of betacarotene (partially recalculated)
Beta-carotene concentration Plasma/serum (nmol/L) Liver (ng/g) Adipose (ng/g) Corpus luteum (ng/g) Lung (ng/g) Milk (nmol/L) Ref.

Species

Humans

1705 360 () 640750 7.511.2 310340 215 55.9 1 751 4 80610 673 279017830 63386 11870 6801 800 1 5 750 088 0.1106 93125 137

() 42910 415 () ND 156 13 368 0487 ~100 ~3 000 () 10740 534 172338 04 700 644 16142 305 18497 () () ()

620 () () n. d. () () () () () 4832 684 () 0300 (~14) 0752 46 () () ()

() () () () () 25172 ~100 ~14 200 () () () () () () () () () ()

() 54859 () () () 0 12 () () () () () () 23 1698 1162 () () ()

() () 3150 () () () () () 147209 () () () () () () () () ()

1, 2 3 4* 5 6 7 8 8 9 10 11 5 12 13 14 15 16 17

Rat Pig Cow/cattle Preruminant calf Ferret

Gerbil Cat

() , 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, Caldern 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 betacarotene 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.

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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
Default values Body Feed weight intake (kg) (g/day) 2 100 400 100 200 650 12 20 2 2 15 40 2 000 8 000 3 000 6 000 20 000 400 1 000 120 120 250 Maximum recommended beta-carotene (mg/kg feed) 30 100 30 30 100 70 30 30 30 30 30 Intake of a 10 % additive (mg/day) 12 2 000 2 400 900 6 000 14 000 120 300 36 36 75 TPPO intake Safe Additive with amount* 100 mg (g/day) TPPO/kg (g/day) 40 1.2 2 000 8 000 2 000 4 000 13 000 240 400 40 40 300 200 240 90 600 1 400 12 30 3.6 3.6 7.5

Animal category

Salmonids Veal calves (milk replacer) Cattle for fattening Pigs for fattening Sows Dairy cows Turkeys for fattening Piglets Chickens for fattening Laying hens Dogs

*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 % betacarotene-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. 3.3.1. Safety for the consumer 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.

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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 betacarotene/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
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(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 betacarotene 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 fr Risikobewertung) on the use of vitamins in foods (2005).32 The authors compared the ATBC trial and the CARET trial with the Physicians 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 4.25.6 mol/L showed an increased lung cancer frequency as a result of beta-carotene supplementation; studies in which plasma levels were in the range of 1.22.2 mol/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 57 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 (510 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 05 mg/kg bw (JECFA, 1975). However, as a consequence of the first observations on lung cancer in intervention studies with betacarotene, 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 12 mg/day in addition to an average 25 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 1.13.9 mg/day for children aged 46 years and 1.45.6 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

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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 3564 years; the P95 is always above 10 mg/day and above 13 mg/day in women aged 2564 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 betacarotene 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 mol/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. Caldern 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 betacarotene 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 cows 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 g/day for calves, 600 g/day for sows, 1400 g/day for dairy cows), assuming 100 mg TPPO/kg additive. However, certain beta-carotenecontaining additives contain only 618 mg TPPO/kg, which would reduce the values to 618 % 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 betacarotenes as food additives is considerably higher than any potential intake from food from beta33 34

http://www.was-esse-ich.de/uploads/media/NVSII_Abschlussbericht_Teil_2.pdf OJ L 83 22.03.2012, p. 1.

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Beta-carotene for all animal species

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 betacarotene. 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 m) 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.

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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 betacarotene, 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 betacarotene/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; Iwaska et al., 1985; Daniel et al., 1991a; Coffey and Britt, 1993; Pre et al., 1993; Chew et al., 2001;
36

OJ L 133, 22.5.2008, p. 1.

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Beta-carotene for all animal species

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-carotenealthough absorbedcannot 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 betacarotene 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 betacarotene. 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.

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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.

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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.

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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% BetaCarotene. 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 BetaCarotene 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-FAD2009-0046.pdf

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APPENDIX B

,-Carotene 15,15 -monooxygenase

Retinal Retinyl esters Retinol

beta-Apo-8 -, 10 - or 12 -carotenals
Storage

Retinol

beta-Apo-10 -carotenoic acid

beta-Apo-12 -carotenoic acid Retinal

Retinal oxidase

Retinoic acid

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 apocarotenals are subsequently oxidised to the apo-carotenoic acids and further shortened to retinoic acid (Bachmann et al., 2002)

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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 m); 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 Time of exposure Inhaled air during exposure (Ia), m3 Active substance in air (Asa), g/m3 40 (batches) 1 (fraction of batches containing additive) = 40 (batches) 40 20 seconds = 800 seconds An uncertainty factor of 2 should be introduced 1.25 m3 per hour 2 800/60/60 in hours = 0.55 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 Case 1: 0.057 (Asa) 0.55 (Ia) 1 000 = 31 Case 2: 0.002 (Asa) 0.55 (Ia) 1 000 = 1 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

Active substance inhaled (Asi), mg/day Reduced by filter mask (Asir), mg/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 m.

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