Mycofix Secure Bentonite (dioctahedral montmorillonite) (FAD ; CRL/090044)

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1 Ref. Ares(2013) /02/2013 EUROPEAN COMMISSION JOINT RESEARCH CENTRE Institute for Reference Materials and Measurements (Geel) Standards for Food Bioscience Unit European Union Reference Laboratory for Feed Additives - Authorisation JRC.D.5/SFB/CvH/PRO/mds/ARES European Union Reference Laboratory Evaluation Report on the Analytical Methods submitted in connection with the Application for Authorisation of a Feed Additive according to Regulation (EC) No 1831/2003 Mycofix Secure Bentonite (dioctahedral montmorillonite) (FAD ; CRL/090044) Retieseweg 111, B-2440 Geel - Belgium. Telephone: (32-14) JRC-IRMM-CRL-FEED-ADDITIVES@ADDITIVES@ec.europa.eu

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3 EUROPEAN COMMISSION JOINT RESEARCH CENTRE Institute for Reference Materials and Measurements (Geel) Standards for Food Bioscience European Reference Laboratory for Feed Additive Authorisation JRC.D.5/SFB/CvH/PRO/mds/Ares Addendum to the EURL report FAD (JRC.DG.D6/CvH/RMO/ag/ARES(2010)593553) Background In the scientific opinion on the safety and efficacy of bentonite (dioctahedral montmorillonite) as feed additive for all species, the FEEDAP panel recommended to introduce the aflatoxin B 1 (AfB1) binding capacity in the corresponding registry entry [1]. The Applicant proposed then to add the following specification: "Bentonite E558 with an AfB1-binding capacity of min 90%". Consequently, the Commission requested the EURL to evaluate the analytical method to determine the AfB1-binding capacity of bentonite [2]. Evaluation of the analytical method for the determination of the AfB1-binding capacity of Mycofix Secure The Applicant submitted a single-laboratory validated and further verified method based on adsorption experiment [3-5] to assess the AfB1-binding capacity. One gram of bentonite was added to 1 L acetate buffer solution (ph 5); the solution was stirred to homogenise. An aliquot (100 µl) was then added to 5 ml of the acetate buffer containing 4 mg AfB1/L. The mixture was first shaken and incubated for 1 hour at 37 o C, then centrifuged for 15 min. The supernatant (100 L) was injected in a high performance liquid chromatograph with diode array detection (HPLC-DAD) and the measurements were performed with an excitation wavelength of 365 nm and an emission wavelength of 800 nm. This experimental protocol was tested on using three products: - a bentonite purchased from Sigma and considered by the Applicant as his "laboratory-reference bentonite" (Ref-Bent); a hydrated sodium calcium aluminosilicate (HSCAS) from Novasil; and the feed additive under investigation (Mycofix Secure). According to the Applicant [6], the binding capacity (BC AfB1 ) is derived from the difference in mycotoxin concentration in the blank solution which contained the initial AfB1 concentration without binder (B) and the supernatant of the incubated binder sample (A). A relative BC AfB1 can then be computed as: BC AfB1 % = (1-A/B)*100 i

4 The Applicant reported that the limit of detection of the HPLC-DAD method was of the order of 10 µg AfB1/L. The experimental results obtained in the frame of the validated and verified study are reported in the Table 1, showing satisfactory precisions (a maximum relative standard deviation for intermediate precision of 2.1%) and binding capacities above 90%. Based on the experimental evidence presented, the EURL considers the method submitted by the Applicant suitable for the monitoring of AfB1-binding capacities above 90%. Table 1: Binding capacities (BC AfB1 ) and relative standard deviations for repeatability (RSD r ) and intermediate precision (RSD ip ) obtained in the frame of the validation [4] and the verification [5] studies Mycofix Secure Ref-Bent, Sigma HSCAS, Novasil [4] [5] [4] [5] [4] [5] RSD r (%) 0,1 0,1 2,1 0,4 RSD ip (%) 1,5 1,0 2,1 0,4 BC AfB1 (%) 95,3 97,1 96,9 98,2 92,5 93,5 Recommended text for the registry entry (analytical method) For the determination of the AfB1-binding capacity: Adsorption experiment followed by high performance liquid chromatograph with diode array detection (HPLC-DAD) Document - Prepared by Gilda D'Arco and Piotr Robouch - Reviewed and approved by Christoph von Holst (EURL-FA) Geel, 12/02/2013 [1] Scientific Opinion - EFSA Journal 2011; 9(2):2007 [2] SANCO Request - ARES(2013)92753, dated 25/01/2013 [3] FAD : Supplementary Information Standard-Operating-Procedure-Binding-Capacity.pdf [4] FAD : Supplementary Information Validation-Report-Binding-Capacity.pdf [5] FAD : Supplementary Information Verification-Report-Binding-Capacity.pdf [6] E. Vekiru et al. Mycotoxin Research 23 (2007) ii

5 EUROPEAN COMMISSION JOINT RESEARCH CENTRE Institute for Reference Materials and Measurements Community Reference Laboratory for Feed Additives JRC.DG.D6/CvH/RMO/ag/ARES(2010) CRL Evaluation Report on the Analytical Methods submitted in connection with the Application for Authorised as Feed Additive according to Regulation (EC) No 1831/2003 Dossier related to: Name of Product: Active Agent (s): Rapporteur Laboratory: Report prepared by: Report checked by: Date: Report approved by: Date: FAD CRL/ Mycofix Secure Bentonite (Dioctahedral Montmorillonite) Community Reference Laboratory for Feed Additives (CRL-FA) Geel, Belgium Roberto Molteni, Christoph von Holst and Piotr Robouch (CRL-FA) Dijana Mitic (CRL-FA) 15/09/2010 Christoph von Holst (CRL-FA) 15/09/2010

6 EXECUTIVE SUMMARY Mycofix Secure is a product for which authorisation as feed additive is sought under the category "Technological feed additive", functional group 1(m) "substances for the reduction of the contamination of feed by mycotoxins" according to Annex I of Regulation (EC) No 1831/2003. The active substance of Mycofix Secure is bentonite (dioctahedral montmorillonite). In the current application submitted according to Article 4(1) of Regulation (EC) No 1831/2003, the authorisation is sought for all animal species and categories. The proposed inclusion level of bentonite in complete feedingstuffs is 0.5 g/kg for the minimum content and 3 g/kg for the maximum content. For the determination of the mineralogical and geological parameters of the bentonite in the feed additive, the Applicant proposed several chemical and mineralogical methods, commonly used in geological studies. The X-ray diffraction (XRD) analysis indicates that the product contains a minimum of 70% dioctahedral montmorillonite. The CRL recommends for official control the X-ray diffraction (XRD) method proposed by the Applicant for the determination of bentonite in the feed additive. The direct determination of the bentonite content added to premixtures or feedingstuffs is not achievable by analysis. The Applicant proposed instead an indirect method for the determination of the bentonite in premixtures and feedingstuffs, based on the addition of a non-nutrient marker in the feed additive. The method consists in counting coloured stainless steel particles extracted from the samples. This method was originally used to monitor the homogeneity of the product. The transferability of the method was further investigated through a collaborative trial organised by the Applicant. The following satisfactory performance characteristics were re-calculated by the CRL based on the experimental data provided: - a relative standard deviation for repeatability and reproducibility ranging from 10.8 to 12.2%; and - a recovery rate ranging from 87 to 103%. Based on the above mentioned performance characteristics, the CRL conditionally considers the indirect method (using non-nutrient marker particles) proposed by the Applicant as suitable for the determination of the bentonite in premixtures and feedingstuffs for official control only, when (1) the content of the specific marker is established - expressed as number of particle per gram of feed additive and (2) the specific marker proposed in this dossier is exclusively used for this feed additive. In addition, EFSA requested the CRL to evaluate the analytical method applied by the Applicant to monitor the stability of the active substance in the product, premixtures and 2/9

7 feedingstuffs. In this dossier the Applicant used the capability of bentonite to adsorb aflatoxin B1 (AfB1) from buffer solutions as indicator for its stability and measured this parameter after having applied different storage conditions. For the analysis the Applicant used liquid chromatography coupled to a diode array detector (LC-DAD). Based on the obtained sensitivity of the LC-DAD method and due to the high AfB1 concentration in the butter solution, the CRL considers the LC-DAD method suitable for the purpose of the stability study. KEYWORDS Mycofix Secure, bentonite, dioctahedral montmorillonite, substances for the reduction of the contamination of feed by mycotoxins 1. BACKGROUND Mycofix Secure is a product for which authorisation as feed additive is sought under the category "Technological feed additive", functional group 1(m) "substances for the reduction of the contamination of feed by mycotoxins" according to Annex I of Regulation (EC) No 1831/2003 [1]. The active substance of Mycofix Secure is bentonite (dioctahedral montmorillonite) [2]. In the current application submitted according to Article 4(1) of Regulation (EC) No 1831/2003, the authorisation is sought for all animal species and categories [1]. The major component of bentonite (> 70%) is a particular smectite (dioctahedral montmorillonite), belonging to the mineralogical group of bentonites (E558) [3]. Bentonite has a minimum Aflatoxin B1 (AfB1) binding capacity of 100 mg/g. The proposed inclusion level of bentonite in complete feedingstuffs is 0.5 g/kg for the minimum content and 3 g/kg [2] for the maximum content. 2. TERMS OF REFERENCE In accordance with Article 5 of Regulation (EC) No 378/2005, as last amended by Regulation (EC) No 885/2009, on detailed rules for the implementation of Regulation (EC) No 1831/2003 of the European Parliament and of the Council as regards the duties and the tasks of the Community Reference Laboratory concerning applications for authorisations of feed additives, the CRL is requested to submit a full evaluation report to the European Food Safety 3/9

8 Authority for each application or group of applications. For this particular dossier, the methods of analysis submitted in connection with Mycofix Secure, and their suitability to be used for official controls in the frame of the authorisation, were evaluated. Furthermore, EFSA requested [4] the CRL to evaluate the analytical method used by the Applicant during the stability study of the product - according to Chapter of Annex II of Commission Regulation (EC) No 429/ EVALUATION Identification/Characterisation of the feed additive Quantitative and quantitative composition of impurities in the additive When required by EU legislation, analytical methods for official control of undesirable substances in the additive (such as arsenic, cadmium, lead, mercury, dioxins, microbiological agents and mycotoxins) are available from the respective Community Reference Laboratories [5]. Description of the analytical methods for the determination of the active agents in the feed additive, premixtures and feedingstuffs For the determination of the mineralogical and geological parameters of the bentonite in the feed additive, the Applicant proposed several chemical and mineralogical methods [6], commonly used in geological studies. The physical analysis of the bulk bentonite identified three fractions: (i) clay - ranging from 76 to 81%; (ii) sand and (iii) silt. The X-ray fluorescence (XRF) analysis of the clay fraction (< 2 μm) provides the following elemental composition: 57% SiO 2 ; 16% Al 2 O 3 ; 5% Fe 2 O 3 and a total of 6% of MgO, CaO and Na 2 O. Finally, the X-ray diffraction (XRD) analysis indicates that the clay fraction consists of smectite only. A minimum content of dioctahedral montmorillonite of 70% in the product can therefore be derived. The use of all the above mentioned techniques is not necessary in the frame of official control; an XRD analysis of the product may be sufficient for the characterisation of the bentonite (by reference to patterns from International Centre for Diffraction Database) and for the quantification (by means of normalised full pattern reference intensity ratio). The CRL recommends for official control the X-ray diffraction (XRD) method proposed by the Applicant for the determination of bentonite in the feed additive. 4/9

9 The Applicant is aware that the direct determination of the bentonite content added to premixtures or feedingstuffs is not achievable by analysis [3]. The Applicant proposed instead an indirect method for the determination of the bentonite in premixtures and feedingstuffs [7], based on the addition of a non-nutrient marker in the feed additive. This marker consists of stainless steel particles (Microtracers TM FS), with a particle distribution ranging from 40 to 65 particles (p) per milligram, with a mean value of 50 p/mg [8]. These marker particles are coated with a unique colour, to be easily distinguished from other iron particles present in the sample. The inclusion of such marker particles in the feed additive, premixtures and feedingstuffs was originally intended to monitor the homogeneity of a production batch after mixing. The analytical method proposed by the Applicant requires an experimental setup comprising a rotation detector, a demagnetiser and filter papers [9]. The weighed sample is transferred on a filter paper in the rotation detector. The iron particles are first demagnetised and transferred on a filter paper, sprayed with a developing agent (ethanol/water mixture). The filter is then dried before counting the coloured particles. The sample intake must be properly chosen to achieve a total number of particles to be counted on the filter ranging from 25 to 200. For example: When using a tracer containing particles per gram, and adding 10 grams of tracer per tonne of feed, the feed would contain 2000 particles per kg. In a feed sample of 40 gram there would be 80 particles to be counted. The conditions described in the example above were used by the Applicant in the frame of a collaborative trial attended by seven laboratories [10]. A batch of 40 kg feedingstuffs was prepared containing particles; a 15 kg portion was used as "meal" samples and the rest was pelleted without use of steam. The following performance characteristics - derived from the five satisfactory set of results - were recalculated by the CRL [11] for the "meal" and "pelleted" samples: - a relative standard deviation for repeatability and reproducibility ranging from 10.8 to 12.2%; and - a recovery rate ranging from 87 to 103%. These performance characteristics were further confirmed by the Applicant when analysing additional premixture and feedingstuffs samples containing from 0.05% to 50% feed additive - starting from a feed additive containing 92 particles per gram 1 [12,13]. The precision (i.e. repeatability and reproducibility) of the indirect method is satisfactory. The recovery rate could only be calculated knowing the amount of marker included in the feed additive. Without this information, the number of counted coloured particles is meaningless. 1 One kilogram of feed sample including 0.05% feed additive (cf. feed 3 in [12]) would contain 46 particles. Average number of particles counted in replicate analyses = [13], resulting in a recovery rate of 99.6% 5/9

10 Upon request from the CRL, the Applicant confirmed that the reference samples sent to the CRL includes 81 non-nutrient marker particles per gram of product [14]. Based on the above mentioned performance characteristics, the CRL considers the indirect method proposed by the Applicant as suitable for the determination of the bentonite in premixtures and feedingstuffs for official control only, when (1) the content of the specific marker is established - expressed as number of particle per gram of feed additive and (2) the specific marker proposed in this dossier is exclusively used for this feed additive,. 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 Evaluation of the analytical method used to monitor the stability of the product Chapter of Commission Regulation (EC) No 429/2008 specifies that stability is generally measured by the analytical follow-up of the active substance. In this specific dossier the Applicant selected the capability of the product to adsorb aflatoxin B1 (AfB1) from an aqueous solution as surrogate test to establish stability. Experiments to establish the stability have been conducted for the product as such and when added to premixtures and feedingstuffs. The storage conditions were modified in terms of duration of the storage and the temperature, depending on which of these matrices was tested. The adsorption capability of the test samples was monitored depending on the specific storage conditions. The experimental design to assess the adsorption capability of the test samples consisted of the following steps: (1) Addition of the sample (product, premixtures or feedingstuffs,) to a buffer solution containing AfB1 at 4 μg/ml. The added amount of the samples was adjusted to obtain a target concentration of the product in the buffer solution of: %, when the product or the premixtures were analysed, and %, when the feedingstuffs were analysed. (2) Shaking of the mixture at 37 o C for one hour, to allow the adsorption of the AfB1 on the product. While the standard operating procedure [15] prescribes to perform the analysis at ph 5 and 7, the experimental data provided refer to ph 7 ([3], cf ). (3) Injecting an aliquot of the aqueous supernatant without further clean-up into the liquid chromatograph (LC) coupled to diode array detector (DAD) adjusted at 365 nm. 6/9

11 Additional samples without the binder (blank samples) consisting of buffer solutions spiked with AfB1 were treated and analysed in the same way as the actual samples. (4) The adsorption capability of the product was calculated from the peak areas obtained from the LC analyses of the samples (PA sample ) and the blanks (PA blank ) as follows: PA Adsorption (%) = 1 PA sample blank 100 The reversed phase LC method coupled to DAD (LC-DAD) utilised in this study is different from the ISO method [16], which shows a very high sensitivity and selectivity and which is based on immunoaffinity clean-up and fluorescence detection after post-column derivatisation. On request of the CRL the Applicant provided supplementary information of the method performance profile of the LC-DAD method showing that its limit of quantification (LOQ) was μg/ml (AfB1 in buffer). In order to establish, whether this value for the LOQ was sufficient for the purpose of the stability study, the theoretical concentration of AFB1 in the buffer solution was calculated assuming that (1) the buffer solution was originally containing 4μg/ml AFB1 and (2) the measured adsorption of the samples was 95 %, which was the maximal adsorption observed in the stability study [3]. The estimation revealed that the theoretical AFB1 concentration after the treatment would be 0.2 μg/ml. Since the LOQ of μg/ml was below this level, the CRL considers the LC-DAD method fit for the intended purpose. 4. CONCLUSIONS AND RECOMMENDATIONS In the frame of this authorisation the CRL recommends for official control the X-ray diffraction method submitted by the Applicant for the determination of bentonite in the feed additive. The CRL conditionally considers the indirect method (using non-nutrient marker particles) proposed by the Applicant as suitable for the determination of the bentonite in premixtures and feedingstuffs for official control only, when (1) the content of the specific marker is established - expressed as number of particle per gram of feed additive and (2) the specific marker proposed in this dossier is exclusively used for this feed additive. As for the LC-DAD method used by the Applicant for the monitoring of the product stability in the feed additive per se, premixtures and feedingstuffs, the CRL concluded that the Liquid Chromatography with Diode Array Detector (LC-DAD) method is fit for the intended purpose of the stability study. 7/9

12 Recommended text for the register entry (analytical method) For the determination of bentonite in the feed additive: X-ray diffraction (XRD) 5. DOCUMENTATION AND SAMPLES PROVIDED TO CRL In accordance with the requirements of Regulation (EC) No 1831/2003, reference samples of Mycofix Secure have been sent to the Community Reference Laboratory for Feed Additives. The dossier has been made available to the CRL by EFSA. 6. REFERENCES [1] Reference SANCO/D/2 Forw. Appl. 1831/ [2] *Application, Proposal for Register Entry [3] *Technical dossier, Section II [4] *Correspondence, EFSA additional request - FEEFADP/JG/nb (2010): out [5] Commission Regulation (EC) No 776/2006 amending Annex VII to Regulation (EC) No 882/2004 of the European Parliament and of the Council as regards to Community Reference Laboratories [6] *Technical dossier, Section II ANNEX_II_02 [7] *Technical dossier, Section II ANNEX_II_34 [8] *Technical dossier, Section II ANNEX_II_04 [9] *Technical dossier, Section II ANNEX_II_36 [10] *Technical dossier, Section II ANNEX_II_49 [11] *Supplementary Information, CRL-Recalculated-Precision [12] *Technical dossier, Section II ANNEX_II_50 [13] *Technical dossier, Section II ANNEX_II_35 [14] *Supplementary Information, CoA QC [15] *Technical dossier, Section II ANNEX_II_31 [16] ISO 17375:2006: Animal feeding stuffs -Determination of aflatoxin B1 * Refers to Dossier No. FAD RAPPORTEUR LABORATORY The Rapporteur Laboratory for this evaluation was Community Reference Laboratory for Feed Additives, IRMM, Geel, Belgium. This report is in accordance with the opinion of the consortium of National Reference Laboratories as referred to in Article 6(2) of Commission Regulation (EC) No 378/2005, as last amended by Regulation (EC) No 885/ /9

13 8. ACKNOWLEDGEMENTS The following National Reference Laboratories contributed to this report: - Ústřední kontrolní a zkušební ústav zemědělský (ÚKZÚZ), Praha (CZ) - RIKILT-Instituut voor Voedselveiligheid, Wageningen (NL) - Instytut Zootechniki w Krakowie, Krajowe Laboratorium Pasz, Lublin (PL) - Schwerpunktlabor Futtermittel des Bayerischen Landesamtes für Gesundheit und Lebensmittelsicherheit (LGL), Oberschleißheim (DE) - Laboratoire de Rennes, SCL L35, Service Commun des Laboratoires, Rennes (FR) The CRL wishes to thank Jörg Stroka (JRC-IRMM) for his valuable comments related to the evaluation of the analytical method used in the frame of the stability study. 9/9

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