SCIENTIFIC OPINION. Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed. (Question No EFSA-Q )

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1 The EFSA Journal (2008) 839, 1-40 SCIENTIFIC OPINION Safety and efficacy of Advastat (containing 10 % acarbose produced by Actinoplanes utahensis CBS ) as feed additive for cattle for fattening and dairy cows 1 Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed PANEL MEMBERS (Question No EFSA-Q ) Adopted on 22 October 2008 Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Bogdan Debski, Noël Dierick, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Joop de Knecht, Lubomir Leng, Sven Lindgren, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Miklós Mézes, Carlo Nebbia, Walter Rambeck, Guido Rychen, Atte von Wright and Pieter Wester SUMMARY Following a request from the European Commission, the European Food Safety Authority (EFSA) was asked to deliver a scientific opinion on the safety and efficacy of Advastat (containing acarbose as active substance) as feed additive for cattle for fattening and dairy cows, under the category zootechnical and functional group digestibility enhancer. The applicant claims two benefits from using Advastat : an effect on animal welfare (reduction of the risk of acidosis) and on animal performance (more favourable ruminal fermentation). Acarbose is already used in human medicine as an oral first-line or adjunctive treatment for reducing postprandial hyperglycaemia in Type II diabetes mellitus. Advastat contains 10 % acarbose, a tetrasaccharide analogue produced by fermentation with Actinoplanes utahensis, which inhibits the activity of gastrointestinal amylases and glucosidases. Advastat is intended to be used in feed of ruminating beef and dairy cattle to maintain optimal rumen ph (>5.5) by slowing down starch fermentation in the rumen. The maximum dose proposed by the applicant for Advastat use is that equivalent to 1.2 mg acarbose kg -1 body weight (bw), corresponding approximately to 70 mg kg -1 and 35 mg 1 For citation purposes: Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) on a request from the European Commission on the safety and efficacy of Advastat for cattle for fattening and dairy cows. The EFSA Journal (2008) 839, 1-40 One member of the Panel did not participate in the discussion on the subject referred to above because of possible conflicts of interest European Food Safety Authority, 2008

2 acarbose kg -1 complete feedingstuffs (dry matter) for cattle for fattening and dairy cows, respectively. In vitro studies (six) used to evaluate the effects of acarbose either on rumen fluid or ruminal metabolism, indicated that acarbose dose-dependently reduces rumen fermentation and contributes to stabilising ruminal ph. In vivo trials included fistulated beef cattle fed acidotic rations and non-fistulated dairy cattle receiving high starch diets. Five short-term studies (two to six days) showed that acarbose reduced the risk of acidosis, as characterised by ruminal ph or lactate concentration. Four trials were conducted on dairy cows, with a cross-over design with three-week periods. The animals were fed a diet expected to induce sub-optimal rumen ph with or without acarbose. Although those studies showed that acarbose increased milk fat and yield but also decreased protein percentage, they were considered of too short duration. Upon EFSA s request, three additional trials (84-87 days duration) were performed at in commercially operating dairy farms in the EU in order to study the effects of acarbose in early lactation ration using high amounts of fermentable carbohydrates on milk yield. Acarbose-treated animals showed positive or negative changes, sometimes significant, in milk production and composition compared to control animals. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) cannot conclude on the efficacy of Advastat for cattle for fattening and dairy cows because benefits in healthy animals, under common farming practices in the EU, could not be demonstrated. However, the former conclusion does not exclude that acarbose might be efficacious in cases where cattle for fattening and dairy cows are at a specific risk or suffering of acidosis due to the particular feeding regime. A tolerance study, which aimed at evaluating the safety of acarbose in lactating cows when added in feed at 0, 35 and 350 mg acarbose kg -1 complete feed DM, was provided; this study did not provide evidence of toxicity with a 10-fold acarbose overdose. The FEEDAP Panel concludes that acarbose is safe at the level of 35 mg kg feed -1 DM in dairy cows. The FEEDAP Panel also considers likely that the conclusion for dairy cows is valid for cattle for fattening. Orally administered labeled acarbose is metabolised extensively in the digestive tract before absorption to a limited extent. No unchanged acarbose or C2 (both pharmacologically active) are found in excreta, milk and edible tissues from cattle administered acarbose. From the different toxicity studies provided, it can be concluded that acarbose is not genotoxic and most effects observed, even at the lowest dose tested (decrease in body weight, 45 mg kg -1 bw), can be related to its pharmacological activity. No NOEL, and consequently no ADI, could be derived from the data provided. However, the consumer will not be exposed to pharmacologically active acarbose or its metabolite (C2) following consumption of food from treated animals. Consequently, the FEEDAP Panel concludes that no safety concerns for the consumer would result from the use of acarbose at the proposed level in beef and dairy cattle. No marker residue could be identified, nor is it considered necessary. In the absence of data on eye and dermal irritation potential, and on skin/respiratory sensitisation, the default approach is to consider the compound as a potential irritant and sensitiser. In the absence of information on potential effects of metabolites on the environment, the FEEDAP Panel cannot fully assess the risk for the environment arising from the use of Advastat in feedingstuffs for cattle. The EFSA Journal (2008) 839, 2-40

3 The FEEDAP Panel made some recommendations for modifications to the Register entry proposed by the applicant. Key words: zootechnical additive, digestibility enhancers, cattle for fattening, dairy cows, Advastat, acarbose, ruminal acidosis, animal welfare The EFSA Journal (2008) 839, 3-40

4 TABLE OF CONTENTS Panel Members... 1 Summary... 1 Table of Contents... 4 Background... 6 Terms of reference... 6 Acknowledgements... 6 Assessment Introduction Characterisation of the product Product identity Characterisation of the active substance Physical properties Purity Purity of Advastat Purity of acarbose Manufacturing process Acarbose Stability Shelf life of the additive Stability in feed formulations Stability after pelleting Homogeneity Proposed mode of action Physiological significance of disorders in rumen fermentation and their prevalence Ruminal acidosis Prevalence of SARA Evaluation of the analytical methods by the Community Reference Laboratory (CRL) Efficacy In vitro studies Reading Pressure Technique (RPT) study Rumen Fermentation Profiling Laboratory (RFPL) study Conclusions from in vitro studies In vivo studies Short-term acidotic challenge Efficacy studies in dairy cows Conclusions on efficacy Safety Safety for the target species Study in beef cattle Study in dairy cows Microbiological Safety Conclusions on safety for target species Metabolism and residues Metabolism Residues Conclusions on metabolism and residues Studies on laboratory animals Acute toxicity Genotoxicity/Mutagenicity Sub-chronic (90-day) oral toxicity Chronic toxicity/carcinogenicity...32 The EFSA Journal (2008) 839, 4-40

5 Reproduction toxicity/teratogenicity Conclusions on laboratory animal studies Consumer safety Safety for the worker/user Safety for the environment Postmarket monitoring Conclusions and Recommendations Conclusions Recommendations Documentation provided to EFSA References Appendices The EFSA Journal (2008) 839, 5-40

6 BACKGROUND Regulation (EC) No 1831/ establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lies down that any person seeking an 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 the company Pfizer Limited 3 for authorisation of the product Advastat 4 (containing 10 % acarbose produced by Actinoplanes utahensis CBS ) to be used as a feed additive for cattle for fattening and dairy cows (category: zootechnical additive; functional group: digestibility enhancer) under the conditions mentioned in Table 1. According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 4.1 (authorisation of a feed additive or new use of a feed additive). EFSA received directly from the applicant the technical dossier in support of this application. 5 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 30 October The product Advastat has not been previously authorised as feed additive at Community level. 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 efficacy and the safety for the target animal(s), user and consumer and the environment, of Advastat (containing 10 % acarbose produced by Actinoplanes utahensis CBS ) when used under the conditions described in Table 1. ACKNOWLEDGEMENTS The European Food Safety Authority wishes to thank the members of the Working Group on Advastat as well as Andrew Chesson for the preparation of this opinion. 2 OJ L 268, , p.29 3 Pfizer Limited. Sandwich, Kent, CT13 9NJ, UK 4 According to the applicant the final name of the product will be ACITEK (see Documentation provided to EFSA) 5 Dossier reference: FAD The EFSA Journal (2008) 839, 6-40

7 Table 1. Register entry as proposed by the applicant Additive Advastat Registration number/ec No/No Category of additive Functional group of additive Pending Zootechnical Digestibility enhancer Composition, description Premixture of acarbose in carrier Chemical formula Acarbose, 10% Up to 1% light liquid paraffin Soybean mill feed to 100% Description Purity criteria (if appropriate) Complies with EU law on undesirable substances Method of analysis (if appropriate) HPLC Trade name Advastat Name of the holder of authorisation Pfizer Animal Health Conditions of use Species or category of animal Maximum Age Minimum content Maximum content mg kg -1 of complete feedingstuffs Withdrawal period Cattle for fattening ppm dry matter of total feed Nil Dairy cows for milk production ppm dry matter of total feed Nil Other provisions and additional requirements for the labelling Specific conditions or restrictions for use Specific conditions or restrictions for handling Post market monitoring Specific conditions for use in complementary feedingstuffs Advastat should be added to the cereal concentrate part of the ration to provide a maximum dose of 1.2 mg of acarbose per kg body weight. This will require Advastat concentrations in the region of ppm in the cereal concentrate, depending upon class of stock and daily concentrate intake (see following table of example dosage calculations). Store in original closed packaging, in a cool, dry place. Avoid direct contact and use suitable protective clothing For user safety: breathing protection during handling, and safety glasses. Pfizer Animal Health will conduct post-marketing monitoring in compliance with EU law on feed hygiene, namely by use of HACCP and Traceability systems, routine post-marketing sampling and analysis, and formal monitoring of customer feedback through product or service complaints The cereal concentrate containing Advastat may be fed separately or may be incorporated into a total mixed ration, according to normal farm practice. The EFSA Journal (2008) 839, 7-40

8 Marker residue Maximum Residue Limit (MRL) (if appropriate) Species or category of animal Not applicable Target tissue(s) or food products Maximum content in tissues Example dosage calculations BW (kg) Acarbose daily dose (mg) DMI (kg/day) Acarbose (ppm DM) Acarbose (ppm) in cereal concentrate Advastat (ppm) in cereal concentrate Fattening cattle (at 1.2 mg/kg) Daily intake of cereal concentrate 3 kg/day 4 kg/day 5 kg/day 3 kg/day 4 kg/day 5 kg/day ,400 1, ,600 1, ,800 1,350 1,080 Daily intake of cereal concentrate Dairy cows (at 1.1 mg/kg) 6 kg/day 8 kg/day 10 kg/day 6 kg/day 8 kg/day , , , , Notes: BW = body weight; DMI = dry matter intake; DM = dry matter. 10 kg/day The EFSA Journal (2008) 839, 8-40

9 ASSESSMENT 1. Introduction Advastat is the brand name of a product containing 10 % acarbose as active substance. Acarbose is a tetrasaccharide analogue of microbial origin which inhibits the activity of gastrointestinal amylases and glucosidases. Advastat is intended to be used in feed of ruminating beef and dairy cattle to maintain optimal rumen ph (>5.5) by slowing down starch fermentation in the rumen. Thus, the applicant claims two benefits from using Advastat : effect on animal welfare (reduction of the risk of acidosis) and on animal performance (more favourable ruminal fermentation). The maximum amount of acarbose from Advastat proposed by the applicant is 1.2 mg acarbose kg -1 body weight (bw), corresponding approximately to 70 mg kg -1 and 35 mg acarbose kg -1 complete feedingstuffs (dry matter) for cattle for fattening and dairy cows, respectively. Acarbose is used in human medicine as an oral first-line or adjunctive treatment for reducing postprandial hyperglycaemia in Type II diabetes mellitus (dosages of 150 to 600 mg person -1 day -1 ). It is shown to slow down the degradation of starch and disaccharides in the small intestine (Chiasson et al., 1994). 2. Characterisation of the product 2.1. Product identity Advastat is based on the active substance acarbose, a fermentation product, diluted in a soybean mill feed (10 % acarbose, 90 % soybean mill feed). To control product dust, up to 1 % light liquid paraffin may be included in the formulation. Batch-to-batch variation of six lots of Advastat ranged from 99 to 105 % of the acarbose content declared on the label, with a relative standard deviation (RSD) of 2 %. All six batches were within the product specification (90 to 110 % of the labelled amount of acarbose). The soybean mill feed used in the manufacture of Advastat is routinely assayed for the absence of genetically modified soybean Characterisation of the active substance Acarbose, the active substance in Advastat, is a white or yellowish, hygroscopic, amorphous powder which is very soluble in water and in methanol and practically insoluble in methylene chloride. The empirical formula is C 25 H 43 NO 18, with MW of 646 daltons, CAS number and EC (EINECS) number The structural formula of acarbose is given in Figure 1. The systematic IUPAC nomenclature is (2R,3R,4S,5R,6R)-5-[(2R,3R,4S,5R,6R)-5- [(2R,3R,4S,5R,6R)-3,4-dihydroxy-6-methyl-5-[[(1S,4S,5S,6S)-4,5,6-trihydroxy-3- hydroxymethyl)-1-cyclohex-2-enyl]amino]oxan-2-yl]oxy-3,4-dihydroxy-6- (hydroxymethyl)oxan-2-yl]oxy-6-(hydroxymethyl)oxane-2,3,4-triol. The EFSA Journal (2008) 839, 9-40

10 Figure 1. Structural formula of acarbose 2.2. Physical properties The bulk density of Advastat not measured is likely to be similar to that of the soybean mill feed (0.52 to 0.64 g (cm 3 ) -1 ), which constitutes 90 % of the product. 6 Over 90 % of Advastat particles are large (2460 to 3500 μm, typical of the soybean mill feed). 7 No further information on the particle size of Advastat was provided. However, an analysis of particle size of acarbose itself, which constitutes 10 % of Advastat, showed a mean diameter of 50 μm. Around 70 to 75 % of acarbose particles exhibit a diameter inferior to 100 μm, which is considered inhalable. Approximately 30 % of acarbose particles are inferior to 25 μm in diameter and around 15 % inferior to 10 μm in diameter, the latter fraction considered respirable. The dusting potential of Advastat was measured using the Stauber-Heubach method. The mean of two lots was 0.73 %. The data suggest that Advastat is a product with low dusting potential Purity Purity of Advastat Three batches of Advastat were tested for impurities (microbial quality, heavy metals, aflatoxins and dioxins), using internationally recognised analytical methods. 8 All three batches exhibited adequate microbial quality. 9 Heavy metals and fluorine contents were low and not of concern. The sum of dioxins and dioxin-like PCBs was below 0.3 ng WHO PCDD/F- PCBTCQ kg Purity of acarbose Acarbose has a purity of % on DM content 11 (Ph.Eur. Monographs, 2005). Acarbose contains several other glucopyranosides and glucopyranoses specified as impurities; the maximum level of each impurity is specified in the Ph.Eur. Monograph Manufacturing process Advastat is manufactured by mixing acarbose with soybean mill feed and light liquid paraffin (up to 1 % may be added to control dust) Technical Dossier Section II, Annex II.1.c Technical Dossier Section II, Annex II.6 Technical Dossier Section II, Annexes II.10a to II.10h 9 Technical Dossier Section II, Table II.6 10 According to Commission Directive 2006/13/EC, 1.5 ng kg -1 are permitted in ruminant feed 11 Technical Dossier Section II, Annex II.2 The EFSA Journal (2008) 839, 10-40

11 Acarbose The active substance acarbose is produced by fermentation of a non-gmo strain of Actinoplanes utahensis (strain SE 50), deposited as strain no. CBS by Bayer AG in 1970 at the Centraalbureau voor Schimmelcultures in The Netherlands. The acarbose used in Advastat is produced by Bayer Healthcare AG under GMP conditions. The applicant has provided sufficient evidence that the current production strain is a natural variant of the strain originally deposited. 12 The fermentation process is described in detail in the dossier. 13 Acarbose is obtained from the final fermentation broth by several steps of isolation (ion-exchange), concentration and purification (ion-exchange chromatography). The resulting product fulfils the criteria laid down in the Ph.Eur. Monograph (2005) Stability Shelf life of the additive Formulations of Advastat with and without light liquid paraffin were evaluated at different storage conditions (2-8 C; 25 ºC/60 % RH; 30 ºC/65 % RH; 40 ºC/75 % RH). 14 Two batches without liquid paraffin (stored in paper bags and foil sachets, respectively) showed satisfactory stability at 25 C over 24 months (95.2 and 95.6 % of the acarbose intended level). Only the 18 months data for the batch produced with liquid paraffin were submitted (104 %). The data support the proposed shelf-life of two years (storage in closed bags in a cool ( 25ºC), dry environment) Stability in feed formulations The stability data below, as well as the homogeneity data, were calculated applying an extraction efficiency of 90 % without providing data to justify this. Each reported result is the average of two (occasionally more) replicates from a single sample. A sample of Advastat diluted in soybean meal (nominal concentration 215 mg acarbose kg -1 ) was evaluated for stability through eight weeks at 25ºC/60% RH and 30ºC/65% RH. 15 After storage for eight weeks, samples stored at 25 ºC/60 % RH had an average acarbose content of 79 % compared to the initial content, but the result may be influenced by the high initial value (248 mg, nominal 215 mg kg -1 ). Results from samples stored for eight weeks at 30 ºC/65 % RH showed 93 % of the initial concentration. Separate samples from two batches of wheat supplemented with Advastat (nominal concentration 500 mg acarbose kg -1 ) were evaluated for stability through eight weeks at 25 ºC/60 % RH and 30 ºC/65 % RH, respectively. 16 After storage for eight weeks, both samples stored at 25 ºC/60 % RH and 30 ºC/65 % RH still contained between 92 and 112 % of the initial concentration. Samples from eight batches of Advastat in cereal concentrate with minerals (wheat, barley, soybean meal, sugar beet pulp, 2 % minerals) from feeding trials, were tested for acarbose stability (nominal concentration 500 mg acarbose kg -1 ). 17 Stability data are available for cereal concentrates at different time intervals. Sample age at the time of testing ranged from less than 12 Technical Dossier Section II, Annex II.3 13 Technical Dossier Section II 14 Technical Dossier Section II, Annex II.4a to II.4c 15 Technical Dossier Section II, Annex II.5a 16 Technical Dossier Section II, Annex II.5b 17 Technical Dossier Section II, Annex II.5c The EFSA Journal (2008) 839, 11-40

12 one week to 14 weeks. For the six samples stored for 6-14 weeks, acarbose recovery was % of the nominal content. The data support at least a two-month stability of Advastat in feedingstuffs. However, typical concentrates for cattle/dairy cows were not examined. Stability in premixtures, mineral concentrates and TMRs could not be assessed due to the lack of data Stability after pelleting A representative feed (nominal content 110 mg acarbose kg -1 ) was steam pelleted at C. 18 Recovery of acarbose from pelleted samples was about 90 % of the nominal amount. Samples were tested again about eight months later with a recovery of 85 % of the nominal amount. The data demonstrate that breakdown of acarbose during pelleting and after eight months storage of pelleted feed is not significant. However, the composition of the feed was not given Homogeneity Each reported result is the average of two (occasionally more) replicates from each of two samples. Samples from 24 batches of Advastat in soybean meal were tested for acarbose content. 19 The nominal concentration was 215 mg acarbose kg -1. Five lots showed results below 80 % of nominal while the other 19 lots ranged between 88 and 119 % of nominal. The RSD (except the five batches below 80 %) varied between 4.7 and 18.8 %. Samples from 49 batches of Advastat in wheat were tested for acarbose content. 20 The nominal concentration was 500 mg acarbose kg -1. One lot showed results below 80 % of nominal while the other 48 lots ranged between 84 and 123 % of nominal concentration. The RSD varied between 1.3 and 11.9 %. Acarbose from Advastat is considered to be homogenously distributed in feed materials. However, homogeneity in typical feedingstuffs for beef cattle and dairy cows (complementary feedingstuffs, mineral mixtures, TMRs) could not be assessed due to the lack of data Proposed mode of action There is a well-established mode of action in humans, involving the inhibition of α- glucosidase activity. The applicant claims a similar mode of action in ruminal function. The inhibition of two microbial enzyme systems (amylase and glucosidase) during ruminal starch degradation would result in a slower fermentation, thus reducing the decrease of ph and leading to greater amounts of starch reaching the small intestine. Consequently, digestive problems associated with ruminal ph below 5.5 will be reduced. As a result, Advastat when used as feed additive may favourably affect animal welfare and performance Physiological significance of disorders in rumen fermentation and their prevalence Ruminal acidosis Ruminal acidosis is a well-known digestive disorder. The aetiology of ruminal and systemic acidosis has been described in comprehensive reviews (Baldwin and Allison, 1983; Huntington, 1988; Nocek, 1997). 18 Technical Dossier Section II, Annex II.5d 19 Technical Dossier Section II, Annex II.5a 20 Technical Dossier Section II, Annex II.5b The EFSA Journal (2008) 839, 12-40

13 Acute acidosis results from incidental overconsumption of highly fermentable carbohydrates (Owens et al., 1998; Krause and Oetzel, 2006; Martin et al., 2006). As a consequence, the rumen ph decreases below ph 5.0 and lactic acid (LA) accumulates in the rumen (up to 300 mmol L -1 rumen fluid) and blood (Enemark et al., 2002; Enemark et al., 2004; Krause and Oetzel, 2006). Subacute (also called latent or subclinical) ruminal acidosis (SARA) is a more frequent situation in high yielding cows fed high starch-containing diets showing suboptimal rumen ph. SARA is not related to LA but to the accumulation and changes of the profile of volatile fatty acid (VFA) in rumen (lowered acetate/propionate ratio) as a consequence of a decreased cellulolytic and increased amylolytic ruminal flora. Besides a lowered milk fat content, those ruminal events can have pathological consequences such as abomosal displacement, liver abscesses and locomotor complications. A schematic view of factors which may indirectly affect the occurrence of SARA is given in Appendix B Prevalence of SARA Literature on the prevalence of SARA is very scarce (Table 2). Limited knowledge of the symptomatology as well as of the quality of the diagnostic methods normally applied in practice is likely to be among the most important reasons for that. SARA is a common problem in well-managed dairy herds and should be regarded as a herd rather than an individual problem (Enemark et al., 2004; Kleen, 2004). Both authors attributed SARA to a depression of rumen ph to ; others (Sauvant et al., 2006) characterised SARA using a wider ph range (between 5.5. and 6.2). Under those conditions, the total concentration of VFA is increased, with a shift in the VFA profile towards propionic acid, butyric acid and valeric acid, and the LA concentration in the rumen does not exceed 5-10 mmol L -1. Table 2. Location Prevalence of SARA during early and mid lactation in lactating cows (ph, VFA and lactic acid measured after ruminocentesis) Herds (n) Total animals (Animals examined) Overall prevalence (%) Prevalence per individual herd (%) Reference The Netherlands (197) Kleen, 2004 Italy (120) Morgante et al., 2007 Ireland 12 (144) O Grady et al., 2008 USA 15 19/26 1 Garrett et al., (737) 20 Oetzel, 2004 Australia 100 (797) 10.2 Bramley et al., first figure: early lactation, second figure: mid lactation Table 2 may indicate that the prevalence of SARA during early or mid lactation in Europe may be lower than in the USA. However, as much as 38 % of individual dairy cows may be affected in a given herd in Europe Evaluation of the analytical methods by the Community Reference Laboratory (CRL) EFSA has verified the CRL report concerning the analytical method(s) for Advastat. The executive summary of the report is attached in Appendix A. The EFSA Journal (2008) 839, 13-40

14 3. Efficacy Six in vitro studies were performed in order to evaluate the effects of acarbose on rumen fluid. Eleven in vivo studies (seven in cows and four in cattle for fattening) were also submitted In vitro studies 21, 22, 23, 24 Four studies with rumen fluid and applying different concentrations of acarbose showed that acarbose could reduce α-amylase and glucosidase activity in the fluid but also propionic acid production and could contribute therefore to maintaining stable rumen ph above 5.5. Because of the lack of proper statistical analysis, those studies were not further considered as demonstration of efficacy. Two other studies (two trials each) are described below Reading Pressure Technique (RPT) study Two trials were conducted in order to examine the effects of acarbose at different concentrations on various starch sources and substrates, using the Reading Pressure Technique (RPT) (Mauricio et al., 1999). 25 This system uses an in vitro batch culture that mimics rumen fermentative activity and simultaneously estimates degradation and fermentation kinetics. Gas production and volatile fatty acid production were examined in those experiments. Data of both experiments were statistically analysed using the GLM procedure (SAS Edition 6.1). Trial 1 Different starch sources (wheat, maize) were incubated in the presence of 0, 0.1, 0.5, 1.0, 2.5, 5.0, 10, 25, 50 and 100 μg acarbose ml -1 fluid. Samples were taken at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 30, 36 and 48 hours post-inoculation. Table 3. Influence of acarbose on gas production (ml g -1 organic material) Substrate Maize Wheat Acarbose levels Hours post-incubation (µg ml -1 ) a a a 24.6 bc 81.9 b bc 23.6 bc 78.2 b bc 20.1 d 58.7 d cd 16.0 e 33.1 f f a abc bc ab d 61.2 a 54.4 b 47.8 bcd 44.0 cde 25.8 g abc a abc d 88.7 g ab a ab ab c a a a a a a-g: For a given substrate, means within columns without common letters are significantly different (P <0.05) Table 3 indicates that acarbose has the potential to significantly reduce (dose-related effect) gas production mainly in the first hours following incubation. A much greater treatment effect was found with maize compared to wheat. Trial 2 The second trial (protocol similar to trial 1) used four inclusion levels of acarbose (0, 1, 10 and 100 μg ml -1 ) to examine the inhibition of starch degradation on various substrates to represent 21 Technical Dossier Section III, Annex III.1 22 Technical Dossier Section III, Annex III.2 23 Technical Dossier Section III, Annex III.3 24 Technical Dossier Section III, Annex III.4 25 Technical Dossier Section III, Annexes III.5 (Trial 1) and III.6 (Trial 2) The EFSA Journal (2008) 839, 14-40

15 specific groups of ruminant feeds, such as (a) cereal grains: barley, wheat, maize, flaked maize, hominy; (b) high pectin by-products: sugar beet pulp, citrus pulp; (c) low value byproducts: almond hulls, brewer s grains, rice bran; (d) protein supplements: field beans, blackeyed beans; (e) root starch: cassava, yam, potato and (f) pure carbohydrates: glucose, maltose and cellulose. This trial indicates that acarbose has the potential to significantly reduce gas production for the various studied substrates, mainly in the first hours following incubation. Significant effects were found for all the substrates except sugar beet and citrus pulp Rumen Fermentation Profiling Laboratory (RFPL) study The in vitro model applied, similar to the RUSITEC technique (long-term rumen continuous fermentation simulation technique), measured starch, protein and fibre digestion as well as microbial growth. 26 Two trials were carried out. Sodium bicarbonate concentration was maintained at a level of 3.5 mg ml -1 to produce a minimum ph range of post feeding in the control fermenters. Each addition of acarbose was fermented in triplicate nine-day fermentation periods. Effluent samples analysis were taken during the last three days; the parameters measured were: ph, fibre, starch, sugar, propionate and butyrate production. Data were analysed using the General Linear Model Procedure of SAS. In addition, a Duncan's Multiple Range Test was used to compare individual treatment means. Trial 1 Three concentrations of acarbose (2.8, 28 and 280 μg ml -1 ) were tested using a high starch ration. Inoculum donor cows were maintained on a high starch diet (28.4 %) consisting of 22.8 % corn silage, 12.6 % mixed haylage, 10.1 % alfalfa hay and 54.5 % grain mix (ground corn, soybean meal, soy hulls, corn gluten meal). Table 4. Effects of various acarbose concentrations on VFA production and ph (high starch diet) VFA Production Acarbose (μg ml -1 ) (Mmoles d -1 Control ) Total VFA 277 a 244 b 251 b 239 b Acetic acid 143 a 129 b 135 b 127 b Propionic acid 82 a 61 b 47 c 45 c Butyric acid 28 d 35 c 45 b 53 a Average ph 5.57 c 5.65 b 5.67 b 5.88 a a-d: in a same row, values with different superscripts differ significantly, P <0.05 The results demonstrated that the three concentrations tested were effective in moderating fermentation and stabilising ph in relation to the buffer control, with the highest dose resulting in an almost constant ph for 12 hours after feeding. With increasing concentration of acarbose, VFA production showed a significant decrease in propionate and acetate and a large increase in butyrate while proportions of acetate remained unchanged (Table 4). Digestion data for fibre, starch and sugar were not considered due to methodological inadequacies. The product had no significant effect on microbial growth and proteolytic activity did not appear to be affected by acarbose. Trial 2 Inoculum donor cows were fed either medium (26.02 %) or low (22.79 %) starch diets. The results confirmed significant dose-related effects on ph associated with reduced starch 26 Technical Dossier Section III, Annexes III.7 (Trial 1) and III.8 (Trial 2) The EFSA Journal (2008) 839, 15-40

16 degradation and improved fibre digestion in the medium starch diet (Table 5). VFA production was significantly reduced with the medium starch ration but not with the low starch ration. There was no effect on ph with the low starch ration (Table 6). Table 5. Table 6. Effects of acarbose on VFA production and ph (medium starch diet) VFA Production Control Acarbose (μg ml -1 ) (Mmoles d -1 ) Total VFA 314 a 291 b 271 b 279 b Acetic acid (%) 52.8 a 62.2 b 65.1 b 63.5 b Propionic acid (%) 34.2 a 21.2 b 16.1 b 17.8 b Butyric acid (%) 8 a 12 b 15 b 13.9 b Average ph 5.72 a 5.78 a 5.90 b 5.97 b a,b: in a same row, values with different superscripts differ significantly, P <0.05 Effects of acarbose on VFA production and ph (low starch diet) VFA Production Control Acarbose (μg.ml -1 ) (Mmoles.d -1 ) Total VFA Acetic acid (%) 65.1 a 65.8 ab 58.6 b 62.7 ab Propionic acid (%) Butyric acid (%) 10.3 a 11.2 ab 18.7 a 13.6 ab Average ph a,b: in a same row, values with different superscripts differ significantly, P < Conclusions from in vitro studies The first four in vitro studies were not considered further. The remaining two studies show that acarbose reduces rumen fermentation (VFA production) and contributes to stabilising ph value in a dose-dependent manner. The acarbose effect is inversely related to the starch content of the fermentation substrate. Those studies give valuable information on the mode of action of acarbose and may be used in support of efficacy studies In vivo studies Based on the results of the in vitro studies with the RUSITEC model and the RPT, it was considered that a concentration of acarbose in rumen fluid of 10 μg ml -1 was sufficient to produce the desired response in those models. The dose used in the majority of the in vivo studies was calculated to produce a similar concentration. A concentration of 10 μg ml -1 is equivalent to 750 mg in 75 litres of rumen fluid, which equates approximately to mg kg -1 bw per day. A number of in vivo bovine models where animals were fed diets that induced a fall in rumen ph were developed. An acute in vivo model was developed to determine if acarbose could control ruminal ph when animals are fed highly acidotic rations. Acidosis was induced using a challenge feed (12.5 g kg -1 bw) containing predominantly corn starch and ground corn in warm water that was poured directly into the rumen via a fistula for two consecutive days. Once a ph of 4.5 was reached, animals were removed from the study on welfare grounds and the rumen washed out with water and inoculated with rumen fluid from donor cattle. In a second model, fistulated beef cattle were fed a diet designed to cause rumen ph to drop consistently below 5.5. A ph of 5.5 is considered the lowest ph at which the rumen may function normally. Rumen ph was monitored continuously with automatic data recording and The EFSA Journal (2008) 839, 16-40

17 storage. This model was used to demonstrate that acarbose controls rumen ph and to identify its minimum effective application rate. In the third model, acarbose was tested in non-fistulated dairy cattle receiving diets previously shown to provoke mildly acidotic conditions in the rumen of fistulated cattle. Milk production parameters were assessed in cows supplemented or not with acarbose Short-term acidotic challenge Three acute and two sub-acute challenge trials were submitted Acute challenge study Three trials were conducted. 27 The acute challenge model in fistulated beef cattle used a dietary starch overload to induce rapid fermentation, resulting in lactic acid accumulation and a rapid fall in rumen ph. Rumen ph was continuously measured using a probe inserted through a fistula. Lactate and VFA concentration were analysed in fluid samples collected at certain intervals after the challenge. Acute acidosis was defined as a ph of <4.5 and >50 mm total lactate, and acidosis as a ph of <5 and >25 mm total lactates. Trial 1 Fourteen cannulated steers (475 kg bw) were administered a challenge dose of 12.5 g carbohydrate/protein mixture (48.4 % cornstarch, 48.4 % ground corn, 2.1 % sodium caseinate, 1.1 % urea) kg -1 bw for two consecutive days. The animals were divided in two treatments groups receiving 0 (control) or 1.07 mg acarbose kg -1 bw. Acute acidosis was induced after the second challenge in four control animals and one acarbose-treated animal (P <0.1). Acidosis was observed in five control animals and one acarbose-treated animal (P <0.05). Total VFA and percent propionate were lower and percent butyrate was higher in acarbose-treated compared with control animals. Feed intakes of acarbose-treated animals were higher than those of control animals during the first three days after the second challenge. Trial 2 The same design as in trial 1 was used but the challenge dose was added to 2 kg of the basal diet (total mixed ration). Acute acidosis was induced after the second challenge in six out of seven control animals and in two acarbose-treated animal (P <0.1). Acidosis was observed in six control animals and six acarbose-treated animals. Trial 3 The same challenge protocol as described in trial 1 was applied to 29 cannulated Holstein steers (620 kg bw) divided in four experimental groups: control (no additive, n = 7), acarbose (1.07 mg kg -1 bw, n = 8), bicarbonate (1 % dry matter, n = 7) or monensin (12 mg kg -1 DMI, n = 7). Three out of eight animals showed signs of acidosis in the acarbose group, while all animals in the control, bicarbonate and monensin groups were acidotic. Incidence of acidosis was less in the acarbose group than in each of the other groups (P = 0.03). Median time to acidosis after the first challenge was about twice as long after acarbose (36 hrs) as control (18 hrs) and monensin (16 hrs), with bicarbonate being intermediate (26 hrs). 27 Technical Dossier Section III, Annexes III.9 to III.11 The EFSA Journal (2008) 839, 17-40

18 Sub-acute challenge study Rumen fistulated steers were fed diets based on cereals and barley straw (composition not given) with increasing amount of concentrate and decreasing quantity of straw in order to lower the rumen ph. 28 The ph levels were monitored daily and the diet adjusted on an animal basis with the objective of producing a rumen ph profile in the range of 5.0 to 5.5 for approximately four hours. The animals were then maintained on this kind of diet for periods with and without acarbose. Acarbose (1.1 mg kg -1 bw) was administered through the cannula. ph kinetics and VFA concentrations in rumen fluid were measured. A repeated measure ANOVA was then performed using all the data over time. Trial 1 Ten animals (average bw of 328 kg) were fed the challenge diet for 19 days, acarbose being administered from day 6 to day 14. The number of hours in which the rumen ph remained below 5.5 was significantly lower (1.65 hours d -1 vs hours d -1 ) during the acarbose period than the control periods, which were not different from each other. VFA concentrations were significantly lower (88 mm vs. 103 mm) during the acarbose period than the control periods. Trial 2 Eleven animals (average bw of 461 kg) were fed the challenge diet for 19 days, acarbose being administered from day 6 to day 13. Due to the considerable variation among animals in the extent of acidosis between the two control periods, acarbose effects were investigated by only analysing data from the first two periods. The number of hours in which the rumen ph remained below 5.5 was significantly lower (2.05 hours d -1 vs hours d -1 ) during the acarbose period than the control period. No significant differences could be noted for VFA production Conclusions from the acute and sub-acute challenge studies Three short-term studies (two days) in which cattle for fattening were subjected to a high starch dietary challenge showed that acarbose at a dose of 1.1 mg kg -1 bw reduces the risk of acidosis, as characterised by ruminal ph and lactate concentration. Two short-term trials (six days) in which cattle for fattening were subjected to an acidotic diet showed that acarbose at a dose of 1.1 mg kg -1 bw reduces the risk of acidosis, as characterised by ruminal ph. Such effects in beef cattle can be extrapolated to dairy cows Efficacy studies in dairy cows Efficacy study in lactating cows fed a diet expected to induce sub-optimal rumen ph Four trials were conducted in early- to mid-lactation dairy cows. All were conducted with a cross-over design with three-week supplement periods. 29 The animals were fed a diet expected to induce sub-optimal rumen ph (corn silage, low ph grain mix), with or without mg acarbose kg -1 bw day -1. Animals were adapted to the supplemented diet for two weeks and daily milk yield, plus yields (and percentages) of fat, protein, lactose and solids were recorded for each animal during the third week of each period. Weekly mean values per animal were subjected to statistical analysis (linear mixed model for a cross-over design). 28 Technical Dossier Section III, Annexes III.12 and III Technical Dossier Section III, Annexes III.14 to III.17 The EFSA Journal (2008) 839, 18-40

19 Table 7. Zootechnical effects of acarbose in lactating dairy cattle Trial #* (No of animals) Acarbose concentration (mg kg -1 day -1 ) Parameters measured Control 1 (26) DMI (kg d -1 ) Milk yield (kg d -1 ) fat yield (g d -1 ) protein yield (g d -1 ) fat (%) protein (%) Acarbose 2 (30) DMI (kg d -1 ) Milk yield (kg d -1 ) % fat corrected milk yield (kg d -1 ) protein yield (g d -1 ) fat yield (g d -1 ) fat (%) protein (%) (30) DMI (kg d -1 ) Milk yield (kg d -1 ) protein yield (g d -1 ) fat yield (g d -1 ) lactose yield (g d -1 ) fat (%) protein (%) lactose (%) (30) DMI (kg d -1 ) Milk yield (kg d -1 ) % fat corrected milk yield (kg d -1 ) protein yield (g d -1 ) fat yield (g d -1 ) lactose yield (g d -1 ) fat (%) protein (%) lactose (%) P value (*) All trials followed a cross-over design (two treatments, two periods). The effects of high-starch concentrate fed in control (0) and acarbose-supplemented (see doses in table) diets were studied. The main results of the four trials are presented in Table 7. They indicate a significant effect of acarbose ( mg kg -1 day -1 ) on increasing fat percent (four studies), fat yield (three studies) and fat-corrected milk yield (two studies), and on decreasing protein (three studies) and lactose (two studies) percents. Dry matter intake was improved in two out of the four studies. All those differences were measured and observed only in the third week of the three-week treatment, conducted under feeding conditions which allow only the secretion of a low fat milk ( % for control animals). Those results were obtained under feeding conditions and with a milk composition which do no not reflect EU production practices. Although the FEEDAP Panel considers a decrease in milk fat as indicative of a ruminal dysfunction, full conclusions can only be based on additional criteria (e.g. urea in milk). Although the observed changes in milk fat should be considered as beneficial, the correlative reduction in milk protein reduces milk quality, thus it can be considered an adverse effect. The EFSA Journal (2008) 839, 19-40

20 Efficacy study in lactating cows fed rations with starch concentrations typical of European diets Three additional trials performed at commercially operating dairy farms in the EU, aimed to study the effects of acarbose in early lactation ration using high amounts of fermentable carbohydrates on milk yield, were provided by the applicant in a supplementary dossier. 30 The experimental design was similar for each trial. Prior to the start of the experiments, the animals were randomised and distributed into two pens based on a previous 305-day lactation yield for multiparous animals or early lactation yield for primiparous animals with similar distribution of parity and stage of lactation. One pen received an early lactation farm ration (R1) and the other pen received a test ration (R2) for 84 to 87 days, depending on the experiment. The test ration was a reformulation of the farm ration, using more ground wheat (including 0.75 g acarbose cow -1 day -1 ) and other carbohydrate sources and less sugar beet pulp or grass or maize silage as fiber sources to increase ME density by 0.48 to 0.56 MJ kg DM -1 (Table 8); this was predicted to increase the 3.5 % fat-corrected milk (FCM) yield by 2.5 kg d -1, assuming that feed intakes were equal. Daily milk yields and biweekly milk fat and protein percents were measured in the study. General health observations (tail scores, locomotion scores) were carried out over the whole experimental period. Table 8. Experimental design applied to the dairy farm experiments Trial # Treatment (1) Animals per treatment 1 R1 32 R R1 203 R R1 74 R2 76 (1) R1: Control diet; R2: Test diet Duration (days) Energy density of the ration (MJ ME kg -1 ) Starch content (%) Acarbose (g cow -1 d -1 ) In the first trial, milk yield and fat percent were numerically increased 1.3 kg d -1 and 0.09 %, respectively for R2 animals compared with R1 animals. Protein percent was increased 0.04 % (P = ) and protein yield was numerically increased 0.07 kg d -1 for R2 compared with R1. A summary of energy balance calculations showed that energy intake was increased by 10 MJ d -1 for R2 compared with R1, primarily due to the increased energy density. No differences were found in health observations between the two experimental groups. In the second trial, milk yield increased to 2.9 kg d -1 (P = ), fat percent decreased to 0.51 % (P <0.0001) and fat yield decreased to 0.98 kg d -1 (P = ), for R2 compared with R1. The consequence was that 3.5 % FCM yield was the same. Although protein percent numerically decreased to 0.03 %, protein yield increased to 0.08 kg d -1 (P = ) for R2 compared with R1. A summary of energy balance calculations showed that even though energy density was increased for R2 compared with R1, the energy intake was the same due to a 1.1 kg d -1 decrease in dry matter feed intake. Thus, both 3.5 % FCM yield predicted from energy intake and actual 3.5 % FCM yield were the same for R1 and R2. No differences were found in health observations between the two experimental groups. In the third trial, milk yield, fat percent and 3.5 % FCM yield were numerically decreased 0.2 kg d -1, 0.23 % and 1.4 kg d -1, respectively, for R2 compared with R1. Milk fat yield was decreased 0.09 kg d -1 (P = ). Milk protein percent and yield were not affected. A summary of energy balance calculations showed that energy intake was decreased 5 MJ for R2 30 Dossier Advastat. Supplementary information. June 2007 The EFSA Journal (2008) 839, 20-40

21 compared with R1, primarily due to a 1.3 kg d -1 decrease in dry matter feed intake. In addition, the predicted 3.5 % FCM yield from energy intake was decreased 0.9 kg d -1 for R2 compared with R1, whereas the actual 3.5 % FCM yield was decreased 1.4 kg d -1. No differences were found in health observations between the two experimental groups. In conclusion, those trials demonstrated that feeding a higher starch ration (test diet, containing acarbose) resulted in positive or negative changes, sometimes significant, in milk production and composition parameters compared to a control ration (not acarbosesupplemented) (Table 9). A benefit from feeding the test diet did not become evident. Table 9. Summary of the results obtained in the efficacy trials described in this section Milk yield FCM Milk fat Milk protein (kg day -1 ) (kg day -1 ) percentage kg day -1 percentage kg day -1 Trial ± +* ± Trial 2 +* ± * * + Trial 3 * ± ± + means increase in R2 compared to R1 (R2: test diet, acarbose supplemented; R1: control diet) means decrease in R2 compared to R1 ± means no differences between R2 and R1 * Statistically significant differences between R2 and R1 In all the experiments, two non identical diets (different in ME content) were used as a base for the preparation of the control and the test (acarbose-supplemented) diets; thus, the study design does not allow a separate assessment of the two factors (i.e. the ME and the acarbose). No conclusion on the efficacy of acarbose can be drawn from those studies Conclusions on efficacy The in vitro studies show that acarbose has a potential to reduce rumen fermentation and contributes to stabilising rumen ph; those effects are confirmed by in vivo short-term challenge studies on cattle for fattening, where acarbose (1.1 mg kg bw -1 ) reduces the incidence and severity of acidosis. Efficacy studies in dairy cows fed acidotic diets for three weeks show that acarbose ( mg kg -1 day -1 ) increases fat percent and yield but also decreases protein percentage in the third week of the treatment. Those studies were considered of too short duration. The experimental design of the efficacy studies performed at commercially operating dairy farms (starch levels of the studied rations from 18 to 30 %) did not allow to draw any conclusions on the efficacy of acarbose. Therefore, the FEEDAP Panel cannot conclude on the efficacy of Advastat for cattle for fattening and dairy cows because benefits in healthy animals, under common farming practices in the EU (standard conditions under which the additive should show efficacy), could not be demonstrated. However, the former conclusion does not exclude that acarbose might be efficacious in cases where cattle for fattening and dairy cows are at a specific risk or suffering of acidosis due to the particular feeding regime. 4. Safety 4.1. Safety for the target species Tolerance of the target animals to Advastat was determined in four trials (one in beef cattle and three in lactating dairy cows). All trials were conducted using three experimental groups: control, maximum recommended dietary dose of acarbose and a group with at least ten times that dose. The EFSA Journal (2008) 839, 21-40