Technical guidance. Compatibility of zootechnical microbial additives with other additives showing antimicrobial activity 1

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1 The EFSA Journal (2008) 658, 1-5 Technical guidance Compatibility of zootechnical microbial additives with other additives showing antimicrobial activity 1 Prepared by the Panel on Additives and Products or Substances used in Animal Feed BACKGROUND (Question EFSA-Q ) Adopted on 5 March 2008 Products containing one or more strains of viable micro-organisms are commonly authorised as zootechnical additives for use in animal nutrition. Such microbial additives are often intended to improve animal performance (e.g., weight gain, feed efficiency) or produce welfare benefits (e.g., survivability). At the same time, other products authorised as feed additives show an antimicrobial activity (e.g., coccidiostats, organic acids). When a microbial additive is introduced in feed containing an additive showing antimicrobial activity, the effect of the microbial additive on animal health or performance may be totally or partially impaired. Therefore, it is necessary to establish the compatibility of those two types of additives when used simultaneously. At present there is no consistency in the data presented to establish compatibility between microbial additives and other authorised additives possessing antimicrobial activity within the digestive tract of livestock. Some applicants base their studies on in vivo trials but measure only performance characteristics, others rely on in vitro data. TERMS OF REFERENCE In view of the above, the FEEDAP Panel is requested to prepare a guidance document on the demonstration of compatibility of microbiological additives with other authorised additives showing antimicrobial activity. 1 For citation purposes: Technical guidance prepared by the Panel on Additives and Products or Substances Used in Animal Feed (FEEDAP) on the compatibility of zootechnical microbial additives with other additives showing antimicrobial activity. The EFSA Journal (2008) 658, 1-5 European Food Safety Authority, 2008

2 1. Introduction The efficacy of a microbial additive may be adversely affected by the conditions encountered in the feed or, subsequently, in the digestive tract; particularly, by the presence of agents or compounds with an antimicrobial activity. However, there are circumstances in animal production when the use of compounds with a known antimicrobial effect is necessary or, at least, desirable. This may be triggered by the need to treat an individual animal for a particular condition, or may arise when a group of animals is at risk from an endemic disease. In the latter case, the use of a prophylactic may be advised. One of the most commonly occurring situations where a prophylactic treatment could adversely affect the efficacy of a microbial additive arises from the routine use of ionophores as coccidiostats. Coccidiosis, a debilitating infection of livestock caused by protozoa, is managed by the introduction in feed of compounds selected for their anti-protozoal activity but most of them incidentally demonstrating some level of antibacterial activity. When it is intended to give a microbial additive in conjunction with another additive with a potential for antimicrobial activity there is an evident need for compatibility. This is formally recognised in the case of coccidiostats. However, there are other additives with known antimicrobial properties (e.g. organic acids) which might trigger a need for an assessment of compatibility of a microbial additive. The purpose of this guidance document is not to define the conditions under which microbial additives can or might be used in conjunction with other additives with antimicrobial activity, but to suggest how compatibility should be assessed if needed. The FEEDAP Panel proposes a stepwise approach to establishing compatibility in which an in vitro screen is used to exclude cases where incompatibility is very unlikely, reserving in vivo studies for situations where in vitro results suggest that an incompatibility may exist. 2. Criteria for assessing the compatibility of microbial additives with other authorised additives possessing antimicrobial activity If it can be demonstrated that the efficacy of the microbial additive does not depend on the viability of the microbial cells, then evidence of compatibility is not required. Otherwise, a stepwise approach to assessing the compatibility of additives should be followed as illustrated in the decision tree below (Figure 1). The EFSA Journal (2008) 658, 2-5

3 Compatibility in feed/water Is the microbial additive stable in feed/water in presence of the additive with antimicrobial activity? (See section 2.1) In vitro assessment Is MIC * > 4 times the maximum concentration of antimicrobial in feed? (See section 2.2) In vivo assessment Can equivalence be established between gastrointestinal counts of the microbial additive in the presence/absence of the antimicrobial additive? (See section 2.3) Compatibility assumed t compatible * MIC: minimum inhibitory concentration Figure 1. Decision tree for the assessment of compatibility between additives 2.1. Compatibility in feed/water The combination of the two additives should not adversely affect the viability of the microbial cells in feedingstuffs or water. In dry feed, it is generally assumed that no interaction between the two additives occurs and therefore no effects on compatibility are expected. When interaction is possible (e.g. both additives administered in water or in wet or liquid feeding) then stability should be assessed reflecting the conditions of practical use, particularly the duration for which the additives remain in contact. To demonstrate compatibility under those circumstances, two treatments should be used the microbial additive and the microbial additive plus the product with antimicrobial activity and microbial cell numbers measured. Studies should be designed using the lowest proposed dose of the The EFSA Journal (2008) 658, 3-5

4 microbial additive and the maximum proposed dose of the product showing antimicrobial activity. Compatibility is assumed when mean differences in numbers between the two treatments are no greater than ± 0.5 log order. For products containing multiple microbial strains, the viability should be separately assessed for each strain In vitro studies The purpose of the in vitro studies is to establish whether the viability of the microbial additive is likely to be affected at the probable concentration of the antimicrobial additive in the digestive tract, and consequently whether in vivo studies are necessary. This is done by determining the minimum inhibitory concentration (MIC) of the antimicrobial additive. The MIC should be determined, according to standardised procedures, by using two-fold dilution procedures in agar or broth of the active antimicrobial substance. After incubation, the MIC is defined as the lowest concentration of the substance that inhibits microbial growth. The existing body of scientific information on the same or related microbial species must be considered when the procedure for MIC determination (dilution method, growth media and incubation conditions) is chosen, keeping in mind the possible interference of media and growth conditions (e.g. buffering effect in case of organic acids). In case of micro-organisms producing spores, the MIC should be calculated with vegetative cells. For products composed by multiple microbial strains, the MIC should be determined for each individual strain and the results interpreted in terms of the most sensitive component. If the MIC is greater than four times the maximum concentration of the antimicrobial in feed/water, compatibility is assumed and no in vivo tests are required. If the MIC is equal to or below four times the maximum concentration of the antimicrobial in feed/water, incompatibility cannot be excluded and should be assessed in vivo In vivo compatibility studies in target species To demonstrate compatibility in vivo, one short-term experiment comparing two treatments (microbial additive and microbial additive plus product with antimicrobial activity) should be performed. Studies should be designed using the lowest proposed dose of the microbial additive and the maximum proposed dose of the product showing antimicrobial activity. The trials should be conducted ensuring that the health of animals and the husbandry conditions (e.g. veterinary intervention) do not adversely affect the interpretation of the results. Care should be taken to avoid cross-contamination of feed, and this should be demonstrated experimentally. The experimental design should have adequate statistical power. Compatibility should be determined by analysing counts (or other means of estimating cell numbers) of microbial cells of the additive in gastrointestinal contents. Faecal counts are the minimum requirement, but preferably counts should be made in ileal/caecal contents. To avoid the possible interference of the animal s gut microbiota in the determination of cell counts, recognition of the active agent at strain level should be achieved. For products composed of multiple microbial strains, each strain should be individually enumerated. In the case of Bacillus and other spore formers, there is evidence that spores are able to germinate in the gastrointestinal tract of animals (Hong et al., 2005, Tam et al., 2006, Leser et al., 2007). Vegetative cells are more likely to be affected by the substances showing antimicrobial activity than the spores. Therefore, in case of products containing bacterial spores, both vegetative cells and spores should be enumerated. The EFSA Journal (2008) 658, 4-5

5 Compatibility is demonstrated between the two groups if the gastrointestinal counts (or estimated numbers) of vegetative cells (and spores when present) are equivalent. Estimating equivalence should take account of the variability of the experimental data but, as a guide, equivalence could be assumed when counts differ by no more than ±1.0 log order. In the absence of estimates of microbial numbers, performance data alone are not considered sufficient to establish compatibility. 3. Extrapolation between animal species Compatibility determined in vitro can generally be extrapolated to all species provided the doses used would allow the same interpretation of the MIC values. Compatibility determined in vivo in one species can be extrapolated to other physiologically similar species provided the doses used are essentially similar. REFERENCES Hong, H.A., Duc, L.H. and Cutting, S.M., The use of bacterial spore formers as probiotics. FEMS Microbiology Reviews. 29, Tam, N.K., Uyen, N.Q., Hong, H. A., Duc, L. H., Hoa, T.T., Serra, C.R., Henriques, A.O., Cutting, S.M., The intestinal life cycle of Bacillus subtilis and close relatives. J. Bacteriol. 188(7), Leser, T.D., Knarreborg, A., Worm, J., Germination and outgrowth of Bacillus subtilis and Bacillus licheniformis spores in the gastrointestinal tract of pigs. J. Appl. Microbiol. [Epub ahead of print] The EFSA Journal (2008) 658, 5-5