Safety and efficacy of bentonite as a feed additive for all animal species
2017; Wiley; Volume: 15; Issue: 12 Linguagem: Inglês
10.2903/j.efsa.2017.5096
ISSN1831-4732
AutoresGuido Rychen, Gabriele Aquilina, Giovanna Azimonti, Vasileios Bampidis, Maria de Lourdes Bastos, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Gerhard Flachowsky, Jürgen Gropp, Boris Kolar, Maryline Kouba, Marta López‐Alonso, Alberto Mantovani, Baltasar Mayo, Fernando Ramos, Maria Saarela, Roberto Edoardo Villa, Robert John Wallace, Pieter Wester, Giovanna Martelli, Derek Renshaw, Gloria López‐Gálvez, Secundino López Puente,
Tópico(s)Thallium and Germanium Studies
ResumoEFSA JournalVolume 15, Issue 12 e05096 Scientific OpinionOpen Access Safety and efficacy of bentonite as a feed additive for all animal species EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP)Search for more papers by this authorGuido Rychen, Guido RychenSearch for more papers by this authorGabriele Aquilina, Gabriele AquilinaSearch for more papers by this authorGiovanna Azimonti, Giovanna AzimontiSearch for more papers by this authorVasileios Bampidis, Vasileios BampidisSearch for more papers by this authorMaria de Lourdes Bastos, Maria de Lourdes BastosSearch for more papers by this authorGeorges Bories, Georges BoriesSearch for more papers by this authorAndrew Chesson, Andrew ChessonSearch for more papers by this authorPier Sandro Cocconcelli, Pier Sandro CocconcelliSearch for more papers by this authorGerhard Flachowsky, Gerhard FlachowskySearch for more papers by this authorJürgen Gropp, Jürgen GroppSearch for more papers by this authorBoris Kolar, Boris KolarSearch for more papers by this authorMaryline Kouba, Maryline KoubaSearch for more papers by this authorMarta López-Alonso, Marta López-AlonsoSearch for more papers by this authorAlberto Mantovani, Alberto MantovaniSearch for more papers by this authorBaltasar Mayo, Baltasar MayoSearch for more papers by this authorFernando Ramos, Fernando RamosSearch for more papers by this authorMaria Saarela, Maria SaarelaSearch for more papers by this authorRoberto Edoardo Villa, Roberto Edoardo VillaSearch for more papers by this authorRobert John Wallace, Robert John WallaceSearch for more papers by this authorPieter Wester, Pieter WesterSearch for more papers by this authorGiovanna Martelli, Giovanna MartelliSearch for more papers by this authorDerek Renshaw, Derek RenshawSearch for more papers by this authorGloria López-Gálvez, Gloria López-GálvezSearch for more papers by this authorSecundino López Puente, Secundino López PuenteSearch for more papers by this author EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP)Search for more papers by this authorGuido Rychen, Guido RychenSearch for more papers by this authorGabriele Aquilina, Gabriele AquilinaSearch for more papers by this authorGiovanna Azimonti, Giovanna AzimontiSearch for more papers by this authorVasileios Bampidis, Vasileios BampidisSearch for more papers by this authorMaria de Lourdes Bastos, Maria de Lourdes BastosSearch for more papers by this authorGeorges Bories, Georges BoriesSearch for more papers by this authorAndrew Chesson, Andrew ChessonSearch for more papers by this authorPier Sandro Cocconcelli, Pier Sandro CocconcelliSearch for more papers by this authorGerhard Flachowsky, Gerhard FlachowskySearch for more papers by this authorJürgen Gropp, Jürgen GroppSearch for more papers by this authorBoris Kolar, Boris KolarSearch for more papers by this authorMaryline Kouba, Maryline KoubaSearch for more papers by this authorMarta López-Alonso, Marta López-AlonsoSearch for more papers by this authorAlberto Mantovani, Alberto MantovaniSearch for more papers by this authorBaltasar Mayo, Baltasar MayoSearch for more papers by this authorFernando Ramos, Fernando RamosSearch for more papers by this authorMaria Saarela, Maria SaarelaSearch for more papers by this authorRoberto Edoardo Villa, Roberto Edoardo VillaSearch for more papers by this authorRobert John Wallace, Robert John WallaceSearch for more papers by this authorPieter Wester, Pieter WesterSearch for more papers by this authorGiovanna Martelli, Giovanna MartelliSearch for more papers by this authorDerek Renshaw, Derek RenshawSearch for more papers by this authorGloria López-Gálvez, Gloria López-GálvezSearch for more papers by this authorSecundino López Puente, Secundino López PuenteSearch for more papers by this author First published: 18 December 2017 https://doi.org/10.2903/j.efsa.2017.5096Citations: 9 Correspondence: feedap@efsa.europa.eu Requestor: European Commission Question number: EFSA-Q-2016-00561 Panel members: Gabriele Aquilina, Giovanna Azimonti, Vasileios Bampidis, Maria de Lourdes Bastos, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Gerhard Flachowsky, Jürgen Gropp, Boris Kolar, Maryline Kouba, Marta López-Alonso, Secundino López Puente, Alberto Mantovani, Baltasar Mayo, Fernando Ramos, Guido Rychen, Maria Saarela, Roberto Edoardo Villa, Robert John Wallace and Pieter Wester. Acknowledgements: The FEEDAP Panel wishes to thank the following for the support provided to this scientific output (in alphabetical order of the last name): Jaume Galobart, Matteo L. Innocenti, Paola Manini, Manuela Tiramani and Maria Vittoria Vettori. Amendment: An editorial correction was carried out that does not materially affect the contents or outcome of this scientific output. The sentence in Section 2.1 referring to the Bentonite authorisation as food additive, and the footnote linked to it, have been amended. To avoid confusion, the older version has been removed from the EFSA Journal, but is available on request, as is a version showing all the changes made. Adopted: 29 November 2017 Amended: 31 January 2018 AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) received a request from the European Commission to assess the safety and efficacy of bentonite when used as a technological feed additive (substances for reduction of the contamination of feed by mycotoxins) for all animal species. The applicant, EUBA aisbl (European Bentonite Association) representing six companies, submitted to EFSA a technical dossier to support the application. The applicant proposes to use bentonite at the maximum level of 20,000 mg/kg complete feed. The additive apparently interferes with the analysis of aflatoxin B1 in feed. The safety of the additive was already evaluated by the Panel in an opinion delivered in 2012. Bentonites are safe for all animal species, the consumers and the environment when used at a maximum level of 20,000 mg/kg complete feed. The results of a new genotoxicity study reinforced the previous conclusion that smectites are non-genotoxic. Bentonites are not skin irritants but might be mildly irritant to the eye; based on a new study submitted, the additive is not a skin sensitiser. Owing to its silica content, the additive is a hazard by inhalation for the users. The in vitro study showed that the di- and tri-octahedral smectites tested can adsorb aflatoxin B1 at different concentrations and at pH 5; however, no adequate in vivo studies were available. Therefore, the Panel cannot draw conclusions on the additive's efficacy. The Panel further considers the safety and efficacy conclusions to apply equally to the di- and tri-octahedral smectites under assessment. The FEEDAP Panel posted some recommendations regarding the maximum content of other minerals in the additive and the incompatibilities of the additive with other medicinal substances. The Panel also drew a remark concerning the denomination of the additive and the current regulatory definition of Bentonite. 1 Introduction 1.1 Background and Terms of Reference Regulation (EC) No 1831/20031 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. The European Commission received a request from EUBA aisbl (European Bentonite Association)2 for authorisation of the product bentonite, when used as a feed additive for all animal species (category: technological additives; functional group: substances for reduction of the contamination of feed by mycotoxins). 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). EFSA received directly from the applicant the technical dossier in support of this application. The particulars and documents in support of the application were considered valid by EFSA as of 24 October 2016. According to Article 8 of Regulation (EC) No 1831/2003, 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. EFSA shall deliver an opinion on the safety for the target animals, consumer, user and the environment and on the efficacy of the additive bentonite, when used under the proposed conditions of use (see Section 3.1.3). 1.2 Additional information Bentonite is authorised as a feed additive3 under the category technological additives and the functional groups: – 'binder', 'substance for control of radionuclide contamination' and 'anticaking agent' (1m558i), for all animal species and – 'substance for reduction of the contamination of feed by mycotoxins (aflatoxin B1 (AfB1))' for ruminants, poultry and pigs (1m558). The authorisation of Bentonite as food additive expired on the 31st of May 2013.4 The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) has delivered several scientific opinions on the safety and/or efficacy of bentonites when used as feed additives (EFSA FEEDAP Panel, 2011a,b, 2012a5, 2013, 2014, 2016. 2 Data and methodologies 2.1 Data The present assessment is based on data submitted by the applicant in the form of a technical dossier6 in support of the authorisation request for the use of bentonite as a feed additive. The technical dossier was prepared following the provisions of Article 7 of Regulation (EC) No 1831/2003, Regulation (EC) No 429/20087 and the applicable EFSA guidance documents. The FEEDAP Panel used the data provided by the applicant together with data from other sources, such as peer-reviewed scientific papers or other scientific reports. EFSA has verified the European Union Reference Laboratory (EURL) report as it relates to the methods used for the control of bentonite in animal feed. The Executive Summary of the EURL report can be found in Annex A.8 2.2 Methodologies The approach followed by the FEEDAP Panel to assess the safety and the efficacy of Bentonite is in line with the principles laid down in Regulation (EC) No 429/2008 and the relevant guidance documents: Guidance on technological additives (EFSA FEEDAP Panel, 2012b), Technical guidance: Tolerance and efficacy studies in target animals (EFSA FEEDAP Panel, 2011c), Technical Guidance for assessing the safety of feed additives for the environment (EFSA, 2008), Guidance for establishing the safety of additives for the consumer (EFSA FEEDAP Panel, 2012c) and Guidance on studies concerning the safety of use of the additive for users/workers (EFSA FEEDAP Panel, 2012d). 3 Assessment The applicant is representing the consortium of the European Bentonite Association (EUBA aisbl – composed by six companies and founded as the official body representing the European bentonite producers) and is now seeking the authorisation of bentonite when used as AfB1 binder for all animal species (category: technological additives; functional group: substances for reduction of the contamination of feed by mycotoxins). 3.1 Characterisation 3.1.1 Characterisation of the additive The applicant used in the dossier the term 'bentonite' as a synonym for both di-octhaedral and tri-octahedral smectites while the term bentonite generally describes any highly colloidal and plastic clay material composed largely, but not exclusively, of montmorillonite (a species of di-octahedral smectite) without reference to a particular origin. Smectites are phyllosilicates characterised by a sheet structure made of layers of polyhedra of silicon oxide with tetrahedral coordination between which there is an octahedral layer. The octahedral layers contain atoms of aluminium, iron (II or III) or magnesium in their interior. When the cation of the octahedral layer is trivalent, as for example in the case of aluminium, one of every three cation positions is unoccupied; the octahedral layer has the structure of gibbsite, Al(OH)3, and the smectite is di-octahedral: this group includes montmorillonite, beidellite and nontronite. In tri-octahedral smectites, the cation in the octahedral layer is divalent and, as a result, all the cation positions are occupied; this gives rise to an octahedral layer with the geometry of brucite, Mg(OH)2, and it is the case for the saponites and hectorites (Hurlbut and Klein, 1982). The smectite content analysed by X-ray diffraction and batch-to-batch variation of the samples from all companies of the consortium are shown in Table 1.9 It should be noted that some samples contained di-octahedral smectite (montmorillonite), whereas others contained tri-octahedral smectite (saponite). A full mineralogical analysis was provided by five companies, detailing the mineral composition in addition to smectite (Table 1). Table 1. Mineralogical analysis (including content of smectite and other minerals) of the batches provided by each company Company No. of batches (Cation occupancy) Mineralogical analysis (%)a Smectite Other minerals A1 2 (di-octahedral) 85/97 Opal 1/0 Illite 0/7 Plagioclase feldspar 0/3 Quartz 1/2 Calcite 0/2 A2 3 (di-octahedral) 70.9–86.5 Quartz 4.3–14.7 Plagioclase 1.4–6.7 Calcite 0.4–1.3 Muscovite 0–11 Kaolinite 0–6.2 Dolomite 0–1.7 Opal-cristobalite 0–1.6 K-feldspar 0–1.1 Phlogopite 0–1.2 2 (tri-octahedral) 85.5/94.0 Quartz 2.5/3 K-feldspar 1.4 Calcite 0.4/1.3 Plagioclase 0.6/0.8 A3 2 (di-octahedral)b 71.9/76.0 Not analysed A4 1 (di-octahedral) 74.7 Cristobalite 11.6 Calcite 3.4 Feldspar and illite 3.2 Clinoptilolite 3.1 Quartz 0.4 Dolomite 0.4 A5 3 (tri-octahedral) 92–94 Quartz 2–3 Feldspar 1 Plagioclases 1 Calcite 0–3 Illite 0–2 A6 1 (tri-octahedral) 88.0 Dolomite 5 Quartz 4 Feldspar 3 1 (di-octahedral) 89.0 Feldspar 5 Quartz 3 Gypsum 3 a Individual values (when up to two figures) or range (more than two figures). b As determined by the magnesium and aluminium oxides content of the chemical analysis. According to Regulation 1060/2013 (concerning the authorisation of bentonite as a feed additive for all animal species), bentonite used as a mycotoxin binder should contain ≥ 70 % smectite (di-octahedral montmorillonite), < 10 % opal and feldspar and < 4% quartz and calcite. Although all samples contained more than 70% smectite, two batches from company A2, three batches from company A5 and one batch from company A6 containing tri-octahedral smectite do not meet the current legal specification for di-octhaedral montmorillonite minimum level. The FEEDAP Panel also notes that one sample of di-octahedral smectite from company A1, three samples of di-octahedral smectite provided by A2, two samples of tri-octaedral smectite from A510 and one sample of tri-octahedral smectite from A611 exceeded the limit for quartz and calcite set by Regulation 1060/2013 (< 4%) for bentonite when used as a mycotoxin binder. The di-octahedral smectite from the company A4 has a high content of cristobalite, a polymorph of quartz. The main chemical elements which characterise the various clay samples are silicon dioxide (SiO2), aluminium oxide (Al2O3) and magnesium oxide (MgO). They were determined using X-ray fluorescence analysis, inductively coupled plasma optical emission spectrometry or atomic absorption spectrometry.12,13 The results of the analyses are summarised in Table 2. Table 2. Chemical analysis (% values) of the main elements found in the clay samples Company No. of batches (Cation occupancy) Mineral oxides (%)a SiO2 Al2O3 MgO Na2O Fe2O3 A1 1 (di-octahedral) 59.0 17.8 2.2 2.7 5.7 A2 3 (di-octahedral) 57.0–63.0 18.4–19.3 2.4–4.9 0.4–2.1 1.2–1.5 2 (tri-octahedral) 55.6/58.5 4.7/6.7 21.8/24.6 0.3/0.4 0.3/0.4 A3 3 (di-octahedral) 53.4–55.0 16.6–17.0 2.9–4.0 2.2–3.1 4.0–5.3 A4 5 (di-octahedral) 55.1–58.4 22.0–26.1 2.1–3.1 1.0–1.4 3.5–3.9 A5 3 (tri-octahedral) 53.8–54.5 7.6–9.4 18.3–20.4 2.2–2.4 2.9–3.3 A6 1 (tri-octahedral) 53.97 4.02 25.5 2.0 1.2 1 (di-octahedral) 61.70 18.29 2.45 3.3 4.8 a Individual values (when up to two figures) or range (more than two figures). From the comparison between di-octahedral (montmorillonite) and tri-octahedral smectites (saponites), it can be noted that the latter characteristically have lower Al2O3 and higher MgO contents. Values regarding the other compounds are comparable between the two smectite types. At least three batches of the different products from all companies were each analysed for lead and cadmium (25 samples analysed).14 In all cases, values were below the maximum levels specified in the Directive on Undesirable substances15 of 30 mg Pb/kg and 2 mg Cd/kg for additives classified as binders. Arsenic was analysed in at least three batches from five companies (21 samples analysed). Mercury was analysed in at least three batches from four companies (18 samples analysed). Although arsenic and mercury are not specifically regulated for binders, Directive 2002/32/EC sets maximum contents for arsenic in mineral feed of 12 mg/kg and 20 mg/kg in magnesium oxide, which can be taken as indicative. With the exception of one sample (company A5, 21.2 mg/kg), the analysed values for arsenic (range 0.1–19.9 mg/kg) comply with this latter maximum limit. The arsenic content in feed resulting from the use of the additive produced by company A5 would be 0.53 mg As/kg feed; this concentration is below the maximum limit of arsenic (2 mg/kg) in complete feed set by Directive 2002/32/EC. Mercury contents were low and would result in feed concentrations below the value accepted for complete feedingstuffs (0.1 mg Hg/kg). Dioxins were measured in at least three batches from each company (except company A4 and A6 which assayed only one and two batches, respectively) and ranged between 0.08 and 0.55 ng PCDD/F (polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofuran WHO toxic equivalent (TEQ))/kg. Dioxin and dioxin-like polychlorinated biphenyls (PCBs) were measured in at least three batches from four companies and levels were found to range between 0.12 and 0.44 ng PCDD/F-PCBs (WHO-TEQ)/kg.16 All analysed levels were below the limits set for 'clays' in Directive 2002/32/EC. 3.1.1.1 Physical properties The bulk density of the smectite samples shows a wide variation (600–1260 kg/m3), as might be expected given the differences in mineral composition. All samples examined were characterised by a low particle size,17 as determined by laser diffraction, and, where measured, a correspondingly high dusting potential,18 as measured by the Stauber–Heubach method (Table 3). Table 3. Particle size distribution and dusting potential of the smectite samples (n=number of batches) Company Particle size distribution Dusting potential (g/m3) A1 < 10 μm: 17.6–18.6% < 50 μm: 63.8–67.7% < 100 μm: 88.3–92.8% (n = 3) ND A2 < 10 μm: 11.4–18.9% < 50 μm: 46.3–64.2% < 100 μm: 71.8–84.5% (n = 3) 11.4 (n = 1) A3 < 10 μm: 12.4–25.3% < 50 μm: 67.0–78.6% < 100 μm: 91.2–95.8% (n = 3) ND A4 < 10 μm: 3.0–3.3% < 50 μm: 14.0–15.3% < 100 μm: 100% (n= 3) 1.6–2.0 (n = 3) A6 < 45 μm: 67.0–68.8% (n = 3) 8.8–12.3 (n = 3) ND: Not determined 3.1.1.2 Production process The additives are obtained by mining from a variety of locations, including some within the European Union. Extraction is followed by crushing, drying, grinding and packaging for shipment. Some producers use a 'soda activation' process with calcium bentonites, using sodium carbonate to increase the swelling and adsorption behaviour and to raise the pH. 3.1.2 Stability and homogeneity 3.1.2.1 Shelf-life Studies demonstrating stability are generally not required for mineral-based products which are assumed stable. However, three studies were provided by two of the companies. In the two studies from company A3, the smectite content (determined by the methylene blue method) showed no variation after three or four years' storage.19 The third study from company A6 showed that the mineral composition measured by X-ray diffraction of three batches of bentonite remained constant after six years' storage.20 3.1.2.2 Homogeneity A single sample of smectite (from company A6), which included cobalt (100 mg/kg feed) as a microtracer, was mixed with an unspecified feed.21 A total of 15 samples were collected and assayed for the cobalt content. Using this method, the microtracer showed good distribution with a coefficient of variation of 4.2%.22 3.1.2.3 Interference with the analysis of mycotoxins in feed The applicant provided a study in which a feed for dairy cows was spiked with 5 μg/kg aflatoxin B1 in the presence or absence of the samples of bentonite provided from all companies (supplemented feed with 20,000 mg/kg of bentonite, either di- or tri-octahedral). In the absence of smectites, aflatoxin was recovered at the expected concentration. However, in the presence of both di- and tri-octahedral smectites, recoveries of aflatoxin were reduced by approximately 40%. 3.1.3 Conditions of use The applicant proposes the use of bentonite as an aflatoxin B1 binder in feed for all animal species at a maximum inclusion level 20,000 mg/kg complete feedingstuffs. 3.2 Safety In the previous opinion based on a technical dossier on bentonite submitted by the same applicant of the current submission, the safety of the additive was assessed (EFSA FEEDAP Panel, 2012a).23 3.2.1 Safety for the target species The FEEDAP Panel concluded that bentonite at 20,000 mg/kg complete feed was safe for all animal species (EFSA FEEDAP Panel, 2012a). The conclusion was based on tolerance studies with a tri-octahedral smectite24 in lactating cows, piglets and chickens for fattening, and two literature studies25 on laying hens and fish. In the previous opinion, the Panel also noted that the data available suggested that addition of bentonites to diets was incompatible with the use of robenidine as a coccidiostat, and that levels of bentonite higher than 0.5% are also expected to reduce the effectiveness of other coccidiostats (EFSA FEEDAP Panel, 2012a). Moreover, the FEEDAP Panel drew a recommendation concerning the simultaneous use of bentonites with other medicinal substances. No new data or studies have been provided in the current application. 3.2.2 Safety for the consumers The FEEDAP Panel concluded in its previous opinion that there was no concern for the safety of consumers of food products derived from animals fed diets containing bentonite" (EFSA FEEDAP Panel, 2012a). In the current submission, the applicant has provided a new genotoxicity study. Bentonite (di-octahedral smectite) was examined for the potential to induce gene mutations using mouse lymphoma L5178Y cells (OECD guideline 476) both with and without metabolic activation with S9 from livers of phenobarbital/β-naphthoflavone treated rats.26 The dose tested under both conditions ranged from 312.5 to 5,000 μg/mL. No increases in mutant frequency were seen at any dose with or without activation thus it is concluded that bentonite is not mutagenic in vitro under the conditions used. 3.2.3 Safety for the users The FEEDAP Panel concluded in its previous opinion that bentonites were not skin irritants but might be mildly irritant to the eye. Skin sensitisation was not considered, but the Panel noted that bentonites are widely used in cosmetics. The Panel also considered it prudent to assume that all bentonite dusts posed a hazard to those handling the additive (EFSA FEEDAP Panel, 2012a). In the current submission, the applicant has provided two studies relating to users safety, which address the data gaps identified previously. An acute inhalation toxicity (nose only) study was performed using six Wistar rats (three males and three females) exposed to 0 or 5.3 mg bentonite (di-octahedral smectite)/L air for 4 h followed by an observation period of 14 days (OECD guideline 436).27 One death occurred at 170 min after exposure. Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur. Surviving animals recovered to appear normal on day 4 post-exposure. With the exception of one female, animals had reasonable body weight gain during the observation period. No macroscopic abnormalities were detected at necropsy among animals that survived until the end of the 14-day observation period. Pale lungs were noted at necropsy in the animal that died during the course of the study. A local lymph node assay (LLNA) was performed using female CBA/Ca mice (OECD guideline 429).28 The concentration of bentonite (di-octahedral smectite) at which no toxic signs were observed (25%) was selected in a preliminary screening test and used as the highest dose investigated. Three groups (five mice/group) were treated with 50 μL (25 μL per ear) of the test item as a suspension in propylene glycol at concentrations of 25%, 10% or 5% w/w. No proliferation response was elicited by the treatment. No clinical signs, mortality or changes in body weight were observed. The test item under assessment is not considered a skin sensitiser. The FEEDAP Panel also notes that the additive contains crystalline silica (range 1–14.7%). Inhalation of silica is known to be hazardous and is associated with increased risk of lung cancer and the industrial disease, silicosis. The EC Scientific Committee on Occupational Exposure Limits (SCOEL) concluded that silicosis is the main effect of respirable crystalline silica (European Commission, 2003) and proposed an occupational exposure limit of 0.05 mg/m3 of respirable silica dust. 3.2.4 Safety for the environment The FEEDAP Panel concluded in its previous opinion that bentonites are ubiquitous in the environment, being natural components of soil; therefore, it was not expected that its use as a feed additive would adversely affect the environment. No new data or studies have been provided in the current submission. 3.2.5 Conclusions on the safety of the additive The FEEDAP Panel reiterates its previous conclusion that bentonites are safe for all animal species, the consumers and the environment when used at a maximum level of 20,000 mg/kg complete feed. The results of a newly submitted genotoxicity study reinforced the previous conclusion that smectites are non genotoxic. The FEEDAP Panel maintains its previous conclusion that bentonites are not skin irritants but might be mildly irritant to the eye. Based on a new study submitted, the additive is not a skin sensitiser. Owing to its silica content, the additive is a hazard by inhalation for the users. The Panel considers these conclusions to apply equally to the di- and tri-octahedral smectites under assessment. 3.3 Efficacy As the substances used for the reduction of the contamination of feed by mycotoxins do not affect the characteristics of feed but produce their effects in the animal, efficacy can only be fully demonstrated by in vivo studies. In vitro studies are considered as a screening tool for the potential of substances to act as mycotoxin binders. However, in vitro studies alone cannot be used to demonstrate efficacy under practical conditions (EFSA FEEDAP Panel, 2012b). In vitro study The binding capacity of the additive towards AfB1 has been studied in vitro, according to the provisions set by Regulation (EU) No 1060/2013.29 For each batch of smectite from the different producers (one sample each from companies A1 and A3, two samples from company A6, three samples each from companies A4 and A5, five samples from company A2), an adsorption isotherm was prepared with a concentration of 4 μg AfB1/mL at pH 5. The inclusion level of the additive was 0.2 mg/mL (0.02% w/v) in all cases. A control (a solution with mycotoxin without binder) and test solutions containing both mycotoxin and binder were prepared. The suspensions were shaken and incubated at 37°C for 60 min and then centrifuged for 15 min. AfB1 concentrations were determined in the supernatants by high performance liquid chromatography. The binding capacity was calculated from the difference in mycotoxin concentration in the control and the supernatant of the incubated binder sample. The results indicate that the efficacy of the additive in adsorbing AfB1 varied from 90.0% to 95.3% at pH 5. In vivo study The applicant submitted only one study on dairy cows performed with the product from company A6.30 The study reports a survey carried out in ten Italian dairy farms with a total of 742 animals. The cows ingested naturally contaminated feed ingredients; analysis of corn and complementary concentrates from three farms
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