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EFSA Journal 2011;9(11):2413

Suggested citation: EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP); Scientific Opinion on the safety and efficacy of vitamin B1 (thiamine mononitrate and thiamine hydrochloride) as a feed additive for all animal species based on a dossier submitted by DSM Nutritional Products. EFSA Journal 2011;9(11):2413. [17 pp.]. doi:10.2903/j.efsa.2011.2413. Available online: www.efsa.europa.eu/efsajournal

European Food Safety Authority, 2011

SCIENTIFIC OPINION

Scientific Opinion on the safety and efficacy of vitamin B1 (thiamine mononitrate and thiamine hydrochloride) as a feed additive for all animal

species based on a dossier submitted by DSM Nutritional Products1

EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP)2,3

European Food Safety Authority (EFSA), Parma, Italy

ABSTRACT Thiamine mononitrate administered via feed and thiamine hydrochloride via water for drinking are safe for the target animals having a wide margin of safety. The use of thiamine mononitrate and thiamine hydrochloride as additives in animal nutrition is safe for consumers. Since no data on inhalation toxicity of thiamine mononitrate and thiamine hydrochloride has been provided, inhalation of dust is considered as potentially hazardous. Diluted solutions of thiamine mononitrate were not irritant to skin and eyes, the pure substances have not been tested. Thiamine mononitrate and thiamine hydrochloride are regarded as skin and eye irritants. On the basis of published literature, thiamine should be considered as a sensitiser. A risk for the environment resulting from the use of thiamine mononitrate and thiamine hydrochloride in animal nutrition is not foreseen. Thiamine mononitrate and thiamine hydrochloride are regarded as effective sources of vitamin B1.

European Food Safety Authority, 2011

KEY WORDS Nutritional additive, vitamins and provitamins, vitamin B1, thiamine hydrochloride, thiamine mononitrate, safety

1 On request from the European Commission, Question No EFSA-Q-2011-00255, adopted on 11 October 2011. 2 Panel members: Gabriele Aquilina, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Nol

Dierick, Mikolaj Antoni Gralak, Jrgen Gropp, Ingrid Halle, Christer Hogstrand, Reinhard Kroker, Lubomir Leng, Anne-Katrine Lundebye Haldorsen, Secundino Lopez Puente, Alberto Mantovani, Giovanna Martelli, Mikls Mzes, Derek Renshaw, Maria Saarela, Kristen Sejrsen, Johannes Westendorf. Correspondence: [email protected]

3 Acknowledgement: The Panel wishes to thank the members of the Working Group on Vitamins, including Annette Schuhmacher, for the preparatory work on this scientific opinion.

Vitamin B1 (thiamine mononitrate and thiamine hydrochloride) for all animal species

2 EFSA Journal 2011;9(11):2413

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 vitamin B1 in the form of thiamine mononitrate as an additive to feed and of thiamine hydrochloride as an additive to water for drinking for all animal species.

Thiamine is present in the organism predominantly as thiamine pyrophosphate, followed by thiamine triphosphate and thiamine monophosphate. Vitamin B1 mainly acts in -ketoacid decarboxylation (e.g. pyruvate, -ketoglutarate and branched-chain -ketoacid acids) and in transketolation (e.g. among hexose and pentose phosphates). Main deficiency symptoms are characterised as disorders in neural function.

Oral administration routes of thiamine hydrochloride via feed or water for drinking are considered as bioequivalent.

Thiamine mononitrate and thiamine hydrochloride are safe for the target animals having a wide margin of safety.

The use of thiamine mononitrate and thiamine hydrochloride as additives in animal nutrition is safe for consumers.

Since no data on inhalation toxicity of thiamine mononitrate and thiamine hydrochloride has been provided, inhalation of dust is considered as potentially hazardous. Diluted solutions of thiamine mononitrate were not irritant to skin and eyes, the pure substances have not been tested. Thiamine mononitrate and thiamine hydrochloride are regarded as skin and eye irritants. On the basis of published literature, thiamine should be considered as a sensitiser.

Thiamine occurs widely in nature. Its use in animal nutrition is not expected to substantially increase the concentration in the environment. Therefore, a risk for the environment resulting from the use of thiamine mononitrate and thiamine hydrochloride in animal nutrition is not foreseen.

Thiamine mononitrate and thiamine hydrochloride are regarded as effective sources of vitamin B1.

To guarantee the quality of thiamine mononitrate and thiamine hydrochloride, the FEEDAP Panel recommends that the specifications detailed in the European Pharmacopeia should be applied to both compounds when used as feed additives.


3 EFSA Journal 2011;9(11):2413

TABLE OF CONTENTS Abstract .................................................................................................................................................... 1Summary .................................................................................................................................................. 2Table of contents ...................................................................................................................................... 3Background .............................................................................................................................................. 4Terms of reference ................................................................................................................................... 4Assessment ............................................................................................................................................... 71.Introduction ........................................................................................................................................... 72.Characterisation ..................................................................................................................................... 8

2.1. Thiamine mononitrate ............................................................................................................. 82.2. Thiamine hydrochloride .......................................................................................................... 82.3. Manufacturing process ............................................................................................................ 9

2.3.1. Thiamine mononitrate ...................................................................................................... 92.3.2. Thiamine hydrochloride ................................................................................................... 9

2.4. Stability and homogeneity .................................................................................................... 102.4.1. Stability of thiamine mononitrate ................................................................................... 10

2.4.1.1. Shelf life ................................................................................................................ 102.4.1.2. Stability of the additive when added to premixtures and feed............................... 10

2.4.2. Stability of thiamine hydrochloride ................................................................................ 102.4.2.1. Shelf life ................................................................................................................ 102.4.2.2. Stability of the additive when added to water for drinking ................................... 10

2.4.3. Homogeneity .................................................................................................................. 102.5. Physico-chemical incompatibilities in feed .......................................................................... 112.6. Conditions of use .................................................................................................................. 112.7. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) .............................................................................................................................................. 11

3.Safety ................................................................................................................................................... 113.1. Safety for the target species .................................................................................................. 113.2. Safety for the consumer ........................................................................................................ 12

3.2.1. Absorption, distribution, metabolism and excretion of thiamine ................................... 123.2.2. Assessment of consumer safety ...................................................................................... 12

3.3. Safety for the user ................................................................................................................. 133.3.1. Inhalation exposure and effects on the respiratory system ............................................. 133.3.2. Effects on the eyes and skin ........................................................................................... 133.3.3. Sensitisation potential ..................................................................................................... 13

3.4. Safety for the environment .................................................................................................... 134.Efficacy ............................................................................................................................................... 135.Post-market monitoring ....................................................................................................................... 14Conclusions and recommendations ........................................................................................................ 14Documentation provided to EFSA ......................................................................................................... 15References .............................................................................................................................................. 15Appendix ................................................................................................................................................ 17


4 EFSA Journal 2011;9(11):2413

BACKGROUND Regulation (EC) No 1831/20034 establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7; in addition, Article 10(2) of that Regulation specifies that for existing products within the meaning of Article 10(1), an application shall be submitted in accordance with Article 7, at the latest one year before the expiry date of the authorisation given pursuant to Directive 70/524/EEC for additives with a limited authorisation period, and within a maximum of seven years after the entry into force of this Regulation for additives authorised without time limit or pursuant to Directive 82/471/EEC.

The European Commission received a request from the company DSM nutritional Products Ltd5 for (i) authorisation of a new use (i.e. use in water for drinking) of vitamin B1 in the form of thiamine hydrochloride and (ii) re-evaluation of authorisation of vitamin B1 in the form of thiamine mononitrate, 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 1a,b.

According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 4(1) (authorisation of a feed additive or new use of a feed additive) and under Article 10(2) (re-evaluation of an authorised feed additive). EFSA received directly from the applicant the technical dossier in support of this application.6 According to Article 8 of that Regulation, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. The particulars and documents in support of the application were considered valid by EFSA as of 20 April 2011.7

The Scientific Committee on Food expressed an opinion on the tolerable upper intake level of vitamin B1 (SCF, 2001). The Panel on Food Additives and Nutrient Sources added to food issued a scientific opinion on benfotiamine, thiamine monophosphate chloride and thiamine pyrophosphate chloride, as sources of vitamin B1 added for nutritional purposes to food supplements (EFSA, 2008) and an opinion on the inability to assess the safety of thiamine-enriched yeast added for nutritional purposes as a source of thiamine in food supplements and the bioavailability of thiamine from this source, based on the supporting dossier (EFSA, 2009a). The Panel on Dietetic Products, Nutrition and Allergies (NDA) issued an opinion on substantiation of several health claims pursuant to Article 13(1) of Regulation (EC) No 1924/2006 (EFSA, 2009b) and another opinion on the substantiation of a health claim related to thiamine and carbohydrate and energy-yielding metabolism pursuant to Article 14 of Regulation (EC) No 1924/2006 (EFSA, 2010a).

Vitamins B1 in the form of thiamine hydrochloride and thiamine mononitrate has been authorised without a time limit under Council Directive 70/524/EEC8 for its use for all animal species as a nutritional additive.

TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/2003, EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the safety for the target animal(s), consumer, user and the environment and the efficacy of vitamin B1 in 4 OJ L 268, 18.10.2003, p. 29. 5 DSM Nutritional Products Ltd, Switzerland, represented in the European Union by DSM Nutritional Products SP. Z.o.o.,

Tarczynska 113, 96.320 Mszczonow Poland. 6 EFSA Dossier reference: FAD-2010-0140. 7 A new mandate was received in EFSA in March 2011. 8 OJ C 50, 25.2.2004, p. 1.


5 EFSA Journal 2011;9(11):2413

the form of thiamine hydrochloride and thiamine mononitrate, when used under the conditions described in Tables 1a/b.

Table 1a: Description and conditions of use of the additive as proposed by the applicant

Additive Vitamin B1 - Thiamine hydrochloride Registration number/EC No/No (if appropriate) -

Category(ies) of additive Nutritional Additives

Functional group(s) of additive Vitamins, provitamines and chemically well defined substances having a similar effect

Description

Composition, description Chemical formula Purity criteria (if appropriate) Method of analysis

(if appropriate)

Thiamine hydrochloride C12H17ClN4OS.HCl

> 93.5 %

< 5 % water

European Pharmacopeia 0303 (potentiometric

titration)

European Pharmacopeia 20512 (semi-micro

determination)

Trade name (if appropriate) -

Name of the holder of authorisation (if 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 of complete

feedingstuffs (select what applicable) All animal species and categories

- - - -

Other provisions and additional requirements for the labelling

Specific conditions or restrictions for use (if appropriate) for use in water for drinking

Specific conditions or restrictions for handling (if appropriate) -

Post-market monitoring (if appropriate)

No specific requirements other than the traceability and complaint system implemented in compliance with the requirements 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 - - - -


6 EFSA Journal 2011;9(11):2413

Table 1b: Description and conditions of use of the additive as proposed by the applicant

Additive Vitamin B1 - Thiamine mononitrate Registration number/EC No/No (if appropriate) -

Category(ies) of additive Nutritional Additives

Functional group(s) of additive Vitamins, provitamines and chemically well defined substances having a similar effect

Description

Composition, description Chemical formula Purity criteria (if appropriate) Method of analysis

(if appropriate)

Thiamine mononitrate C12H17N4O4S.NO3

> 97 %

< 1 % loss on drying

European Pharmacopeia 0531 (potentiometric

titration)

European Pharmacopeia 20232

Trade name (if appropriate) -

Name of the holder of authorisation (if 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 of complete

feedingstuffs (select what applicable) All animal species and categories

- - - -

Other provisions and additional requirements for the labelling

Specific conditions or restrictions for use (if appropriate) for use in premixtures and in feedingstuffs

Specific conditions or restrictions for handling (if appropriate) -

Post-market monitoring (if appropriate)

No specific requirements other than the traceability and complaint system implemented in compliance with the requirements 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 - - - -


7 EFSA Journal 2011;9(11):2413

ASSESSMENT This opinion is based in part on data provided by a single company involved in the production of vitamin B1 in the form of thiamine mononitrate and thiamine hydrochloride. It should be recognised that this data from a single applicant covers only a fraction of existing additives containing thiamine mononitrate and thiamine hydrochloride. The composition of the additives is not subject of the application. The FEEDAP Panel has sought to use the data provided together with data from other sources to deliver an opinion and to produce recommendations for the authorisation, which would secure the safety of future uses of vitamin B1 in the form of thiamine mononitrate and thiamine hydrochloride as feed additives.

1. Introduction Thiamine is present in the organism predominantly as thiamine pyrophosphate, followed by thiamine triphosphate and thiamine monophosphate. Vitamin B1 mainly acts in -ketoacid decarboxylation (e.g. pyruvate, -ketoglutarate and branched-chain -ketoacid acids) and in transketolation (e.g. among hexose and pentose phosphates). Main deficiency symptoms are characterised as disorders in neural function.

Vitamin B1 in the form of thiamine mononitrate and thiamine hydrochloride 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 it use in all animal species as a nutritional additive and in the case of thiamine hydrochloride as a sensory additive (feed flavouring).

The applicant asks for the re-evaluation of the use of vitamin B1 in the form of thiamine mononitrate as additive to feed for all animal species and categories without restrictions on age, (withdrawal) time and content in feedingstuff. The applicant is also seeking for a new use of vitamin B1 in the form of thiamine hydrochloride in water for drinking.

Vitamin B1 as thiamine mononitrate or thiamine hydrochloride 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 to processed cereal based foods and baby foods for infants and young children (Directive 2006/125/EC, Annex IV)14 and to infant formulae and follow-on formulae when reconstituted as instructed by the manufacturer (Directive 2006/141/EC, Annex III).15 Vitamin B1 is also listed as a pharmacologically active substance in veterinary medicinal products and it is not subject to maximum residue levels when used in food-producing animals (Commission Regulation (EC) No 37/2010).16 Thiamine mononitrate and thiamine hydrochloride are authorised in cosmetics as skin conditioners (Commission Decision 2006/257/EEC).17

Thiamine mononitrate and thiamine hydrochloride are described in the European Pharmacopeia (PhEur) as Monographs (MG) 0531 and 0303, respectively.

9 OJ C 50, 25.2.2004, p. 1. 10 OJ L 404 30.12.2006, p. 26. 11 OJ L 314 1.12.2009, p. 36. 12 OJ L 183 12.7.2002, p. 51. 13 OJ L 269, 14.10.2009, p. 9. 14 OJ L 339 6.12.2006, p. 16. 15 OJ L 401 30.12.2006, p. 1. 16 OJ L 15, 20.1.2010, p. 1. 17 OJ L 97 5.04.2006, p. 1.


8 EFSA Journal 2011;9(11):2413

2. Characterisation

2.1. Thiamine mononitrate Thiamine mononitrate (IUPAC name 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium nitrate, synonyms: vitamin B1, aneurine) is identified by the CAS number 532-43-4 and the EINECS number 208-537-4. The structural formula of thiamine mononitrate is shown in Figure 1.

Figure 1: Structural formula of thiamine mononitrate

The molecular formula of thiamine mononitrate is C12H17N4OSNO3 and its molecular weight is 327.36. It has a melting point of 198 C (decomposition), shows a bulk density of approximately 0.35 g/mL and is soluble in water (2.7 g dissolves in about 100 mL at 25 C and approximately 30 g in 100 mL at 100 C), slightly soluble in methanol, ethanol and chloroform, insoluble in ether.

Thiamine mononitrate is a white or almost white to yellowish crystalline powder, with a faint, characteristic odour and a bitter taste. The analysis of five batches of the product showed an average ( SD) content of thiamine mononitrate of 99.7 0.2 % (with a water content of 0.010.08 %)18 which meets the minimum specifications of PhEur MG 0531 (98 % thiamine mononitrate in the dried substance). However, the applicant proposes a minimum content of 97 % thiamine mononitrate in the additive.

The applicant reported that the total amount of substance-related impurities is very low, maximum 1 % for each individual impurity and maximum 1.5 % for total impurities. This statement would comply with the corresponding requirements of PhEur. However, no data on these impurities has been provided. The substance complies with PhEur concerning sulphated ash (< 0.1 %) and heavy metals (< 20 mg/kg) expressed as lead. Data for residual solvents (ethanol) indicate that VICH thresholds are not exceeded. Control measures are in place.

Three batches of thiamine mononitrate were analysed for particle size distribution determined by laser diffraction. The fraction of particles below 50 m ranged between 18 % and 28 % (v/v). The substance was examined for its dusting potential which was 3.3 g/kg.19

2.2. Thiamine hydrochloride Thiamine hydrochloride (IUPAC name 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium chloride hydrochloride, synonyms: vitamin B1, aneurine) is identified by the CAS number 67-03-8, the EINECS number 200-641-8 and the FLAVIS number 16.027. The structural formula of thiamine hydrochloride is shown in Figure 2.

18 Technical dossier/Section II/Annex 2.11. 19 Technical dossier/Section II/Annex 2.12.


9 EFSA Journal 2011;9(11):2413

Figure 2: Structural formula of thiamine hydrochloride

The molecular formula of thiamine hydrochloride is C12H17N4OS+Cl-HCl and its molecular weight is 337.3. It has a melting point of 248250 C (with decomposition), a pKa of 4.8 (20 C), shows a bulk density of approximately 0.4 g/cm3, a tap density of 0.51 g/cm3 and is freely soluble in water (100 g dissolves in 100 mL), soluble in glycerol (ca. 56 g/L), moderately soluble in methanol, sparingly soluble ethanol, insoluble in ethyl ether, acetone, benzene, hexane, and chloroform. The pH of a 2.5 % solution in water is 2.73.3.

Thiamine hydrochloride is a white or almost white crystalline powder, practically odourless. The analysis of five batches of the product showed an average ( SD) content of thiamine hydrochloride of 100.0 0.6 % (with a water content of 3.73.9 %)20 which meets the minimum specifications of PhEur MG 0303 (98.5 % thiamine hydrochloride in the anhydrous substance). However, the applicant proposes a minimum content of 93.5 % thiamine hydrochloride in the additive.

The amount of substance-related impurities as well as other impurities (sulphated ash and heavy metals) comply with the thresholds by PhEur, as demonstrated by the analysis of five batches of each producer. Data for residual solvents (ethanol) indicate that VICH thresholds are not exceeded. Control measures are in place.20

Three batches of thiamine hydrochloride were analysed for particle size distribution determined by laser diffraction. The fraction of particles below 50 m ranged between 55 and 67 % (v/v). The substance was examined for its dusting potential which was 4.5 g/kg.21

2.3. Manufacturing process The active substances are prepared synthetically from thiothiamine which is produced in-house. Thiothiamine is oxidised to thiamine sulphate by use of hydrogen peroxide; then the salts are formed by ion exchange. The applicant provided generic flow-charts of the synthetic processes and the purification steps.

2.3.1. Thiamine mononitrate The nitrate salt of thiamine is obtained by action of sodium hydroxide, ammonia and sodium nitrate on the oxidation product (thiamine sulphate) in aqueous ethanol solution. The precipitate is filtered off and purified with charcoal in nitric acid solution. The final product is then precipitated by action of sodium hydroxide and ammonia, filtered off and dried under vacuum.

2.3.2. Thiamine hydrochloride Thiamine hydrochloride is obtained by use of an ion exchange resin from the oxidation product (thiamine sulphate). The solution is then decolourised with charcoal. Water of the eluate is partly removed by distillation, the solution is acidified by hydrochloric acid and the active is precipitated by addition of ethanol. The final product is then isolated by centrifugation and dried under vacuum.

20 Technical dossier/Section II /Annex 2.10. 21 Technical dossier/Section II/Annex 2.12.


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2.4. Stability and homogeneity

2.4.1. Stability of thiamine mononitrate

2.4.1.1. Shelf life

Thiamine mononitrate (eight batches, stored in aluminium-coated polyethylene bags) was demonstrated to have a shelf life of 36 months at 25 C. Shelf life under accelerated conditions at 40 C was shown for three batches of the product for six months.22

2.4.1.2. Stability of the additive when added to premixtures and feed

According to the data published in the literature (Coelho, 2002), the stability of the additive in premixtures depends on the presence of trace elements and choline. Recovery of thiamine mononitrate after six months of storage in a vitamin premixture without choline chloride was 99 %, with choline chloride 73 %, in a vitamin trace element premixture without choline chloride 80 %, with choline chloride 48 %. These data indicate a reduced stability of thiamine mononitrate in the presence of choline chloride and of inorganic compounds of trace elements.

The retention of thiamine mononitrate during feed processing is inversely correlated with the temperature applied during pelleting or extrusion. The recovery of thiamine mononitrate after pelleting was 96 % at 6670 C and 66 % at 116120 C, after extrusion 93 % at 9195 C and 66 % at 141145 C (Coelho, 2002). Similar results were obtained by Dozier (2002) for thiamine (form not given).

The average monthly loss of thiamine mononitrate in pelleted and extruded feed can be estimated according to Coelho (1996) to be 4.5 %.

Three batches of a broiler premixture containing 500 mg thiamine mononitrate/kg (with choline chloride) showed a 13 % reduction on the content of thiamine mononitrate after six months when stored at 20 C.23

The stability of three batches of thiamine mononitrate was also examined in mash and pelleted poultry feed (supplementation level: 6 mg/kg) at 25 C. After three months, no significant losses were found in either feed type (% of initial value: 97 % in mash feed, 93 % in pelleted feed). Thiamine mononitrate was also stable during feed processing (pelleting at 75 C).23

2.4.2. Stability of thiamine hydrochloride

2.4.2.1. Shelf life

Thiamine hydrochloride (three batches, stored in aluminium-coated polyethylene bags) was demonstrated to have a shelf life of 36 months at 25 C. Shelf life under accelerated conditions at 40 C was shown for three batches of the product for six months.24

2.4.2.2. Stability of the additive when added to water for drinking

The stability of thiamine hydrochloride (two batches) was demonstrated when added at a concentration of 10 mg/L and kept for 48 hours at 25 C.23

2.4.3. Homogeneity The applicant provided a study to evaluate the homogeneous distribution of thiamine mononitrate incorporated in a pelleted feed for broilers (three batches) at the concentration 6 mg/kg. For each

22 Technical dossier/Section II/Annex 2.22. 23 Technical dossier/Section II/Annex 2.12. 24 Technical dossier/Section II/Annex 2.21.


11 EFSA Journal 2011;9(11):2413

batch, six independent samples were taken during the pelleting process. The coefficient of variation of six different determinations ranged from 6.5 % and 11.3 %, with an average value of 9.3 %.

Thiamine hydrochloride is freely soluble in water and homogeneity is not an issue.

2.5. Physico-chemical incompatibilities in feed No physico-chemical incompatibilities or interactions have been reported between thiamine mononitrate or thiamine hydrochloride 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.

2.6. Conditions of use Vitamin B1 in the form of thiamine mononitrate and thiamine hydrochloride is intended for use in all animal species and categories without a maximum content and a withdrawal period. Thiamine mononitrate is intended for use via feed (premixtures, complete or complementary feed), thiamine hydrochloride in water for drinking only.

2.7. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL)

EFSA has verified the EURL report as it relates to the methods used for the control thiamine mononitrate and thiamine hydrochloride in animal feed. The Executive Summary of the EURL report can be found in the Appendix.

3. Safety According to Regulation (EC) No 429/2008, tolerance, metabolism and residue, and toxicological (concerning consumer safety) studies are not required for vitamins, pro-vitamins and chemically defined substances having similar effects which are already authorised as feed additives under Directive 70/524/EEC and which do not have the potential to accumulate, which the FEEDAP Panel considers is the case for vitamin B1.

3.1. Safety for the target species Little information is published concerning thiamine tolerance and toxicity in domestic animal species. Studies in mice, rats, rabbits and dogs indicate that parenteral administration (80400 mg/kg bw) of thiamine (hydrochloride) may be the only route by which signs of thiamine toxicity can be produced. In those animal species thiamine toxicity is characterised by depression of the respiratory centre; however, the mechanism of those effects is unknown. The NRC (1987) concluded that dietary intake of thiamine up to 1000 times the requirement level are apparently safe for most animal species.

Requirements for thiamine (NRC, see McDowell, 2000) are in the range of 0.72.0 mg/kg feed for poultry, 1.01.5 mg/kg for pigs, 115 mg/kg for fish and 0.755 mg/kg for pets. Vitamin supplementation of commercial compound feed is mostly oriented towards recommendations, which are in the range of 14 mg/kg for pigs and poultry, 815 mg/kg for fish and 210 mg/kg feed for pets (AWT, 2002). A survey on vitamin supplementation of commercial feeds for pigs and poultry in Europe (Belgium, Denmark, Germany, Italy, Netherlands, Portugal, Spain, United Kingdom) identified a range of 07 mg vitamin B1/kg as commercial use levels (Gropp, 1994). A range for piglets and pigs of 13 mg/kg feed was reported by Whittemore et al. (2002).

Three classes of thiamine antagonists are known. Structurally-related compounds (e.g. pyrithiamine, oxythiamine and amprolium) exert an antagonistic effect by a competitive mechanism. Structurally unrelated compounds are heat-stable polyphenols in feedingstuffs which oxidise the thiazole ring to non absorbable thiamine disulphide or sulphur which can reduce thiamine synthesis by bacteria. Thiaminases can be present in fresh fish or be produced by rumen microbes. Feed manufacturers are familiar with these thiamine antagonists and consider this in feed formulation.


12 EFSA Journal 2011;9(11):2413

The FEEDAP Panel concludes that thiamine mononitrate and thiamine hydrochloride are safe for the target animals having a wide margin of safety (about 1000 compared to the requirements/recommendations).

3.2. Safety for the consumer

3.2.1. Absorption, distribution, metabolism and excretion of thiamine Free thiamine released in the gastrointestinal tract is absorbed in the proximal intestinal tract by active and passive mechanisms. It is stored predominantly in the liver and kidneys. In humans 80 % of the body depot is found in form of thiamine pyrophosphate, 10 % as thiamine triphosphate and the remaining as thiamine monophosphate. Thiamine is excreted via the kidneys mainly in unchanged form, although several and partially different metabolites were identified in humans and rats. Thiamine is one of the most poorly stored vitamins. Mammals may deplete their body stores within one to two weeks (Ensminger et al., 1983).

The only data found on the relationship between dietary thiamine and thiamine deposition in edible tissues and products refer to studies with laying hens. In one study (Leeson and Caston, 2003), the vitamin B1 content was 49 and 67 g/60 g egg after a three-month feeding period with diets supplemented with 4 and 24 mg thiamine/kg feed, respectively. Johnson and Korver (2008) found at lower dietary supplementation (2.4 and 11.4 mg thiamine/kg) after six weeks of feeding a higher thiamine concentration in the egg (239 and 395 g/60 g egg). In another study (two factorial design, 60-week duration), thiamine in the whole egg was 0.52 and 0.65 mg/kg (corresponding to 31 and 39 g/60 g egg) in groups fed diets supplemented with 0.91 and 3.0 mg/kg, respectively (Prez-Vendrell et al., 2004).

In all studies, no data were provided on the background content of thiamine. Cereals may contain between 1 and 5 mg/kg and yeasts between 40 and 50 mg/kg; however literature data indicate considerable variation. Although all studies demonstrated the dose-dependent effect on thiamine content of eggs and considering the variable concentration in feed, the differences in the absolute values between the studies are so high that a considerable influence of the dietary thiamine level on the egg content of thiamine should not be expected in practice.

It is concluded that vitamin B1 supplementation of feed at recommended levels (see Section 3.1) would not essentially modify the contribution of thiamine from food of animal origin to consumer intake.

3.2.2. Assessment of consumer safety Estimates based on food intake indicate that reported mean intake of vitamin B1 in some European countries varied from 1.10 mg/day to 2.28 mg/day (SCF, 2001). In Europe high level intake (97.5th percentile) varied from 1.90 to 6.35 mg/day.

The Scientific Committee on Food (SCF, 2001) could not identify a numerical tolerable upper intake level of vitamin B1 since no LOAEL and NOAEL could be established due to the lack of systematic oral dose-response intake studies as well as the extreme low toxicity.

The Expert Group on Vitamins and Minerals (EVM, 2003) concluded that dietary supplements up to 100 mg/person/day (equivalent to 1.7 mg/kg bw/day supplemented thiamine for a 60-kg adult) would not be expected to result in adverse effects This conclusion was based on the results of a randomised double-blind placebo-controlled study by Gokhale (1996) on 556 young females (1221 years). No adverse effects were found when a daily oral dose of 100 mg of thiamine hydrochloride was given for 90 days followed by placebo for 60 days or vice versa.

An exposure assessment is consequently not necessary since a contribution of thiamine in feed to the consumer intake is marginal and available data showed that the oral toxicity is very low and there is no


13 EFSA Journal 2011;9(11):2413

UL for vitamin B1 identified. Therefore, the FEEDAP Panel considers that the use of thiamine mononitrate and thiamine hydrochloride as additives in animal nutrition is safe for consumers.

3.3. Safety for the user

3.3.1. Inhalation exposure and effects on the respiratory system Thiamine mononitrate and thiamine hydrochloride showed more than 1 % of particles with diameter < 50 m and a dusting potential of 3.2 g/kg and 4.5 g/kg, respectively (corresponding to 8.2 and 11.1 g/m3). The applicant provided an exposure assessment for persons handling the additives in a premixtures factory,25 based on assumptions also applied by the FEEDAP Panel in some opinions (EFSA, 2009c, 2010b, 2010c). Two main settings are different: (i) all dust particles are considered as respirable (worst case assumption) and (ii) without protection by a mask.26 Maximum exposure under such conditions was calculated to be 3.1 mg/person and day.

Since no data on inhalation toxicity of thiamine mononitrate and thiamine hydrochloride has been provided, inhalation of dust is considered as potentially hazardous.

3.3.2. Effects on the eyes and skin Skin and eye (Draize test) irritation tests for aqueous solutions of thiamine mononitrate in rabbits were performed by the applicant. Undiluted material was not tested. Skin irritation was tested using a 20 % solution of thiamine mononitrate in water applied on intact and abraded skin for four hours. Irritation did not occur.27 In the eye irritation study, a 2.5 % solution of thiamine mononitrate was tested. Eye irritation did not occur; only temporary redness of the eye was found in three out of six rabbits at 1 hour and in one out of six at 24 hours.28

Owing to the acidity and water solubility of thiamine hydrochloride, moderate irritation of eyes and mucous membranes is expected. From experience in workplaces, irritation of unprotected and moist skin is common but not severe.

3.3.3. Sensitisation potential Allergic eczematous reactions to topical exposure to thiamine hydrochloride in pharmaceutical workers have been reported (Larsen et al., 1989). Contact dermatitis may occur with occupational exposure and may cause sensitisation and lead to dermatitis-type reactions after oral or injected administrations. Anaphylactic reactions have only been observed with large parenteral (intravenous, intramuscular, subcutaneous) doses of thiamine hydrochloride, generally after multiple doses (Natural Standard Research Collaboration, 2010). Occupational asthmatic reactions have been reported (Seed and Agius, 2006). Thiamine (vitamin B1) is the vitamin that most frequently induces an allergic reaction (Schalch and Dostalova, 1983 according to Fernandez et al. 1997). On this basis, thiamine should be considered as a sensitiser.

3.4. Safety for the environment Thiamine occurs widely in nature. Its use in animal nutrition is not expected to substantially increase the concentration in the environment. Therefore, a risk for the environment resulting from the use of thiamine mononitrate and thiamine hydrochloride in animal nutrition is not foreseen.

4. Efficacy According to Regulation (EC) No 429/2008, efficacy studies are not required for vitamins, pro-vitamins and chemically defined substances having similar effects which are already authorised as

25 Technical dossier/Section III. 26 Techniccal dossier/Section III. 27 Technical dossier/Section III/Annex 3.11. 28 Technical dossier/Section III/Annex 3.12.


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feed additives under Directive 70/524/EEC and which do not have the potential to accumulate, which the FEEDAP Panel considers is the case for vitamin B1.

Due to the long history of use and its established nutritional role in domestic animals, thiamine mononitrate and thiamine hydrochloride are regarded as an effective source of vitamin B1.

Vitamin B1 has been globally used in animal nutrition for decades. Data on requirement, allowances and recommendations for feed supplementation are easily accessible as standard literature for animal nutrition experts.

5. Post-market monitoring The FEEDAP Panel considers that there is no need for specific requirements for a post-market monitoring plan other than those established in the Feed Hygiene Regulation29 and Good Manufacturing Practice.

CONCLUSIONS AND RECOMMENDATIONS

CONCLUSIONS

Oral administration routes of thiamine hydrochloride via feed or water for drinking are considered as bioequivalent.

Thiamine mononitrate and thiamine hydrochloride are safe for the target animals, having a wide margin of safety.

The use of thiamine mononitrate and thiamine hydrochloride as additives in animal nutrition is safe for consumers.

Since no data on inhalation toxicity of thiamine mononitrate and thiamine hydrochloride has been provided, inhalation of dust is considered as potentially hazardous. Diluted solutions of thiamine mononitrate were not irritant to skin and eyes, the pure substances have not been tested. Thiamine mononitrate and thiamine hydrochloride are regarded as skin and eye irritants. On the basis of published literature, thiamine should be considered as a sensitiser.

Thiamine occurs widely in nature. Its use in animal nutrition is not expected to substantially increase the concentration in the environment. Therefore, a risk for the environment resulting from the use of thiamine mononitrate and thiamine hydrochloride in animal nutrition is not foreseen.

Due to the established nutritional role in domestic animals thiamine mononitrate and thiamine hydrochloride are regarded as effective sources of vitamin B1.

RECOMMENDATIONS

The FEEDAP Panel proposes to use the specifications for thiamine mononitrate and thiamine hydrochloride according to PhEur (MG 0531 and MG 0303) considering purity, substance-related impurities and other impurities (sulphated ash and heavy metals).

When giving a warranty for stability in premixtures, the manufacturer should consider the effect of choline chloride. The FEEDAP Panel recommends including a corresponding warning under other provisions of the authorisation (e.g. Stability of thiamine mononitrate and thiamine hydrochloride may be reduced in premixtures containing choline chloride).

Considering the potential risk of users by inhalation exposure and to guarantee homogeneous distribution in the final feed, the use of thiamine mononitrate and thiamine hydrochloride should be

29 OJ L 35, 8.2.2005, p. 1.


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restricted to premixture manufacturers. Poor distribution of vitamin B1 in feed may result in thiamine deficiency which would affect animal welfare.

DOCUMENTATION PROVIDED TO EFSA 1. Vitamin B1 as a feed additive for all animal species. October 2010. Submitted by DSM

nutritional Products Ltd.

2. Vitamin B1 as a feed additive for all animal species. Supplementary information. September 2011. Submitted by DSM nutritional Products Ltd.

3. Evaluation report of the European Union Reference Laboratory for Feed Additives on the methods of analysis for vitamin B1.

4. Comments from Member States received through the ScienceNet.

REFERENCES AWT (Arbeitsgemeinschaft fr Wirkstoffe in der Tierernhrung e.V.), 2002. Vitamins in animal

nutrition, ISBN 3-86037-167-3.

Coelho M, 1996. Stability of vitamins affected by feed processing. Feedstuffs, 29, 9-14.

Coelho M, 2002. Vitamin stability in Premixes and Feeds. A Practical Approach in Ruminant Diets. Proceedings 13th Annual Florida Ruminant Nutrition Symposium, pp. 127-145.

Dozier WA, 2002. Mash conditioning and vitamin stability. Feed International, May 2002, 20-24.

EFSA (European Food Safety Authority), 2008. Opinion of the Scientific Panel on Food Additive and Nutrient Sources added to food on Benfotiamine, thiamine monophosphate chloride and thiamine pyrophosphate chloride, as sources of vitamin B1 added for nutritional purposes to food supplements. The EFSA Journal, 864, 1-31.

EFSA (European Food Safety Authority), 2009a. Opinion of the Scientific Panel on Food Additive and Nutrient Sources added to food on the inability to assess the safety of thiamine-enriched yeast added for nutritional purposes as a source of thiamine in food supplements and the bioavailability of thiamine from this source, based on the supporting dossier. The EFSA Journal, 1121, 1-6.

EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2009b. Scientific opinion of the on substantiation of health claims related to thiamine and energy-yielding metabolism (ID 21, 24, 28), cardiac function (ID 20), function of the nervous system (ID 22, 27), maintenance of bone (ID 25), maintenance of teeth (ID 25), maintenance of hair (ID 25), maintenance of nails (ID 25), maintenance of skin (ID 25) pursuant to Article 13(1) of Regulation (EC) No 1924/2006. EFSA Journal, 7(9): 1222.

EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2009c. 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 a health claim related to thiamine and carbohydrate and energy-yielding metabolism pursuant to Article 14 of Regulation (EC) No 1924/2006. EFSA Journal, 8(7):1690.

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EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2010c. Scientific Opinion on the safety and efficacy of Maxiban G160 (narasin and nicarbazin) for chickens for fattening. EFSA Journal, 8(4):1574.

Ensminger AH, Ensminger ME, Konlande JE and Robson JRK, 1983. In: Foods and Nutrition Encyclopedia, Vol. 1, A-H. California: Pegus Press, p. 1208.

European Pharmacopeia (PhEur), 2010. Thiamine mononitrate, Monograph (MG) 0531. 7th Edition. Strasbourg, France. Council of Europe (COE) European Directorate for the Quality of Medicines.

European Pharmacopeia (PhEur), 2010. Thiamine hydrochloride, Monograph (MG) 0303. 7th Edition. Strasbourg, France. Council of Europe (COE) European Directorate for the Quality of Medicines.

EVM (Expert group on vitamins and minerals), 2003, online. Safe upper levels for vitamins and minerals. Available from www.food.gov.uk/multimedia/pdfs/vitmin2003.pdf

Fernandez M, Barcel M, Munoz C, Torrecillas M and Blanca M, 1997. Anaphylaxis to thiamine (vitamin B1). Allergy, 52, 958-960.

Gokhale LB, 1996. Curative treatment of primary (spasmodic) dysmenorrhoea. Indian Journal of Medical Research, 103, 227-31.

Gropp JM. 1994. Vitamin Fortification Levels in European Commercial Poultry and Swine Diets. Proceedings of the Arkansas Nutrition Conference, Fayetteville, Arkansas, 105-134.

Johnson ML and Korver DR, 2008, online. The effect of dietary supplementation on egg vitamin content. Metabolism and Nutrition IV: Vitamins and Minerals and Feed Manufacturing, Abstracts of papers, Poultry Science Association, Niagara Falls, Ontario, Canada. Available from http://www.poultryscience.org/psa08/abstracts/050.pdf

Larsen AI, Jepsen JR and Thulin H, 1989. Allergic contact dermatitis from thiamine. Contact Dermatitis, 20, 387-388.

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McDowell LR. 2000. Vitamins in animal and human nutrition, Second Edition, Iowa State University Press. ISBN 0-8138-2630-6, p. 395.

NRC (National Research Council), 1987. Vitamin tolerance of animals. National Academic Press, Washington DC, 43-46.

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Prez-Vendrell M, L. Llaurad L, Brufau J, Hernndez JM, 2004. Improving the nutritive value of eggs by feeding laying hens an Optimum Vitamin Nutrition (OVN). Zootecnica, December, 56-58.

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Seed MJ and Agius RM, 2006. Prediction of asthma hazard of thiamine. Allergy, 61, 648.

Schalch W and Dostalova L, 1983. Vitamins. In: de Week AL, Bundgard H, editors. Allergic reactions to drugs. Vol. 63. Berlin: Springer-Verlag, 657-690.

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APPENDIX Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for vitamin B1

The EURL considered that the conclusions and recommendations reached in the assessment of FAD-2010-0040 and FAD-2010-0052 are valid and applicable also to the current application.30

30 The full reports are available on the EURL website. http://irmm.jrc.ec.europa.eu/SiteCollectionDocuments/FinRep-FAD-

2010-0040.pdf and http://irmm.jrc.ec.europa.eu/SiteCollectionDocuments/FinRep-FAD-2010-0052.pdf