The EFSA Journal (2006) 337, 1-17

Transcription

1 The EFSA Journal (2006) 337, 1-17 Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on the safety and efficacy of the product Amaferm as a feed additive for dairy cows and cattle for fattening in accordance with Regulation (EC) No 1831/2003 (Question N o EFSA-Q ) Adopted on 8 of March 2006 SUMMARY Amaferm is the fermentation product of a non-pathogenic and non-genetically modified strain of Aspergillus oryzae mixed with wheat bran as carrier. It is proposed to be used as feed additive to dairy cows and cattle for fattening at doses between 2 and 5 g head -1 day -1 in order to improve production performance. According to Regulation (EC) No 1831/2003, the European Food Safety Authority (EFSA) should deliver an opinion on the efficacy and the safety of the product Amaferm for the target animals, consumers, users and the environment. Amaferm has been shown to be capable of producing a positive effect on milk yield of dairy cows. However, evidence has only been provided for a dose of 3 g head -1 day -1 instead of the minimum recommended dose of 2 g head -1 day -1 claimed by the Applicant. However, only one out of four studies with cattle for fattening showed a persistent improvement of weight gain and feed conversion with Amaferm. Therefore, the FEEDAP Panel considers the data provided to be insufficient to draw final conclusions on efficacy of Amaferm in cattle for fattening kept under common production conditions. A tolerance study with lactating dairy cows demonstrated the absence of adverse effects of Amaferm on production performance. The FEEDAP Panel regards this study to be representative also for cattle for fattening and considers tolerance of Amaferm to be sufficiently demonstrated for both dairy cows and cattle for fattening. This includes also microbiological safety within the digestive tract of animals supplied with Amaferm. According to the long experience of use of fermentation products from Aspergillus oryzae as components of human food and reassurance of lack of aflatoxin production by the strain, the FEEDAP Panel has no reason for concern for consumer safety deriving from the use of Amaferm. The studies provided with the dossier did not give rise for safety concerns for consumer and user safety. However, the FEEDAP Panel considers that, as with all proteinaceous substances, there might be a risk of sensitisation by inhalation for those handling the product. The FEEDAP Panel recognises that Amaferm consists of natural substances with no environmental concern. No risks associated with the use of the product are foreseen, and therefore the FEEDAP Panel does not see the need for specific requirements for a post-market monitoring plan. Keywords: Aspergillus oryzae, Amaferm, dairy cows, cattle for fattening, safety, efficacy

2 Opinion on Amaferm 2/17 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 Regal 2 for authorisation of the product Amaferm to be used as a feed additive for dairy cows and beef cattle in growing-finishing stage (category: zootechnical additives) under the conditions mentioned under Table 1. This was considered by the Commission as an application under Article 4.1 (authorisation of a feed additive or new use of a feed additive) of Regulation (EC) No 1831/2003. According to Article 7(1) of the same Regulation, the Commission forwarded the application to the European Food Safety Authority (EFSA). EFSA received directly from the applicant the technical dossier in support of this application. According to Article 8 of that Regulation, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. The particulars and documents in support of the application were considered valid by EFSA as of 20 of May of Amaferm is a fermentation product of Aspergillus oryzae. This product has not previously been authorised as feed additive in EU. 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. Therefore, EFSA shall deliver an opinion on the efficacy and the safety for the target animals, user and consumer and the environment of the product Amaferm when used under the conditions described in Table 1. 1 OJ L 268, , p Regal, Wilhelminalaan EP Roermond, The Netherlands.

3 Opinion on Amaferm 3/17 Table 1. Register entry as proposed by the applicant. Additive AMAFERM Registration number/ec No/No - Category of additive Functional group of additive Zootechnical additives Other zootechnical additives Composition, description Extract of Aspergillus oryzae (NRRL 458): 4% Wheat bran as carrier: 96 % Description Chemical formula - Purity criteria Method of analysis - Thiamine: AOAC 17 th Ed α-amylase AOAC 17 th Ed Citric acid AOAC 17 th Ed Trade name AMAFERM Name of the holder of authorisation Trouw Nutrition BV, The Netherlands Species or category of animal Maximum Age Conditions of use Minimum content 3 Maximum content 4 g head -1 day -1 Dairy cows Beef cattle in growing finishing stage Withdrawal period (if appropriate) - 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 Amaferm can be mixed in feeds, dosage to be calculated upon the table above. Feed may be pelleted; avoid high temperature expansion or extrusion. None Tracking and tracing will be in full place, all remarks during the use of Amaferm will be noticed and recorded Amaferm can be mixed in feeds, dosage to be calculated upon the table above Marker residue Maximum Residue Limit (MRL) Species or category of animal Target tissue(s) or food products Maximum content in tissues Not applicable Not applicable Not applicable Not applicable 3 Due to different feeding systems mg kg -1 is less applicable. 4 Due to different feeding systems mg kg -1 is less applicable.

4 Opinion on Amaferm 4/17 ASSESSMENT 1. Introduction Amaferm is a fermentation product of a non-pathogenic and non-genetically modified strain of Aspergillus oryzae (NRRL 458) mixed with wheat bran as carrier. It is proposed for use as feed additive to dairy cows and beef cattle at doses between 2 and 5 g head -1 day -1 in order to improve ruminal fermentation of feed, feed intake and hence production performance. The company seeks authorization of Amaferm to be used as a feed additive for dairy cows and beef cattle in growing-finishing stage (category: zootechnical additives) according to Regulation (EC) No 1831/2003. The following assessment focuses especially on the aspects of efficacy and safety in context with the proposed use of Amaferm. 2. Characterisation of the product Description of the product The fungus A. oryzae is grown in a liquid fermenter system in two stages at closed and sterile conditions using standard production media. After second fermentation, the supernatant (broth and cell solids) is sprayed on food grade wheat bran at 4-5% w/w ratio and air dried at 55 C until moisture content is less than 10% (7.2% on average). Amaferm is added with 1 g kg -1 of a non-nutrient marker (stainless steel grit covered with 5% cobaltous carbonate) in order to confirm proper mixing of Amaferm in the final feed formulations (Table 2). Table 2. Composition of Amaferm % Fermentation product 4-5 Wheat bran Stainless steel grit containing 5% cobaltous carbonate 1 According to the applicant, batch to batch variation of the fermentation is verified and controlled through measurement of salt concentration, broth ph, dry matter, protein concentration, enzyme assays (cellulase and amylase). However, the description appears to reflect more the minimum quality assurance rather than a procedure to compensate batch to batch variation in order to gain a standardized product. The final product retains in general the physical characteristics from the food-grade wheat bran. It is a light brown, fairly odourless powder with a density of 425 kg m -3. Particle size varies between and 2 mm with most particles (>90%) being greater than 0.15 mm and less than 1% being smaller than mm. The dusting potential is low (8 mg from 50 g of product, Steuber-Heubach). The electrostatic properties of Amaferm are stated to be essentially the same as those for wheat bran. Quality control parameters are described, and typical data from one batch of Amaferm was provided [Heavy metal contamination (Cd: not detected (n.d.); Pb: n.d.; Hg: 0.06 µg g -1 ; As: n.d.), mycotoxins (aflatoxin: n.d.; citrinin: n.d.; ochratoxin: 5.0 ng g -1 ; T2 toxin: n.d.; vomitoxin: 1.1 µg g -1 ; zearaleone: n.d.). The results on aflatoxin and vomitoxin were confirmed in an additional batch (n.d. and 1.2 µg g -1, respectively)]. The final product Amaferm is stated to contain about 7 x 10 6 intact Aspergillus oryzae conidiospores and 0.2% citric acid, 2.3 mg riboflavin kg -1, mg niacin kg -1, 23.2 mg pantothenic acid kg -1, 8.5 mg pyridoxine hydrochloride kg -1, 0.9 mg folic acid kg -1, 0.4 mg biotin kg -1, 1.2 µg cobalamin kg -1, as well as 3.0 IU cellulase kg -1 and 40.0 IU amylase kg -1 activities.

5 Opinion on Amaferm 5/ Stability Shelf-life of Amaferm has been tested in four charges for 9, 22, 42 and 104 weeks, respectively (ambient conditions not further specified). In the 104 week study, final ph, dry weight, contents of thiamine, citrate and protein, and activities of cellulase and amylase were 106%, 99%, 86%, 95%, 90%, and 88% of the initial values, respectively. The applicant states that Amaferm is insensitive to atmospheric pressure and light and that it does not react with the packing material and will remain unchanged during all normal transport and storage conditions. Provided the product is stored at temperatures not exceeding 80ºC and at a ph between 5 and 8, the shelf-life of Amaferm is claimed to be two years after production Tests on stability of Amaferm during preparation and storage of premixes and feed mixtures were not reported in the dossier. The company recommends that during feed manufacturing, temperatures in the mixer, conditioner, pellet mill and expander should be maintained below 80ºC, although the basis for this statement is not clear. Provided the same conditions of storage as the sole product, the company claims a stability of Amaferm in premixes or complete fed mixtures of at least three months Proposed mode of action and conditions of use Amaferm is suggested to improve the animals performance mainly through enhancing the microbial fermentation of the feed in the rumen. Another proposed mode action of Amaferm is provision of citric acid and vitamin B complex to the rumen. Furthermore, Amaferm is suggested to enhance ruminal degradation of fibrous feed components by cellulolytic bacteria. Another hypothesized mode of action addresses the facilitated degradation of ruminal lactate, which is an adverse factor of fiber degradation through its strong potential to reduce rumen ph (Frumholtz et al., 1989; Firkins et al., 1990; Newbold et al., 1991a). Amaferm is also mentioned to possibly reduce heat stress (Gomez Alarcon et al., 1991, Higgenbotham et al., 1993). The most consistent ruminal response to Amaferm is an increase in total culturable bacteria seen both in vitro and in vivo (Newbold, 1995). In some studies this has been associated with an increased flow of nitrogen/microbial protein leaving the rumen and an enhanced supply of amino acids entering the small intestine (Caton et al., 1993). The increase in total bacterial numbers is often accompanied by an increase in numbers of cellulolytic bacteria and VFA concentrations in the rumen (Newbold et al., 1992). Protozoal numbers apparently are not affected in the presence of Amaferm, but there are reports of positive effects of the additive on numbers and activity of the rumen fungi (Harper et al., 1996; Chang et al., 1999). It is thought that the increased cell wall degrading activity associated with increased bacterial and fungal numbers accounts, in part at least, for the often observed increase in fibre degradation in the rumen. However, autoclaved Amaferm apparently loses its stimulatory effect, in contrast to the γ-irradiated product where the effect was retained (Newbold et al., 1991b). This suggests that either a heat-sensitive nutrient of importance to the flora was destroyed by heating or that the polysaccharidase actively found in the additive and also destroyed by autoclaving, is an important factor in its action. Overall, it is probable that increased microbial numbers both contribute to and reflect the increase in cell wall degradation. Among the proposed beneficial effects on activity and numbers of ruminal microflora, none of them appear to be dominant. But as an overall result they may be assumed to enhance nutrient supply to the host animal and hence to improve its production performance. For dairy cows, the company suggests a daily dose between 2 and 5 g Amaferm per head and day, mixed in premixes, concentrate feed, total mixed ration or as a topfeeding. For cattle for fattening, the respective suggestions are 2 to 4 g per head and day.

6 Opinion on Amaferm 6/17 Amaferm is claimed to be free of incompatibilities or interactions that could be expected with feedingstuffs, carriers, and other approved additives or with medicinal products Control methods EFSA has verified the report of the Community Reference Laboratory (CRL) as it relates to the methods used for the control of the active agent in animal feeds. The executive summary of the CRL is attached in Annex A. The CRL states that there is not enough supporting experimental evidence to conclude on the suitability of the methods proposed by the applicant for use in official control. 3. Efficacy The company cites a total of 122 publications as a basis for the zootechnical response of Amaferm. Emphasis has been given to six studies, which are described in detail and which form the basis for the present assessment Dairy cows Four studies are given to assess the efficacy of Amaferm in dairy cows. Experiment 1 One hundred fifty high-production early-lactation Holstein cows were randomly allocated into one of three groups (50 cows per group): control diet without and with supplementation of either 3 g Amaferm or another product not relevant for this assessment (data not shown). Mixed ration consisted of corn earlage, alfalfa silage, rolled barley, whole cottonseed and premix. The cows were about 106 days in milk; the lactation number was about 2.5. Two rations were fed during the course of trial period with ration 1 being fed for the first 83 days and ration 2 (lower concentrate levels) being fed during the remaining 170 days. Feed consumption was measured per pen (number of cows per pen not reported), and milk production and milk composition was determined individually. Rectal temperatures were monitored at three different ambient temperature ranges ( 0ºC, 0-20ºC or 20ºC), and reproductive data were also recorded. Table 3. Effect of Amaferm supplementation on zootechnical parameters of Holstein cows (Kellems et al., 1990) Parameters Control Amaferm (3 g day -1 ) Dry matter Intake (kg day -1 ) Ration 1 (83 days) Ration 2 (170 days) Total trial Milk production 3.5% FCM yield (kg day -1 ) (total trial, 253 days) Milk yield (kg day -1 ) (from week 18) 22.2 a 24.5 b Milk fat (%) (from week 18) % FCM yield (kg day -1 ) (from week 18) Initial body weight (kg) Final body weight (kg) Body condition score (BCS) Initial Final Reproductive performance Number of days to conception Number of services per conception Days to first service a, b: Means in a row not sharing a common superscript are significantly different (P 0.05)

7 Opinion on Amaferm 7/17 No differences in feed consumption were noted (Table 3). Amaferm supplementation increased milk production in the second half of the experiment compared to the control group (24.5 vs kg day -1, P 0.05). Average fat content of the milk was not affected by supplementation with Amaferm. Fat corrected milk (FCM) production was numerically higher for the Amaferm group, but the differences were statistically not significant. FCM production corrected for days in milk at the beginning of the trial was higher for the Amaferm group both at the beginning and at the late stages of the lactation. All the other parameters measured such as reproduction performance, body condition score or body weight (BW) changes were not significantly affected (P 0.05) by Amaferm. Experiment 2 Two trials were performed, one in cows at mid lactation (24 Holstein cows; 12 cows per treatment; average 145 days in lactation) and one with cows at early lactation (46 Holstein cows, 23 per treatment; average 42 days in lactation). The animals were randomly assigned to the control treatment (90 g sorghum day -1 ) or to the Amaferm supplemented treatment (87 g sorghum + 3 g Amaferm day -1 ). Both trials lasted for 12 weeks. Milk yield, milk composition and feed intake were recorded. Other measurements included rectal temperature, respiration rate, water consumption and body weight change. Digestibility studies were also performed in 22 and 34 of the cows from the first and second trial, respectively. In the first trial, milk production from cows fed Amaferm was higher than the control, but the differences were only significant during weeks 2 and 8. No differences were found in 3.5% FCM milk yields, dry matter (DM) intake and feed efficiency (Table 4). In the second trial, the Amaferm treated cows produced 7% more milk than the controls and had a better feed efficiency. Digestibility of DM, organic matter (OM), crude protein, neutral detergent fiber and acid detergent fiber were increased by Amaferm. Milk composition was not affected by the treatments (Table 4). Table 4. Effect of Amaferm supplementation (3 g day -1 ) on lactation performance and nutrient digestibility of Holstein cows (12 weeks) (Gomez-Alarcon et al., 1991) Parameter Mid-lactation (n=12) Early lactation (n=23) Control Amaferm Control Amaferm Dry Matter Intake (kg day -1 ) Milk yield (kg day -1 ) a 39.8 b Milk fat (%) % FCM (kg day -1 ) Protein (%) Feed Efficiency (kg milk kg feed -1 ) a 1.57 b Water intake (kg day -1 ) Digestibility (%) Dry matter a 71.9 b Organic matter a 72.9 b Crude protein a 77.6 b Neutral detergent fiber a 57.1 b Acid detergent fiber a 48.6 b a, b: Means in a row not sharing a common superscript are significantly different (P 0.05)

8 Opinion on Amaferm 8/17 In summary, milk yield, efficiency of milk production and nutrient digestibility were higher for early lactating cows fed a high-concentrate diet supplemented with 3 g Amaferm per day. Mid-lactation cows fed a lower-energy diet were less responsive to Amaferm (Table 4). The improved nutrient digestibility in the early lactation may be considered as base for the higher milk yield, but the better digestibility is not always explainable. Experiment 3 A total of 110 cows in mid-lactation (140 days) were used in a 120-day trial (40 days in a non-cooled and 80 days in a cooled ambience), and distributed to a group which received either a basal diet or supplemented with Amaferm (3 g day -1 ). The cows were offered at equal quantities a total mixed ratio composed of alfalfa silage, alfalfa hay, corn silage, rolled barley, whole cottonseed beet pulp and a premix (58% of DM from concentrate). Milk production and milk composition were measured biweekly and rectal temperature weekly. Rumen contents and blood samples were obtained during the last week of the trial. Table 5. Effect of Amaferm on milk production and composition of Holstein cows (Higginbotham et al., 1993) Parameter Control Amaferm Milk yield (kg day -1 ) % FCM (kg day -1 ) Milk composition (%) Fat Protein 3.05 b 3.12 a Solids not-fat 8.51 b 8.63 a Blood urea N (mg %) 18.1 b 13.4 a a, b: Means in a row not sharing a common superscript are significantly different (P 0.05) Milk and FCM production and milk fat proportion were not affected by the use of Amaferm (Table 5). Milk protein and solids-not-fat were higher and urea N in blood lower in the cows fed with Amaferm. Experiment 4 A total of 40 lactating dairy cows were blocked by days in milk and milk yield into four groups (ten cows per group); control and supplemented with 3, 30 and 60 g day -1 of Amaferm for 102 days. Ration consisted of rye grass, wheat and alfalfa silage (43% DM) and 53% of DM as concentrate (corn, soybean meal, etc). There were no health disorders. Amaferm supplementation increased milk production (P 0.05), the highest response was obtained with 3 g day -1 of Amaferm (Table 6). Milk fat and milk protein contents were not affected by Amaferm treatment. Table 6. Effect of various amounts of Amaferm on some zootechnical parameters Parameter Control Amaferm (g day -1 ) Milk yield (kg day -1 ) 26.0 a 28.5 c 27.0 b 27.2 b Milk fat (%) Milk protein (%) % FCM (kg day -1 ) 24.3 a 26.6 c 25.3 b 26.2 c a, b: Means in a row not sharing a common superscript are significantly different (P 0.05)

9 Opinion on Amaferm 9/ Cattle for fattening Four studies with growing cattle were submitted for efficacy assessment of Amaferm. Experiment 1: Grazing steers A total of fourteen Hereford steers (ten ruminally and duodenally cannulated, and four oesophageally fistulated, body weights: 326 and 394 kg) were randomly assigned to control group or supplementation with Amaferm at 2 g steer -1 day -1 via ruminal cannula. Rumen fermentation characteristics, ruminal and total digestive tract protein flow as well as nutrient digestibility were recorded. Ruminal volatile fatty acids (VFA), ph and ammonia concentration were not affected by Amaferm supplementation. On the other hand, forage intake and in vitro dry matter digestibility were increased by the supplementation with Amaferm (Table 7). The response of the animals varied with the season. Table 7. Effect of Amaferm supplementation on in vitro digestibility and forage intake in grazing steers (Caton et al., 1993). Parameters Control Amaferm (2 g steer -1 day -1 ) Intake of feed organic matter (kg day -1 ) 7.74 a 9.77 b Intake of feed organic matter (g kg BW -1 ) 25.4 a 31.5 b In vitro dry matter digestibility (%) 59.3 a b In vivo organic matter digestibility (%) a, b: Means in a row not sharing a common superscript are significantly different (P 0.05) Experiment 2: Growing heifer calves Eighty-four Charolais crossbred heifers (initial BW: 259 kg) were blocked by BW and distributed across 12 pens (7 calves per pen). The pens were randomly assigned to control, Amaferm (2 g head -1 day -1 ), and two further treatment groups supplemented with another substance not relevant for this assessment (data not shown). The treatments were formulated and delivered in a protein supplement. The animals were fed a corn-silage and oat hay based growing ration. The experiment lasted for 84 days. Total daily weight gain and feed conversion were improved (5% and 6%, respectively) by Amaferm supplementation compared to the control (Table 8). Table 8. Effect of Amaferm on performance parameters of heifers fed forage based diets Control Amaferm (2 g head -1 day -1 ) Dry matter intake (kg day -1 ) Final weight (kg) 342 a 346 b Total Gain (kg) 82.6 a 86.6 b Total ADG (kg day -1 ) 0.98 a 1.03 b Feed conversion (kg feed kg -1 ADG) 7.36 a 6.92 b a, b: Means in a row not sharing a common superscript are significantly different (P 0.05) Experiment 3: Growing steer calves Fifty-six Angus and Angus x Hereford steer calves (initial BW: 180 to 225 kg) were blocked by breed and BW to four pens (7 calves per pen). Each two pens were randomly assigned to control or Amaferm (2 g head -1 day -1 ) delivered in the protein mineral mix added to corn silage. The experiment lasted for 126 days. Body weight was recorded in 28 day intervals. Amaferm improved daily weight gain during the first 28 day interval

10 Opinion on Amaferm 10/17 (0.70 vs kg day -1, P<0.05) but was not efficacious for the entire experimental period (1.10 vs kg day -1 ; P>0.05). Experiment 4: Growing yearling steers The study was performed in 1973 and is presented as a brief summary. One-hundredeighty Holstein yearling steers (BW not reported) were distributed among 2 drylots (n = 90) which were assigned to control or Amaferm (2 g head -1 day -1 ) supplemented via concentrate to a corn silage diet. The experiment lasted for 38 days. Amaferm was stated to improve daily weight gain to a statistically significant extent (1.72 vs kg day -1 ). However, further details on statistics were not reported. Moreover, weight gain was calculated from initial body weight before feeding and final body weight after feeding, thus overestimating weight gain by confounding with feed intake Product quality No studies have been provided regarding the effect of Amaferm on the quality of animal products. However, the FEEDAP Panel considers such studies to be not necessary in this case, since feeding Amaferm to ruminants does not cause absorption of substances other than nutrients and compounds naturally occurring in feed Conclusions on efficacy Amaferm has been shown to be capable of producing a positive effect on milk yield (three studies). However, evidence has only been provided for a dose of 3 g head -1 day -1 instead of the minimum recommended dose of 2 g head -1 day -1 claimed by the applicant. Regarding cattle for fattening, one of the four studies submitted could not be used for assessment of efficacy due to methodological reasons. One study showed a positive effect of Amaferm on weight gain and feed conversion over a 84 days period while in another study an improved weight gain was persistent only for the first 28 days. A third study suggested that such zootechnical effects might be due to an enhanced digestibility. Therefore, the FEEDAP Panel considers the data provided to be insufficient to draw final conclusions on efficacy of Amaferm in cattle for fattening kept under common production conditions. 4. Safety for the target species 4.1. Tolerance in dairy cows As described already under section 3.1 (Experiment 4), a tolerance study was carried out with 40 mid-lactating dairy cows supplemented with Amaferm at levels of 0, 3, 30 and 60 g per head and day on top of a uniform for 102 days. This is equivalent to the 0, 0.6, 6 and 12-fold amount of Amaferm compared to the recommended upper level. Among treatment groups, no health disorders or changes in rectal temperature were observed. No adverse effects on milk production were observed when Amaferm was supplemented at 12 times overdose (60 g head -1 day -1 ) (Table 6). Therefore the FEEDAP Panel considers that enough evidence has been provided to demonstrate that Amaferm is tolerated by dairy cows Tolerance in cattle for fattening Tolerance studies with cattle for fattening were not included in this dossier. However, the FEEDAP Panel considers safety studies with high-yielding dairy cows to provide sufficient evidence of safety also for cattle for fattening. Therefore the FEEDAP Panel considers that Amaferm is also tolerated by cattle for fattening.

11 Opinion on Amaferm 11/ Microbiological safety No adverse effects of the product on the microbial flora have been reported in any of the many published studies and all effects detected have been considered to be neutral or beneficial. The product does not show antimicrobial properties and any remaining viable material from the production strain would not be expected to be metabolically active within the ruminant digestive tract. Therefore, the FEEDAP Panel does not expect any microbiological risk due to Amaferm supplementation to dairy cows and cattle for fattening Conclusions on safety for the target species A tolerance study with lactating dairy cows found no adverse effects of Amaferm at a 12-fold dose of the maximum recommended dose. Although no specific tolerance was conducted for cattle for fattening, the FEEDAP Panel regards the study performed in dairy cows to be representative also for cattle for fattening. The FEEDAP Panel does not expect any microbiological risk due to Amaferm supplementation to dairy cows and cattle for fattening. Therefore, the FEEDAP Panel considers Amaferm to be safe for dairy cows and cattle for fattening. 5. Consumer Safety The dossier submitted includes only limited toxicological data, described below under user safety, since it is argued that the long experience of use of such fermentation products as components of human food provides sufficient evidence of human safety from direct consumption thus should be sufficient reassurance for use in animal feed. The producing strain has been found not to produce aflatoxins (Wei and Jong, 1986). The FEEDAP Panel accepts this argument and has no reason for concern for consumer safety deriving from the use of this product as an animal feed additive. 6. User Safety 6.1. Acute oral limit test toxicity The summary of an acute oral limit test toxicity study in rats conducted according to OECD guidelines showed no adverse effects following a single oral dose of 2000 mg kg -1 as an aqueous suspension. Amaferm is therefore considered to be a product of very low acute oral toxicity Inhalation exposure and dusting potential The particle size distribution of Amaferm shows 90% of the particles to be greater than 150 μm and that only 2.1% of particle passed through a 75 μm sieve. Since dusting levels determined using the Stauber Heubach procedure showed only 8 mg dust per 50 g of Amaferm the potential for inhalation exposure to Amaferm is regarded as very low. Coupled with the lack of other acute effects the applicant regarded an inhalation study in animals as unnecessary, the FEEDAP Panel supports this conclusion Genotoxicity Amaferm was tested in an Ames test using Salmonella Typhimurium strains TA98, TA100, TA102, TA1535 and TA 1537 at concentrations from 100 to 5000 μg plate -1 both with and without metabolic activation. There were no signs of cytotoxicity at any dose although at the highest dose there was precipitation of test substance. Amaferm showed no indication of any mutagenic effects either with or without activation. Amaferm was tested for clastogenic activity in human lymphocytes both with and without metabolic activation at concentrations up to 2500 μg L -1, a dose which was highly cytotoxic without activation. Amaferm showed a very slightly increased number of

12 Opinion on Amaferm 12/17 aberrations only at this cytotoxic dose, a known artefact of this test. Otherwise Amaferm showed no evidence of mutagenic effects either with or without activation Skin Irritation The summary of a skin irritancy study in rabbits conducted to OECD guidelines with 5000 mg Amaferm per patch showed no evidence of irritancy after four hours semioccluded exposure. Amaferm is therefore concluded to be non-irritant and requires no labelling Eye Irritation The summary of an eye irritancy study in rabbits conducted to OECD guidelines with 100 mg Amaferm introduced into the conjunctival sac showed slight evidence of irritancy but the effects had all cleared within 48 hours. From these results it is concluded that Amaferm is non-irritant and requires no labelling Dermal sensitisation The summary of a Magnusson & Kligman maximisation study in ten treated guinea pigs with the supernatant of a 25% aqueous suspension of Amaferm showed no evidence of a sensitisation reaction. On the basis of the information provided labelling for sensitisation is not required Safety precautions The applicant s safety data sheet specifies that no means or methods of protection are necessary during the manufacture and handling of this product. However, the FEEDAP Panel considers that, as with all proteinaceous substances, there might be a risk of sensitisation by inhalation and this should be taken into consideration when handling the product. 7. Environmental Safety Amaferm is a fermentation product of the fungus Aspergillus oryzae, universally present in the environment. The FEEDAP Panel concludes that the use of Amaferm will not pose a risk to the environment. 8. Post-market monitoring No risks associated with the use of the product are foreseen, and therefore the FEEDAP Panel does not see the need for specific requirements for a post-market monitoring plan. CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS Amaferm has been shown to be capable of producing a positive effect on milk yield of dairy cows. However, evidence has only been provided for a dose of 3 g head -1 day -1 instead of the minimum recommended dose of 2 g head -1 day -1 claimed by the Applicant. Regarding cattle for fattening, only one study showed a persistent improvement of weight gain and feed conversion through Amaferm. Therefore, the FEEDAP Panel considers the data provided to be insufficient to draw final conclusions on efficacy of Amaferm in cattle for fattening kept under common production conditions. A tolerance study with lactating dairy cows found no adverse effects of Amaferm at a 12-fold dose of the maximum recommended dose. Although no specific tolerance study

13 Opinion on Amaferm 13/17 was conducted for cattle for fattening, the FEEDAP Panel regards results with dairy cows to be representative also for cattle for fattening. The FEEDAP Panel does not expect any microbiological risk due to Amaferm supplementation to dairy cows and cattle for fattening. Therefore, the FEEDAP Panel considers Amaferm to be safe for dairy cows and cattle for fattening. According to the long experience of use of fermentation products from Aspergillus oryzae as components of human food and reassurance of lack of aflatoxin production by the strain, the FEEDAP Panel has no reason for concern for consumer safety deriving from the use of Amaferm. The studies provided in the dossier did not give rise to safety concerns regarding toxicity, mutagenicity, clastogenicity, potential for inhalation, skin and eye irritation, and dermal sensitization through Amaferm. However, the FEEDAP Panel considers that, as with all proteinaceous substances, there might be a risk of sensitisation by inhalation for those handling the product. The FEEDAP Panel recognises that Amaferm consists of natural substances without environmental concern. No risks associated with the use of the product are foreseen, and therefore the FEEDAP Panel does not see the need for specific requirements for a post-market monitoring plan. RECOMMENDATIONS The minimum recommended dose for dairy cows should be increased from 2 to 3 g head -1 day -1. The entry for cattle for fattening should be deleted from the Register entry due to insufficient data supporting efficacy. The definition of the product should be fermentation product instead of extract. The FEEDAP Panel also recommends the applicant to consider reducing the number of items designated for control purposes. It is evident from the available experimental data on mode of action that the product owes most of its activity to the two declared enzyme activities. It would considerably simplify the registration process if the method of analysis in the Register entry recognised this and specified only methods for amylase and cellulose. The functional group would then become digestibility enhancer. A consequence of this is that the product would have then to be considered as unique as any other enzyme product. Exclusion of thiamine and citric acid from the method of analysis would not preclude their concentration being monitored as part of the in-house quality control or any mention of their possible contribution as secondary constituents to any overall effects produced by the product.

14 Opinion on Amaferm 14/17 Table 9. Register entry, as proposed by the FEEDAP Panel Additive AMAFERM Registration number/ec No/No - Category of additive Zootechnical additives Functional group of additive Digestibility enhancer Composition, description Fermentation product of Aspergillus oryzae (NRRL 458): 4% Wheat bran as carrier: 96 % Description Chemical formula - Purity criteria Method of analysis - α-amylase AOAC 17 th Ed Cellulase Trade name AMAFERM Name of the holder of authorisation Trouw Nutrition BV, The Netherlands Conditions of use Species or category of animal Maximum Age Minimum content 5 Maximum content 6 g head -1 day -1 Withdrawal period (if appropriate) Dairy cows 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 None None Not applicable Amaferm can be mixed in feeds, dosage to be calculated upon the table above Marker residue Maximum Residue Limit (MRL) Species or category of animal Target tissue(s) or food products Maximum content in tissues Not applicable Not applicable Not applicable Not applicable 5 Due to different feeding systems mg kg -1 is less applicable. 6 Due to different feeding systems mg kg -1 is less applicable.

15 Opinion on Amaferm 15/17 REFERENCES Caton, J.S., Erickson, D.O., Carey, D.A and Ulmer, D.L Influence of Aspergillus oryzae fermentation extract on forage intake, site of digestion, in-situ degradability, and duodenal amino acid flow in steers grazing cool-season pasture. J. An. Sci. 71, Chang, J.S., Harper, E.M. and Calza, R.E Fermentation extract effects on the morphology and metabolism of the rumen fungus Neocallimastix frontalis EB 188. J. Appl. Microbiol. 86, Firkins, J.L. and Weiss, W.P Effects of feeding fungal culture extract and animalvegetable fat on degradation of hemicellulose and on ruminal bacteria growth in heifers. J. Dairy Sci. 73, Frumholtz, P.P., Newbold, C.J. and Wallace, R.J Influence of Aspergillus oryzae fermentation extract on the fermentation of a basal ration in the rumen simulation technique (Rusitec). J. Agric. Sci. (Camb.) 113, Gomez-Alarcon, R.A., Huber, J.T., Higginbotham, G.E., Wiersma, F., Ammon, D. and Taylor, B Influence of feeding Aspergillus oryzae fermentation extract on the milk yields, eating patterns, and body temperatures of lactating cows. J. Anim. Sci. 69, Harper, E.G., Welch, R.P., Contreras Lara, D., Chang, J.S. and Calza, R.E The effect of Aspergillus oryzae fermentation extract on the anaerobic fungi Neocallimastix frontalis EB 188, Piromyces communis DC 193 and Orpinomyces ssp. RW 206: generalized effects and component analysis. Appl. Microbiol. Biotech. 45, Higginbotham, G.E., Bath, D.L. and Butler, L.J Effect of Feeding an Aspergillus oryzae extract on milk production and related responses in a commercial dairy herd. J. Dairy Sci., 76, Kellems, R.O., Lagerstedt, A. and Wallentine, M.V Effect of feeding Aspergillus Oryzae fermentation extract or Aspergillus oryzae plus yeast culture plus mineral and vitamin supplement on performance of Holstein cows during a complete lactation. J. Dairy Sci. 73, Newbold, C.J., Brock, R. and Wallace, R.J. 1991a. Influence of Aspergillus oryzae fermentation extract in the rumen simulation technique (Rusitec). J. Agric. Sci. (Camb.) 116, 59. Newbold, C.J., Brock, R. and Wallace, R.J. 1991b. Influence of Autoclaved or Irradiated Aspergillus oryzae fermentation extract of fermentation in the rumen simulation technique (Rusitec). J. Agri. Sci. (Camb.) 116, Newbold, C.J., Brock, R. and Wallace, R.J The effect of Aspergillus oryzae fermentation extract on the growth of fungi and cilitate protozoa in the rumen. Letters Appl. Microbiol. 15, Newbold, C.J Microbial feed additives for ruminants. In: Biotechnology in Animal Feeds and Animal Feeding (Wallace, R.J. and Chesson, A. eds.) pp VCH Verlagsgesellschaft, Weinheim. Wei D.L. and Jong S.C Production of aflatoxins by strains of the Aspergillus flavus group maintained in ATCC. Mycopathologia 93, DOCUMENTATION PROVIDED TO EFSA 1. Dossier on Amaferm. December Submitted by Regal. 2. Dossier on Amaferm. Version April Submitted by Regal.

16 Opinion on Amaferm 16/17 3. Supplementary information on Amaferm. May Submitted by Regal. 4. Supplement 1 to FAD regarding dossier Amaferm. Date 6 October Supplement to Section II 2.5 Control methods. Submitted by Regal. 5. Supplement 2 to FAD regarding dossier Amaferm. Date 18 October Supplement to Section II 2.5 Control methods. Submitted by Regal. 6. Evaluation report of the Community Reference Laboratory feed additives authorisation on the methods(s) of analysis of Amaferm 7. Comments received from Member States via the EFSAnet. SCIENTIFIC PANEL MEMBERS Arturo Anadón, Margarita Arboix Arzo, Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Dierick, Gerhard Flachowsky, Anders Franklin, Jürgen Gropp, Anne-Katrine Lundebye Haldorsen, Ingrid Halle, Alberto Mantovani, Kimmo Peltonen, Guido Rychen, Pascal Sanders, Amadeu Soares, Pieter Wester and Wilhelm Windisch APPENDIX ANNEX A. Executive Summary of the Evaluation Report of the Community Reference Laboratory Feed Additives Authorisation on the Method(s) of Analysis of Amaferm

17 Opinion on Amaferm 17/17 ANNEX A. Executive Summary of the Evaluation Report of the Community Reference Laboratory Feed Additives Authorisation on the Method(s) of Analysis of Amaferm In the current application authorisation is sought for the feed additive Amaferm under the category zootechnical additives, functional group 4 other zootechnical additives, according to the categorisation system of Annex I of Regulation (EC) No 1831/2003. Specifically, authorisation is sought to use Amaferm for dairy cows and beef cattle. Amaferm consists of a fermentation extract originating from a non-pathogenic strain of the fungus Aspergillus oryzae (ATCC 10063) (4 %) sprayed onto wheat bran (96 %). A daily dose between 2 and 5 g Amaferm per animal (beef cattle, cows), mixed in concentrate feed, total mixed ratio or as a top-feeding is recommended. Amaferm contains four defined active agents, an alpha-amylase (1,4-α-D-glucan glucanohydrolase), an endo-1,4-β-d-glucan glucano-hydrolase, a vitamin B complex and citric acid. The applicant proposes colorimetric methods for the two enzymes, a fluorometric rapid method for the vitamin B complex (determined as vitamin B1) and a gravimetric method for citric acid. Although the applicant has cited official methods of analysis from the Association Of the Analytical Community (AOAC) International and one publication for the determination of the four active agents of Amaferm in the fermentation extract, the target matrices of these methods do not cover the additive Amaferm, premixtures and feedingstuffs. In summary, taking into account the proposed methods for the four active agents it is not possible to conclude on their suitability for routine analysis of the additive, premixtures or feedingstuffs due to the absence of supporting experimental data. Equally, it is not possible to comment on the suitability of the proposed methods for the use in official control.